Engineered induced stem cell-derived myeloid cells and their differentiation and use methods

Abstract

A composition and method for a cell population comprising macrophages having synthetic cytokine receptors for a non-physiological ligand are provided. The non-physiological ligand activates the synthetic cytokine receptor in engineered stem cells to induce differentiation of the stem cells and induce expansion and / or activation of the resulting cytotoxic macrophages.

Description

Technology Field

[0001] Cross-reference regarding related applications

[0002] This application claims priority to U.S. provisional application No. 63 / 609,821 filed on December 13, 2023 (title of invention “Engineered induced stem cell-derived myeloid cells and their differentiation and use methods”), the contents of which are incorporated by reference in their entirety.

[0003] Included as a reference in the sequence list

[0004] The present application is filed together with a sequence list in electronic form. The sequence list is provided as a file named 260132001440SeqList.xml, which is 97,096 bytes in size and was created on December 13, 2024. The full text of the information in electronic form of the sequence list is incorporated by reference.

[0005] field

[0006] The present disclosure provides a composition and method relating to a cell population comprising engineered myeloid cells derived from stem cells, comprising synthetic cytokine receptors for a non-physiological ligand. The non-physiological ligand activates the synthetic cytokine receptor in the engineered stem cells to induce differentiation of the resulting myeloid cells (e.g., macrophages) and to induce expansion and / or activation. Background Technology

[0007] Macrophages are a class of immune cells that can be used in immunotherapy, including cancer immunotherapy. Macrophages are a cell type generally identified as positive for the cell surface protein CD14 (CD14+) and other markers, and identified as possessing phagocytic and cytotoxic activity.

[0008] Macrophages for use in immunotherapy may be derived from and obtained from primary sources, such as peripheral blood or umbilical cord blood. Artificial sources for macrophages include induced pluripotent stem cells (iPSCs), which are cells derived from somatic cells (typically fibroblasts or peripheral blood mononuclear cells [PBMCs]), and pluripotent stem cells, including human embryonic stem cells (hESCs), which are induced to enable unlimited proliferation and differentiation into other cell types when applied to appropriate differentiation conditions.

[0009] A method for differentiating iPSCs into CD34+ HPCs using the culture of single-cell iPSCs in embryonic bodies (EB) or feeder cells is known. Subsequently, CD34+ HPCs can differentiate into myeloid progenitor cells. Improved methods are needed, including methods for differentiating stem cells into macrophages and using them in immunotherapy.

[0010] In some aspects, engineered cells that are myeloid progenitor cells comprising synthetic cytokine receptors for non-physiological ligands are provided herein, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit gamma (IL-2RG); and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit beta (IL-2RB), an intracellular domain of the interleukin-7 receptor subunit beta (IL-7RB), and / or an intracellular domain of the interleukin-21 receptor subunit beta (IL-21RB). In some of the embodiments provided, the myeloid progenitor cells are granulocyte / monocyte progenitor cells (“GMP”). In some of the provided embodiments, the myeloid progenitor cells are characterized by surface phenotypes CD34+, CD90-, and CD45RA+. In some of the provided embodiments, the myeloid progenitor cells are characterized by surface phenotypes CD34+, CD90-, CD123+, and CD45RA+.

[0011] In some aspects, engineered cells that are myeloid cells comprising synthetic cytokine receptors for non-physiological ligands are provided herein, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. In some of the embodiments provided, the myeloid cells are macrophages, neutrophils, megakaryocytes, monocytes, basophils, eosinophils, and / or erythrocytes.

[0012] In some aspects, engineered cells are provided herein as macrophages comprising synthetic cytokine receptors for non-physiological ligands, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit gamma (IL-2RG); and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit beta (IL-2RB), an intracellular domain of the interleukin-7 receptor subunit beta (IL-7RB), and / or an intracellular domain of the interleukin-21 receptor subunit beta (IL-21RB). In some of the embodiments provided, the macrophage is a mature macrophage expressing CD14.

[0013] In some aspects, engineered cells that are neutrophils comprising synthetic cytokine receptors for non-physiological ligands are provided herein, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. In some of the provided embodiments, the engineered cells are differentiated from stem cells. In some of the provided embodiments, the stem cells are pluripotent stem cells. In some of the provided embodiments, the stem cells are induced pluripotent stem cells. In some of the provided embodiments, the myeloid cells are induced myeloid cells (iMCs) differentiated from stem cells engineered with synthetic cytokine receptors. In some of the provided embodiments, the macrophages are induced macrophages (iMACs) differentiated from stem cells engineered with synthetic cytokine receptors. In some of the provided embodiments, the neutrophils are induced neutrophils (iNEUs) differentiated from stem cells engineered with synthetic cytokine receptors. In some of the provided embodiments, the synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an IL-2RG intracellular domain in the order from N-terminus to C-terminus, and the synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain in the order from N-terminus to C-terminus. In some of the provided embodiments, the first dimerization domain and the second dimerization domain are extracellular domains.

[0014] In some of the provided embodiments, the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 1, or a polypeptide sequence as presented in SEQ ID NO: 1. In some of the provided embodiments, the first transmembrane domain comprises the IL-2RG transmembrane domain. In some of the provided embodiments, the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 8 or 31, or a polypeptide sequence as presented in SEQ ID NO: 8 or 31. In some of the provided embodiments, the beta-chain intracellular domain comprises the IL-2RB intracellular domain. In some of the provided embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, or a polypeptide sequence as presented in SEQ ID NO: 2. In some of the provided embodiments, the beta-chain intracellular domain is the IL-7RB intracellular domain. In some of the provided embodiments, the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as presented in SEQ ID NO: 3. In some of the provided embodiments, the beta-chain intracellular domain comprises the IL-21RB intracellular domain. In some of the provided embodiments, the IL-21RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 4, or a polypeptide sequence as presented in SEQ ID NO: 4. In some of the provided embodiments, the second transmembrane domain comprises a transmembrane domain from the same polypeptide as the beta-chain intracellular domain.In some of the provided embodiments, the second transmembrane domain is a transmembrane domain of IL-2RB comprising a polypeptide sequence that is at least 95% identical to SEQ ID NO: 35 or 36, or a polypeptide sequence as presented in SEQ ID NO: 35 or 36.

[0015] In some of the provided embodiments, the first transmembrane domain of the synthetic gamma-chain polypeptide is an IL-2RG transmembrane domain comprising the sequence presented in SEQ ID NO: 8 or 31, and the IL-2RG intracellular domain comprises the sequence presented in SEQ ID NO: 1; the second transmembrane domain of the synthetic beta-chain polypeptide is an IL-2RB transmembrane domain comprising the sequence presented in SEQ ID NO: 35 or 36, and the beta-chain intracellular domain is an IL-2RB intracellular domain comprising the sequence presented in SEQ ID NO: 2. In some of the provided embodiments, the first transmembrane domain and the IL-2RG intracellular domain of the synthetic gamma-chain polypeptide comprise the sequences presented in SEQ ID NO: 31 and SEQ ID NO: 1; The second transmembrane domain and the beta-chain intracellular domain of the synthetic beta-chain polypeptide include the sequences presented in sequence identification number: 35 and sequence identification number: 2.

[0016] In some of the provided embodiments, the first dimerization domain and the second dimerization domain are heterodimerization domains selected from the FKBP12-rapamycin binding (FRB) domain and the FK506-binding protein (FKBP) of size 12 kD; and the non-physiological ligand is rapamycin or rapalog. In some of the provided embodiments, the first dimerization domain is FKBP and the second dimerization domain is FRB. In some of the provided embodiments, the first dimerization domain is FRB and the second dimerization domain is FKBP. In some of the provided embodiments, the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, the FRB domain comprises the polypeptide sequence presented in SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 30. In some of the provided embodiments, the FKBP domain comprises the polypeptide sequence presented in SEQ ID NO: 5 or SEQ ID NO: 30.

[0017] In some of the provided embodiments, the synthetic gamma-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 28, and the synthetic beta-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 55. In some of the provided embodiments, the synthetic gamma-chain polypeptide has the amino acid sequence presented in SEQ ID NO: 28, and the synthetic beta-chain polypeptide has the amino acid sequence presented in SEQ ID NO: 55. In some of the provided embodiments, the synthetic gamma-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 28, and the synthetic beta-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 55. In some of the provided embodiments, the synthetic gamma-chain polypeptide has the amino acid sequence presented in SEQ ID NO: 28, and the synthetic beta-chain polypeptide has the amino acid sequence presented in SEQ ID NO: 55.

[0018] In some of the provided embodiments, the synthetic gamma-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 56, and the synthetic beta-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 57. In some of the provided embodiments, the synthetic gamma-chain polypeptide has the amino acid sequence presented in SEQ ID NO: 56, and the synthetic beta-chain polypeptide has the amino acid sequence presented in SEQ ID NO: 57.

[0019] In some of the provided embodiments, the first dimerization domain and the second dimerization domain are homomerization domains selected from: i) FK506-binding protein (FKBP) of size 12 kD; ii) cyclopyli A (CypA); or iii) gyrase B (CyrB); the non-physiological ligands are each as follows: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine-A (CsA) or its analogs; or iii) coumermycin or its analogs. In some of the provided embodiments, the engineered cells are resistant to rapamycin-mediated mTOR inhibition. In some of the provided embodiments, the engineered cells express a cytosolic polypeptide that binds to a non-physiological ligand, optionally wherein the cytosolic polypeptide is a cytosolic FRB domain. In some of the provided embodiments, the non-physiological ligand is rapamycin or rapalog, and the engineered cells express a cytosolic FRB domain or a variant thereof.

[0020] In some of the provided embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, the engineered cell comprises a disrupted FKBP12 gene that reduces the expression of FKBP12 within the cell. In some of the provided embodiments, the engineered cell comprises a knockout of the FKBP12 gene. In some of the provided embodiments, the engineered cell comprises a nucleotide sequence encoding a synthetic cytokine receptor inserted into the cell genome. In some of the provided embodiments, the nucleotide sequence encoding the synthetic cytokine receptor is inserted into a non-target locus within the cell genome. In some of the provided embodiments, a nucleotide sequence encoding a synthetic cytokine receptor is inserted into an endogenous gene of a cell. In some of the provided embodiments, the insertion reduces the expression of the endogenous gene within the locus. In some of the provided embodiments, the insertion knocks out the endogenous gene within the locus. In some of the provided embodiments, the insertion is by homology-directed repair. In some of the provided embodiments, the endogenous gene is a housekeeping gene, a blood-lineage specific locus, or an immune-related gene. In some of the provided embodiments, the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB). In some of the provided embodiments, the immune-related gene is selected from the beta-2-microglobulin (B2M) gene and the signal regulatory protein alpha (SIRPA) gene.In some of the provided embodiments, the engineered cell comprises a B2M knockout. In some of the provided embodiments, the engineered cell comprises a B2M knockout and an FKBP12 knockout.

[0021] In some of the provided embodiments, the engineered cells comprise a chimeric antigen receptor (CAR). In some of the provided embodiments, binding of a non-physiological ligand to a synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered cells, thereby inducing expansion and / or activation of the engineered cells in a cell population.

[0022] In some aspects, a population comprising any engineered myeloid progenitor cells provided herein is provided herein. In some aspects, a population comprising any engineered myeloid cells provided herein is provided herein. In some aspects, a population comprising any engineered macrophages provided herein is provided herein. In some aspects, a population comprising any engineered neutrophils provided herein is provided herein.

[0023] In some aspects, a method for generating genetically engineered myeloid cells differentiated from stem cells is provided herein, comprising: a) culturing a population of stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a hematopoietic progenitor (HP) population, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. and b) a step of culturing the cells produced in a) by incubation under conditions for generating myeloid cells, wherein at least part of one or both of steps a) and b) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, the myeloid cells are macrophages or neutrophils.

[0024] In some aspects, a method for generating genetically engineered macrophages differentiated from stem cells is provided herein, comprising: a) culturing a population of stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a hematopoietic progenitor (HP) population, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular interleukin-2 receptor subunit gamma (IL-2RG) domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an intracellular interleukin-2 receptor subunit beta (IL-2RB) domain, an intracellular interleukin-7 receptor subunit beta (IL-7RB) domain, and / or an intracellular interleukin-21 receptor subunit beta (IL-21RB) domain. and b) a step of culturing the cells produced in a) by incubation under conditions for generating macrophages, wherein at least part of one or both of steps a) and b) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor.

[0025] In some of the provided embodiments, culture in a) is performed by a first incubation under conditions for producing an embryoid body (EB), followed by one or more additional incubations in the presence of a non-physiological ligand and one or more myeloid cell differentiation factors optionally selected from one or more of IL-3, M-CSF, and GM-CSF. In some of the provided embodiments, one or more myeloid cell differentiation factors are IL-3, M-CSF, and GM-CSF. In some of the provided embodiments, one or more additional incubations include a second incubation in a second medium containing one or more of IL-3, GM-CSF, and M-CSF, and a third incubation in a third medium containing one or more of IL-3, GM-CSF, and M-CSF, wherein one or both of the second medium and the third medium contain a non-physiological ligand.

[0026] In some of the provided embodiments, step a) comprises: (i) performing a first incubation comprising culturing a population of stem cells engineered with a synthetic cytokine receptor under conditions for forming a first aggregate in a first medium; (ii) contacting the aggregate with a dissociating agent to form a dissociated cell population; (iii) performing a second incubation comprising culturing the dissociated cell population under conditions for forming a second aggregate in a second medium; and (iv) performing a third incubation comprising culturing the cell population of (iii) in a third medium. In some embodiments, the second medium comprises one or more of IL-3, GM-CSF, and M-CSF. In some embodiments, the third medium comprises one or more of IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, the first incubation is performed in a first medium comprising one or more of BMP4, FGF2, VEGF-165, and Rock inhibitor. In some of the provided embodiments, the first incubation is performed in a first medium comprising BMP4, FGF2, VEGF-165, and a Rock inhibitor. In some of the provided embodiments, the Rock inhibitor is Y27632. In some of the provided embodiments, the second medium further comprises a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, the second medium does not comprise a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, culture in the first medium is performed for 1 to 3 days. In some of the provided embodiments, the second medium comprises one or more of BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, the second medium comprises BMP4, FGF2, VEGF, LY294002, IL-3, and M-CSF.In some of the provided embodiments, the second medium further comprises a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, the second medium does not comprise a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, culture in the second medium is performed for 3 to 6 days. In some of the provided embodiments, the third medium comprises one or more of UM729, StemRegenin-1, BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, the third medium comprises UM729, StemRegenin-1, BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, the third medium further comprises a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, the third medium does not comprise a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, culture in the third medium is carried out for 6 to 12 days.

[0027] In some of the provided embodiments, the culture in a) produces myeloid progenitor cells. In some of the provided embodiments, the myeloid progenitor cells are granulocyte / monocyte progenitor cells (“GMP”). In some of the provided embodiments, the myeloid progenitor cells are characterized by surface phenotypes CD34+, CD90-, and CD45RA+. In some of the provided embodiments, the myeloid progenitor cells are characterized by surface phenotypes CD34+, CD90-, CD123+, and CD45RA+. In some of the provided embodiments, the culture in b) is carried out in a medium containing one or more of UM729, SCF, StemRegenin1, IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, the culture in b) is carried out in a medium containing UM729, SCF, StemRegenin1, IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, culture in b) is carried out in a medium further comprising a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, culture in b) is carried out in a medium not comprising a non-physiological ligand of a synthetic cytokine receptor. In some of the provided embodiments, culture in b) is carried out for 12 to 24 days.

[0028] In some aspects, a method for generating myeloid cells genetically engineered to express a synthetic cytokine receptor is provided herein, comprising: culturing a population of hematopoietic progenitor (HP) cells derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a myeloid cell population, wherein at least part of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimeric domain, a first transmembrane domain, and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG); A synthetic beta-chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta-chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain.

[0029] In some aspects, a method for generating myeloid cells genetically engineered to express a synthetic cytokine receptor is provided herein, comprising: culturing a myeloid progenitor cell population derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a myeloid cell population, wherein at least part of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimeric domain, a first transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit gamma (IL-2RG); A synthetic beta-chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta-chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. In some of the provided embodiments, the myeloid cells are macrophages or neutrophils.

[0030] In some aspects, a method for generating macrophages genetically engineered to express a synthetic cytokine receptor is provided herein, comprising: culturing a population of hematopoietic progenitor (HP) cells derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a macrophage population, wherein at least part of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimeric domain, a first transmembrane domain, and an intracellular domain of interleukin-2 receptor subunit gamma (IL-2RG); A synthetic beta-chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta-chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain.

[0031] In some aspects, a method for generating macrophages genetically engineered to express a synthetic cytokine receptor is provided herein, comprising: culturing a myeloid progenitor cell population derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating cells into a macrophage population, wherein at least part of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimeric domain, a first transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit gamma (IL-2RG); A synthetic beta-chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta-chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain.

[0032] In some of the provided embodiments, culture is carried out in a medium comprising one or more of UM729, SCF, StemRegenin1, IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, culture is carried out in a medium comprising UM729, SCF, StemRegenin1, IL-3, GM-CSF, and M-CSF. In some of the provided embodiments, culture is carried out for 12 to 24 days. In some of the provided embodiments, culture of one or both of steps a) and b) is carried out in a bioreactor. In some of the provided embodiments, culture is carried out in a bioreactor. In some of the provided embodiments, the bioreactor is a vertical wheel bioreactor. In some of the provided embodiments, the bioreactor is a vertical wheel bioreactor having a volume of about 10 mL to about 1000 mL. In some of the provided embodiments, the culture in a) is carried out in a bioreactor, wherein the bioreactor is a vertical wheel bioreactor having a volume of about 100 mL. In some of the provided embodiments, the culture in b) is carried out in a bioreactor, wherein the bioreactor is a vertical wheel bioreactor having a volume of about 500 mL. In some of the provided embodiments, the bioreactor is a vertical wheel bioreactor having a volume of about 500 mL. In some of the provided embodiments, the stem cells are pluripotent stem cells. In some of the provided embodiments, the pluripotent stem cells are induced pluripotent stem cells.

[0033] In some of the provided embodiments, the synthetic gamma-chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an IL-2RG intracellular domain in order from N-terminus to C-terminus, and the synthetic beta-chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain in order from N-terminus to C-terminus. In some of the provided embodiments, the first dimerization domain and the second dimerization domain are extracellular domains. In some of the provided embodiments, the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 1, or a polypeptide sequence as presented in SEQ ID NO: 1. In some of the provided embodiments, the first transmembrane domain comprises an IL-2RG transmembrane domain.

[0034] In some of the provided embodiments, the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 8 or 31, or a polypeptide sequence as presented in SEQ ID NO: 8 or 31. In some of the provided embodiments, the beta-chain intracellular domain comprises an IL-2RB intracellular domain. In some of the provided embodiments, the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, or a polypeptide sequence as presented in SEQ ID NO: 2. In some of the provided embodiments, the beta-chain intracellular domain comprises an IL-7RB intracellular domain. In some of the provided embodiments, the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as presented in SEQ ID NO: 3. In some of the provided embodiments, the beta-chain intracellular domain comprises the IL-21RB intracellular domain.

[0035] In some of the provided embodiments, the IL-21RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 4, or a polypeptide sequence as presented in SEQ ID NO: 4. In some of the provided embodiments, the second transmembrane domain comprises a transmembrane domain from the same polypeptide as the intracellular domain.

[0036] In some of the provided embodiments, the second transmembrane domain is a transmembrane domain of IL-2RB comprising a polypeptide sequence that is at least 95% identical to SEQ ID NO: 35 or 36, or a polypeptide sequence as presented in SEQ ID NO: 35 or 36. In some of the provided embodiments, the first transmembrane domain of the synthetic gamma chain polypeptide is an IL-2RG transmembrane domain comprising the sequence presented in SEQ ID NO: 8 or 31, and the IL-2RG intracellular domain comprises the sequence presented in SEQ ID NO: 1; the second transmembrane domain of the synthetic beta chain polypeptide is an IL-2RB transmembrane domain comprising the sequence presented in SEQ ID NO: 35 or 36, and the beta chain intracellular domain comprises the sequence presented in SEQ ID NO: 2.

[0037] In some of the provided embodiments, the first transmembrane domain and the IL-2RG intracellular domain of the synthetic gamma chain polypeptide comprise the sequences presented in SEQ ID NO: 31 and SEQ ID NO: 1; and the second transmembrane domain and the beta chain intracellular domain of the synthetic beta chain polypeptide comprise the sequences presented in SEQ ID NO: 35 and SEQ ID NO: 2. In some of the provided embodiments, the first dimerization domain and the second dimerization domain are heteromerization domains selected from the FKBP12-rapamycin binding (FRB) domain and the FK506-binding protein (FKBP) of size 12 kD; and the non-physiological ligand is rapamycin or rapalog. In some of the provided embodiments, the first dimerization domain is FKBP and the second dimerization domain is FRB. In some of the provided embodiments, the first dimerization domain is FRB and the second dimerization domain is FKBP. In some of the provided embodiments, the FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, the FRB domain comprises the polypeptide sequence presented in SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, the FKBP domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 5 or SEQ ID NO: 30. In some of the provided embodiments, the FKBP domain comprises the polypeptide sequence presented in SEQ ID NO: 5 or SEQ ID NO: 30.

[0038] In some of the provided embodiments, the synthetic gamma chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number: 28, and the synthetic beta chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number: 55. In some of the provided embodiments, the synthetic gamma chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number: 56, and the synthetic beta chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number: 57.

[0039] In some of the provided embodiments, the first dimerization domain and the second dimerization domain are homomerization domains selected from: i) FK506-binding protein (FKBP) of size 12 kD; ii) cyclopyli A (CypA); or iii) gyrase B (CyrB); the non-physiological ligands are each as follows: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine-A (CsA) or its analogs; or iii) cumermycin or its analogs.

[0040] In some of the provided embodiments, cells of a cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) are resistant to rapamycin-mediated mTOR inhibition. In some of the provided embodiments, cells of a cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) express a cytosolic polypeptide that binds to a non-physiological ligand, optionally wherein the cytosolic polypeptide is a cytosolic FRB domain. In some of the provided embodiments, the non-physiological ligand is rapamycin or rapalog, and cells of the population express a cytosolic FRB domain or a variant thereof. In some of the provided embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, the cytosolic FRB domain comprises a polypeptide sequence that is at least 98% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some of the provided embodiments, cells of a cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) comprise a disrupted FKBP12 gene that reduces the expression of FKBP12 within the cells. In some of the provided embodiments, cells of a cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) comprise a knockout of the FKBP12 gene.

[0041] In some of the provided embodiments, a synthetic cytokine receptor is incorporated into the endogenous gene of a cell in a cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) by targeted incorporation of the nucleotide sequence encoding the synthetic cytokine receptor into the endogenous gene. In some of the provided embodiments, the targeted incorporation is by non-homologous end-linking (NHEJ). In some of the provided embodiments, the targeted incorporation is by homologous-directed repair. In some of the provided embodiments, the insertion reduces the expression of the endogenous gene within the locus. In some of the provided embodiments, the insertion knocks out the endogenous gene within the locus. In some of the provided embodiments, the insertion is by homologous-directed repair.

[0042] In some of the provided embodiments, the endogenous gene is a housekeeping gene, a blood-lineage specific locus, or an immune-related gene. In some of the provided embodiments, the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB). In some of the provided embodiments, the immune-related gene is selected from the beta-2-microglobulin (B2M) gene and the signal regulatory protein alpha (SIRPA) gene. In some of the provided embodiments, cells of the cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) contain B2M knockout. In some of the provided embodiments, cells of a cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) include B2M knockout and FKBP12 knockout. In some of the provided embodiments, cells of a cell population (stem cell population, hematopoietic progenitor cell population, or myeloid progenitor cell population) include a chimeric antigen receptor (CAR). In some of the provided embodiments, macrophages are mature macrophages expressing CD14.

[0043] In some of the provided embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some of the provided embodiments, the rapamycin analog is rapalog. In some of the provided embodiments, the non-physiological ligand is in the medium at 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 It is added at concentrations of nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM. In some of the provided embodiments, the non-physiological ligand is added to the medium at a concentration of 10 nM or about 10 nM. In some of the provided embodiments, the non-physiological ligand is added to the medium at a concentration of 100 nM or about 100 nM.

[0044] In some aspects, a myeloid cell population produced by any method provided herein is provided herein. In some aspects, a macrophage population produced by any method provided herein is provided herein. In some of the embodiments provided, the macrophage population expresses CD14. In some aspects, a pharmaceutical composition comprising any engineered myeloid cell population provided herein is provided herein. In some aspects, a pharmaceutical composition comprising any engineered macrophage population provided herein is provided herein. In some of the embodiments provided, the pharmaceutical composition comprises pharmaceutically acceptable excipients. In some of the embodiments provided, the pharmaceutical composition comprises a cryoprotectant.

[0045] In some aspects, a method for expanding myeloid cells is provided herein, comprising contacting any myeloid cell population provided herein or any pharmaceutical composition provided herein with a non-physiological ligand of a synthetic cytokine receptor. In some aspects, a method for expanding macrophages is provided herein, comprising contacting any macrophage population provided herein or any pharmaceutical composition provided herein with a non-physiological ligand of a synthetic cytokine receptor. In some of the embodiments provided, the method is performed in vitro or in vitro.

[0046] In some of the provided embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some of the provided embodiments, the rapamycin analog is rapalog. In some of the provided embodiments, the non-physiological ligand is 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 nM, 100 nM to Contact is made at concentrations of 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM. In some of the provided embodiments, the non-physiological ligand is contacted at a concentration of 10 nM or about 10 nM. In some of the provided embodiments, the non-physiological ligand is contacted at a concentration of 100 nM or about 100 nM. In some of the provided embodiments, the method is performed in vivo on a subject, and the non-physiological ligand is administered to the subject.

[0047] In some aspects, a method for treating a disease or pathological condition in a subject is provided herein by administering to the subject an effective amount of any myeloid cell population provided herein or any pharmaceutical composition provided herein. In some aspects, a method for treating a disease or pathological condition in a subject is provided herein by administering to the subject an effective amount of any macrophage cell population provided herein or any pharmaceutical composition provided herein together with a non-physiological ligand of a synthetic cytokine receptor. In some of the embodiments provided, the disease or pathological condition is cancer. In some of the embodiments provided, the cells express a CAR directed toward an antigen expressed by the cells of the disease or pathological condition. In some of the embodiments provided, the CAR targets a tumor antigen.

[0048] In some of the provided embodiments, the method comprises administering a non-physiological ligand of a synthetic cytokine receptor to a subject. In some of the provided embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some of the provided embodiments, the rapamycin analog is rapalog. In some of the provided embodiments, the non-physiological ligand is administered in doses of 1 mg to 100 mg, optionally 10 to 100 mg, optionally 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, or about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, or any value between any of the above. In some of the provided embodiments, multiple doses of the non-physiological ligand are administered to the subject. In some of the provided embodiments, multiple doses are administered to the subject intermittently or at regular intervals after administration of the macrophage population or its composition for an arbitrarily predetermined period. In some of the provided embodiments, 2 to 8 doses of a non-physiological ligand are administered to the subject. In some of the provided embodiments, a single dose of a non-physiological ligand is administered to the subject.

[0049] In some aspects, a kit is provided herein comprising any engineered cell provided herein or any engineered myeloid cell population provided herein, any macrophage population provided herein or any pharmaceutical composition provided herein and instructions for administering it to a subject requiring it. In some of the embodiments provided, the kit further comprises a container containing a non-physiological ligand and instructions for administering the non-physiological ligand to a subject after administration of the cell population. In some of the embodiments provided, the subject has cancer. Brief explanation of the drawing

[0050] Figure 1a shows a schematic diagram of the CRISPR-Cas-mediated site-specific melting of a construct encoding RACR. Figure 1b shows a histogram panel illustrating the detection of RACR proteins after melting at various promoters. Figure 1c is a graph showing the detection of RACR proteins after melting in various promoters including locus 1 (EEF1A1) and locus 2 (ACTB). Figure 2a shows a schematic diagram illustrating the role of FKBP12 in the inhibition of proliferation by rapamycin via mTOR. Figure 2b is a graph showing protection from rapamycin-mediated inhibition of iPSC proliferation in polyclonal FKBP12 knockout (KO) cell lines (left panel) and phase contrast images of morphology in wild-type and FKBP12 KO cells (right panel). Figure 2c is a graph showing the confluence of wild-type iPSCs after 4 days of treatment with various doses of rapamycin. Figure 2d is a graph showing the confluence of FKBP12 KO iPSC growth during 4 days of treatment with various doses of rapamycin. Figure 2e is a graph showing the ratio of hematopoietic progenitors (HP) to iPSCs of clonal FKBP12 KO iPSCs compared to control iPSCs. Figure 2f is a graph showing the confluence of FKBP12 KO iPSC growth during 4 days of treatment with 25 nM of rapamycin. Figure 3 is a schematic diagram showing the experiment timeline. Figure 4 is a graph showing CD14 expression in cells on days 19-24 of differentiation via flow cytometry. Figure 5 is a graph showing CD19 CAR expression in cells on days 19-24 of differentiation via flow cytometry. Figure 6a is a graph showing the proportion of tumor cells phagocytosed by engineered macrophages over a period of 29 hours. Figure 6b is an image showing the progression of tumor cells phagocytosed by engineered macrophages at 0, 24, and 48 hours. Specific details for implementing the invention

[0051] Genetically engineered myeloid cells, such as macrophages and neutrophils, differentiated from progenitor cells engineered with synthetic cytokine receptors, and a method for the resulting differentiation are provided herein. In some embodiments, the genetically engineered myeloid cells are derived from stem cells or myeloid progenitor cells containing synthetic cytokine receptors. In some embodiments, the synthetic cytokine receptor contains an intracellular domain from a common gamma chain intracellular signaling domain (e.g., interleukin-2 receptor subunit gamma, IL-2RG) and an intracellular domain from an interleukin-2 receptor subunit beta (IL-2RB). In some embodiments, the synthetic cytokine receptor also contains an extracellular domain that can be bound by a non-physiological ligand (e.g., rapamycin or an analog). In this way, the binding of a non-physiological ligand to the extracellular domain of a synthetic cytokine receptor activates cytokine receptor-mediated signaling, including JAK / STAT signaling, which is an important pathway for the differentiation of stem cells, e.g., iPSCs or other pluripotent stem cells, into downstream cell lineages, e.g., myeloid cells (e.g., macrophages or neutrophils). Therefore, in the presence of a non-physiological ligand (e.g., rapamycin), the synthetic cytokine receptor may bind during cell differentiation, thereby eliminating the need for endogenous receptors or exogenous growth factors. In some embodiments, this increases the control of JAK / STAT signaling and reduces its variability during cell differentiation, thereby allowing for the efficient generation of induced myeloid cells.

[0052] Myeloid cells for use in immunotherapy can be derived from and obtained from primary sources, such as peripheral blood or umbilical cord blood. Artificial sources for myeloid cells include induced pluripotent stem cells (iPSCs), which are cells derived from somatic cells (typically fibroblasts or peripheral blood mononuclear cells [PBMCs]), and pluripotent stem cells, including human embryonic stem cells (hESCs), which are induced to enable unlimited proliferation and differentiation into other cell types when applied to appropriate differentiation conditions. Induced pluripotent stem cells (iPSCs) are a renewable, modifiable, and scalable source of material for the manufacture of cell therapies. iPSCs can be created by reprogramming adult cells into a cellular state similar to embryonic stem cells. iPSCs are thought to be capable of differentiating into all cell types found in the human body and possess unlimited scalability, meaning they can reproduce and proliferate indefinitely to generate a nearly endless supply of starting material. Furthermore, iPSCs are processable for precise multi-genome editing, allowing for the safe introduction of multiple genetic modifications. Due to these characteristics, iPSCs provide consistent starting materials derived from a single cell (clone), which enables consistent genomic integrity in process intermediates and final cell products.

[0053] Myeloid cells can be derived from iPSCs by sequentially differentiating iPSCs into hematopoietic progenitor cells (HPCs), also referred to as hematopoietic stem cells (HSCs); HPCs into myeloid progenitor cells; and then myeloid progenitor cells into myeloid cells, such as macrophages (referred to as iPSC-derived macrophages). Generally, iPSC-macrophages express CD14 and possess phagocytic and cytotoxic activity like macrophages; however, iPSC-macrophages may differ from macrophages phenotypically and in other aspects.

[0054] However, current iPSC-based approaches to cell therapy face major challenges. Specifically, current methods for differentiating iPSCs into therapeutic immune cell types require exogenous growth factors and, in some cases, the presence of feeder cells. Overall, iPSC-based cell therapies are generally inefficient at generating the necessary intermediate progenitor cells, resulting in low initial yields of therapeutic cell types (e.g., macrophages) and requiring subsequent feeder cell-driven expansion. Such feeder cell-driven expansion can dramatically reduce the proliferative capacity of the final cell therapy product. Therefore, to achieve the engraftment required for any therapeutic effect, high cell numbers (~1 billion cells) and repeated dosing are necessary, in addition to repeated cycles of lymphocyte-depleting chemotherapy.

[0055] Furthermore, current approaches to the manufacture of cell therapies involve autologous or allogeneic cell starting materials in which disease-targeted therapeutic cell products are engineered. In some embodiments, allogeneic or "off-the-shelf" cell therapies have the potential to shift cell therapy from personalized medicine to routine treatment. However, current "off-the-shelf" cell therapies are struggling to demonstrate the same in vivo cell engraftment and persistence achieved by approved autologous cell therapy products. In some respects, this is due to the foreign nature of allogeneic or even engineered elements of autologous cells, which can be recognized and rejected by the host immune system. To date, cell therapies achieve cell engraftment by treating patients with a highly toxic chemotherapy regimen (referred to as Lymphocyte Depletion (LD)) prior to the administration of the cell therapy product. LD essentially eliminates the host immune system and offers many benefits to the cell therapy product; first, it provides free "homeostatic cytokines" to the ex vivo cell therapy product, as well as reducing the anti-graft response to the foreign graft by the host immune system. However, LD is a transient solution, and the host immune system is rapidly reconfigured. Therefore, to sustain allogeneic cell exposure, multiple rounds of LD and cell infusion are required. Additionally, exogenous cytokines, such as IL-2, are administered, and these cytokine treatments have low exposure times due to the high toxicity associated with their use. Finding better ways to increase cell persistence is crucial for achieving sustained tumor remission and has proven to be a challenging task in the field of allogeneic cell therapy.

[0056] Allogeneic cells can be further degraded into donor- or iPSC-derived cells. Donor-derived cells are typically sourced from the circulating or cord blood of healthy donors, and therapeutic cell types (e.g., myeloid cells, such as macrophages and neutrophils) are generally selected in complex cell culture processes involving multiple cytokines, growth factors, genetic engineering, and feeder cells, subsequently harvested, and expanded to generate large volumes. Alternatively, iPSC-derived cells, which also require multiple complex cell culture conditions, must implement these conditions in a stepwise manner to drive the cells through the progenitor steps necessary to ultimately obtain the intended end-cell product (e.g., immune effector cells). For example, macrophages are classically activated by specific molecular patterns commonly found in pathogenic organisms, such as lipopolysaccharides (LPS) or nucleic acid CpGs. Furthermore, it is known that effective methods for macrophage activation and expansion for clinical-scale purposes require exogenous cytokines, including IL-4 and / or IL-13, as well as antigen molecules, co-stimulatory molecules, and / or cell adhesion molecules. Macrophages are phagocytic cells characterized by the ability to distinguish between self and non-self by monitoring the expression of MHC class I molecules and the release of cytokines, and to directly kill and / or phagocytize non-self or infected target cells. It is known in the relevant art that macrophages do not represent a homogeneous population. Rather, there are many distinguishable subsets of macrophages. Effectively expanding macrophages dependent on large quantities of various exogenous factors using currently known methods often requires complex and expensive manufacturing processes.

[0057] A method for generating and expanding genetically engineered myeloid cells, such as macrophages and neutrophils, by utilizing synthetic cytokine receptors is provided herein, wherein the exogenous factor described in detail above is replenished during the in vitro expansion of macrophages and / or after injection of macrophages into a target.

[0058] Furthermore, the optimal standard for cell therapy is autologous chimeric antigen receptor (CAR) T-cell therapy. Decades of CAR T-cell therapy efforts, ranging from the "first-generation" therapies in the 1990s to the first FDA-approved CAR T-cell therapy in 2017, have resulted in success in the treatment of B-cell malignancies, with long-term remissions achieved in 30–40% of specific patient populations. Importantly, CAR T-cell efficacy requires lymphocyte-depleting chemotherapy to eliminate uptake sources for survival factors, such as IL-15. While CAR T-cell therapy has revolutionized the treatment of malignancies (e.g., hematological), its widespread application is hampered by major limitations. Although the field of allogeneic CAR T-cell therapy has shown promising early clinical results; however, despite the use of continuously increasing intensity LD regimens, sustained response profiles have generally been poor compared to autologous CAR T-cell therapy. This is likely due to limitations in the drug product cell type and manufacturing process, as well as anti-allograft reactions to the therapeutic cells. Therefore, despite the promising clinical efficacy of CAR T cells in hematological malignancies, significant challenges remain, including patient accessibility, complex manufacturing, and high costs.

[0059] Compared to T cells, myeloid cells, such as macrophages, have the advantage of being able to enter solid tumors more easily and are less likely to be suppressed by other types of cells, thus enabling them to play a more suitable role in tumor immunotherapy. Because expressed chimeric antigen receptors are located on the surface of macrophages, macrophages can accurately target tumor cells. The provided engineered macrophages and related methods offer a "ready-to-use" cancer therapy to overcome these challenges.

[0060] iPSCs can also be modified via CRISPR to express CARs, which can overcome challenges associated with, for example, targeting the xenogeneic solid tumor microenvironment.

[0061] Synthetic receptor-available differentiation (ShRED), a directed differentiation and expansion process controlled by a synthetic cytokine receptor, e.g., rapamycin-activated cytokine receptor (RACR), is provided herein. RACR is activated by the addition of its synthetic ligand, rapamycin, which induces JAK / STAT signaling that drives the differentiation and expansion of cells into hematopoietic progenitor cells (HP) and subsequently into immune effector cells (referred to as RACR-induced myeloid cells, e.g., macrophages (RACR-iMAC)). Furthermore, because rapamycin is a safe, effective, and approved therapeutic agent for immunosuppression, RACR can also be bound in vivo through the administration of rapamycin to increase the persistence of RACR-expressing myeloid cells while protecting these cells from allogeneic rejection.

[0062] A platform for producing immune effector cells in the absence of exogenous cytokines and feeder cells is provided herein by genetically modifying iPSCs and iPSC-derived progenitor cells to express synthetic cytokine receptors. Synthetic small molecule ligands (e.g., rapamycin) activate the receptors to drive the differentiation and expansion of immune effector cells. The compositions and methods provided herein comprise myeloid cells (e.g., macrophages and neutrophils) engineered to express synthetic cytokine receptors. The non-limiting advantages of engineered myeloid cells include excellent and controllable expansion when administered to a subject, cytotoxic activity and phagocytosis similar to natural myeloid cells, improved iPSC-derived cell production, and enhanced antitumor activity.

[0063] In the aspects provided, the RACR manipulation platform provided herein improves the production of iPSC-derived cells by controlling cell production. Through the administration of rapamycin and the activation of RACR, a more reproducible differentiation process and uniform cell products are produced. The RACR manipulation platform also reduces manufacturing costs because RACR activation eliminates the need to add expensive growth factors, cytokines, and other raw materials. In certain embodiments, the method disclosed herein may further enhance activation through the ability of the macrophages described herein to expand without exogenous factors or using fewer exogenous factors, such as TGF-β and / or IFN-γ. In some embodiments, the method disclosed herein may further enhance activation and tumor death through the ability of the macrophages described herein to be produced by removing one or more exogenous factors compared to conventional processes. The RACR manipulation platform increases the yield of high-purity intermediates and final cell products. The RACR manipulation platform provided herein generates high-purity hematopoietic progenitors (HP), intermediate progenitor populations, and high-purity, phenotypically mature resulting myeloid cells, thereby eliminating the need for cell sorting after macrophage differentiation. The RACR manipulation platform increases patient-compatibility of cells because there are no feeder cells or xenogeneic cells in the manufacturing process. The RACR manipulation platform is also compatible with cells in suspension, promoting scalability of cell production.

[0064] In another aspect provided, the RACR manipulation platform eliminates the need for additional physical processing of differentiated progenitor cells. In the conventional process of differentiating progenitor cells, residual cell aggregates must be removed before blood cell differentiation. Physical processing includes enzymatic digestion (e.g., collagenase or TrypLE™ enzymes) and filtration (e.g., filtering cells to remove unwanted cell aggregates). In contrast, the RACR manipulation platform results in embryoids that are completely dissociated into pure HP without the need for cell filtration.

[0065] In the aspect provided, the RACR manipulation platform provided herein improves the antitumor activity of iPSC-derived cells by increasing cell engraftment, persistence, and effector function. The RACR manipulation platform provided herein also improves the antitumor activity of iPSC-derived cells by suppressing the host immune response through the administration of rapamycin, which further enables the engraftment of cells (e.g., macrophages). The RACR manipulation platform provided herein also improves the antitumor activity of iPSC-derived cells by activating the RACR system to selectively support RACR cell expansion and survival, thereby eliminating the need for toxic LD.

[0066] In some aspects, the advantages of the RACR system for macrophages containing engineered synthetic cytokine receptors and its activation using rapamycin or rapalog include: the ability to engineer unlimited starting materials characterized by high efficiency in immediate progeny generation and minimized expansion requirements for the final cell type; the ability to efficiently edit cells; the absence of requirements for feeder cells, thereby minimizing complex raw materials; the absence of requirements for lymphocyte depletion in subjects receiving RACR-engineered cells; low or no cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICAN); and the promotion of engraftment, expansion, and persistence by the administration of rapamycin or rapalog.

[0067] Synthetic cytokine receptors that can be applied to support the induction of macrophages are provided herein. Macrophages may be derived from stem or progenitor cells, and such cells are referred herein as “induced macrophage” (iMAC) cells. iMAC cells share distinct cell surface markers and functional properties as described herein. The terms “induced macrophage” or “iMAC” as used herein refer to macrophages produced by inducing the differentiation of progenitor cells. As disclosed herein, iMACs may be produced and / or expanded by expressing synthetic cytokine receptors in stem or progenitor cells and acting on the synthetic cytokine receptors with non-physiological ligands. This process may involve the differentiation of progenitor cells engineered to express synthetic cytokine receptors by activation of the synthetic cytokine receptors. The process may additionally or alternatively involve the expansion of progenitor cells or macrophages by activation of the synthetic cytokine receptors.

[0068] In some embodiments, the present disclosure provides stem cells (e.g., iPSCs) and macrophages engineered to express a rapamycin-activated cytokine receptor (RACR), a synthetic cytokine receptor activated by small molecule rapamycin, or a rapalog. Macrophages containing RACR and activated by rapamycin or a rapalog are referred herein as "RACR-iMAC" cells. Stem cells containing RACR and activated by rapamycin or a rapalog are referred herein as "RACR-SC". RACR has been demonstrated to support the differentiation and / or expansion of RACR-SC and RACR-iMAC cells in a feeder-free manufacturing process. RACR-iMAC cells express multiple innate tumor-targeting receptors and can exert CAR-directed cytolytic activity when engineered to express a chimeric antigen receptor (CAR). Thus, RACR-iMAC cells provide a "pre-made" allogeneic cell therapy.

[0069] The present disclosure relates to a surprising discovery that stem cells engineered to partially express synthetic cytokine receptors differentiate into hematopoietic or myeloid progenitor cells in response to a non-physiological ligand of the receptor. Macrophages differentiated from the engineered stem cells retain the synthetic cytokine receptor and expand in response to a non-physiological ligand of the receptor. The engineered macrophages can be produced in large quantities with functional activity equal to or greater than that of macrophages from other sources.

[0070] Macrophages may be derived from iPSCs, myeloid progenitor cells, or other stem or progenitor cells. Additionally, stem or progenitor cells engineered to express a synthetic cytokine receptor, and a method for differentiating engineered stem or progenitor cells into macrophages by contacting the stem or progenitor cells with a homologous non-physiological ligand for the cytokine receptor are provided herein.

[0071] In certain embodiments, macrophages generated in vitro are provided herein. The engineered cells and related compositions described herein may be used in immunotherapy using ligand-controlled in vitro expansion. Additionally, a method for expanding macrophages by contacting cells with a non-physiological ligand homologous to a synthetic cytokine receptor is provided herein. Furthermore, the engineered macrophages disclosed herein may be further engineered to express a chimeric antigen receptor (CAR), which enables the engineered macrophages to express an antigen recognized by the CAR or to be targeted to cells labeled therewith.

[0072] In some embodiments, the provided engineered macrophages and a method provided for an improved immunotherapy compared to existing strategies. Although chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies, its widespread application is being hindered by major limitations.

[0073] In some embodiments, binding of synthetic cytokine receptors can not only promote differentiation through binding of synthetic cytokine receptors to the provided engineered macrophages but also increase cell growth and promote expansion. In some aspects, the provided engineered macrophages and related methods can be used to increase the in vivo cell growth and expansion of the engineered cell therapy through the administration of rapamycin to the patient following the cell therapy product. In some embodiments, rapamycin simultaneously expands and protects the cells. Expansion is achieved through JAK / STAT signaling activation, and protection is achieved through the suppression of the host anti-graft response by rapamycin. In some embodiments, the need for exogenous cytokine administration as well as lymphocyte depletion is not required. In some embodiments, the provided administration and treatment method using engineered macrophages can be performed without lymphocyte depletion (e.g., without the need for lymphocyte depletion therapy, such as cyclophosphamide and / or fludarabine). In some embodiments, the provided administration and treatment method using engineered macrophages can be performed without the administration of exogenous cytokines.

[0074] In some embodiments, the provided method may also involve administering a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or rapalog) to the subject to expand or reactivate the engineered macrophages in the subject. Therefore, in some embodiments, since cells can be expanded in vivo with a non-physiological ligand, there is no need to re-administer macrophages to the subject. However, re-administration of macrophages is also possible due to low-immunization as described herein, which enables allogeneic cell therapy. Furthermore, since the provided method can be performed without lymphocyte depletion, it offers additional advantages in promoting not only the expansion of the transferred cells but also the host anti-tumor response. This is because the host immune system is maintained without lymphocyte depletion and is not severely depleted. Thus, the immune response generated by the macrophages (e.g., release of cytokines and other pro-inflammatory factors) can stimulate the host's existing immune system against the tumor. Furthermore, the exemplary non-physiological ligand rapamycin not only promotes the expansion of transferred cells through the binding of synthetic cytokine receptors, but the transient mTOR suppression achieved through rapamycin can also reactivate T cells and promote apoptosis in suppressive macrophages. Additionally, while non-physiological ligand rapamycin or its analogs can suppress the host-induced anti-graft response, this is only transient, and a more normal host anti-tumor response is expected to resume once the administration of the non-physiological ligand is discontinued.

[0075] In some embodiments, the engineered cells of the present invention are further modified to be resistant to the effects of rapamycin on inhibiting or reducing cell growth and expansion. In some embodiments, cells may become "rapamycin resistant" by complexing with rapamycin and thereby providing free cytosolic FRBs to the cells to eliminate or reduce rapamycin-mediated growth inhibition of source cells or iMACs. In other embodiments, cells may become "rapamycin resistant" by destroying (e.g., inactivating or knocking out) FKBP12 in the engineered cells. In some cases, overexpression of FRBs may not result in free FRBs capable of completely quenching rapamycin. Therefore, in some cases, editing the endogenous gene within the cell, for example by FKBP12 knockout, may provide complete rapamycin resistance to the cells.

[0076] Accordingly, the provided embodiment using a synthetic cytokine receptor system, such as the rapamycin-activated cytokine receptor (RACR), which can be bound by rapamycin or an analogue, e.g., a rapalog, protects and expands cells with a single technique. Additionally, the further inclusion of genetic disruption of specific immune genes, e.g., beta-2-microglobulin (B2M), e.g., knockout, can also produce "stealth" cells, which have additional advantages in allogeneic cell therapy.

[0077] All publications, including patent literature, scientific papers, and databases mentioned in this application, are incorporated by reference in their entirety for all purposes, to the same extent that each individual publication is incorporated by reference individually. Where a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated by reference herein, the definition set forth herein shall prevail over the definition incorporated by reference herein.

[0078] The section headings used herein are for illustrative purposes only and should not be construed as limiting the subject matter. A person skilled in the art will recognize that various embodiments are possible within the scope and spirit of this disclosure. The following description exemplifies this disclosure and, of course, should not be construed as limiting the scope of the invention described herein in any way.

[0079] I. Definition

[0080] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0081] Unless otherwise indicated by the context, the various features described herein may be used in any combination with any feature or combination of features presented herein, and each feature may be excluded or omitted from the combination.

[0082] As used herein, the singular form includes the plural form unless the context otherwise indicates. The conjugation "and / or" indicates all possible combinations of one or more of the listed items.

[0083] As used herein, "subject" refers to a engineered macrophage or a recipient of another agent. This term includes mammals, such as primates, mice, rats, dogs, cats, cattle, horses, goats, camels, sheep, or pigs, preferably humans.

[0084] As used herein, “to treat,” “treating,” or “treatment” refers to any type of act or administration that provides benefit to a subject having a disease or disorder, including the improvement of the patient’s condition (i.e., improvement, reduction, or remission of one or more symptoms, and a partial or complete response to treatment).

[0085] The term "effective dose" refers to an amount effective in producing a desired biochemical, cellular, or physiological response. The term "therapeutic effective dose" refers to an amount, dosage, or dosage regimen of a therapy effective in inducing a desired therapeutic effect.

[0086] As used herein, “polynucleotide” refers to a biopolymer composed of two or more nucleotide monomers covalently linked by an ester linkage between the phosphoryl group of one nucleotide in the chain and the hydroxyl group of the sugar component of the next nucleotide. DNA and RNA are non-limiting examples of polynucleotides.

[0087] The term "polypeptide" as used herein refers to a polymer consisting of amino acid residues linked together by peptide bonds, which forms part (or all) of a protein.

[0088] A person skilled in the art will understand that due to the degeneracy of the genetic code, numerous different polynucleotides and nucleic acids can code for the same polypeptide. Furthermore, it should be understood that a person skilled in the art can use routine techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed.

[0089] Nucleic acids may include DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides containing synthetic or modified nucleotides. Numerous different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, and the addition of acridine or polylysine chains to the 3' and / or 5' ends of the molecule. It should be understood that for the purposes of use as described herein, polynucleotides may be modified by any method available in the art. Such modifications may be performed to enhance the in vivo activity or lifetime of the polynucleotide of interest.

[0090] The term "variant" means a polynucleotide or polypeptide having at least one substitution, insertion, or deletion in its sequence compared to a reference polynucleotide or polypeptide. A "functional variant" is a variant that retains one or more functions of the reference polynucleotide or polypeptide.

[0091] As used herein, the term “sequence identity,” or “identity” relating to a polynucleotide or polypeptide sequence, refers to the degree of agreement between two optimally aligned polynucleotide or polypeptide sequences at each position of alignment over the entire length of the reference sequence. “Percent identity” is the value obtained by adding the number of matching positions in the optimal alignment to the length of the reference sequence and dividing by the sum of any gap lengths of the reference sequence in the alignment. The optimal alignment is the alignment that results in the maximum percentage identity. Sequence alignment for determining percentage identity can be achieved by numerous widely known methods, including the use of mathematical algorithms, such as those in the BLAST suite or the Clustal Omega sequencing program. Unless otherwise noted, in the claims, the term “sequence identity” refers to sequence identity as calculated by BLAST version 2.12.0 using default parameters. And, unless otherwise noted, alignment is the alignment of all or part of the polynucleotide or polypeptide sequence of interest over the entire length of the reference sequence.

[0092] As used herein, “small molecule” refers to a low molecular weight (<1000 daltons) organic compound. Small molecules can bind to specific biological macromolecules and may have various biological functions or applications, including but not limited to roles as cell signaling molecules, drugs, secondary metabolites, or various other modes of action.

[0093] The term "analogous" in relation to small molecules refers to a compound that has a structure and / or function similar to another compound but differs in specific components. Analogous compounds may differ in one or more atoms, functional groups, or substructures, which are replaced by different atoms, groups, or substructures. Despite high structural and / or functional similarity, analogous compounds may possess different physical, chemical, physicochemical, biochemical, or pharmacological properties.

[0094] The term "rapalog" refers to a group of rapamycin analogs recognized in the relevant technical field that share structural and functional similarities with rapamycin. It is known that certain rapalogs share some, but not all, of the functional properties of rapamycin. For example, some rapalogs promote dimerization but are suitable for use as non-physiological ligands because they substantially lack immunosuppressive activity (e.g., AP21967, AP23102, or iRAP).

[0095] An exemplary rapalog of the present disclosure is AP21967:

[0096]

[0097] An exemplary rapalog of the present disclosure is AP23102:

[0098]

[0099] An exemplary rapalog of the present disclosure is iRAP:

[0100]

[0101] The term "cell population" refers to a mixture of cells suspended in a solution, attached to a substrate, or stored in a container. The characteristics of a cell population as a whole can be studied through bulk measurements of sample volumes containing multiple cells. Flow cytometry methods can be used to reduce background fluorescence issues that occur during bulk cell population measurements.

[0102] The terms "macrophage" or "iMac," as used herein, refer to a class of phagocytic leukocytes that constitute a major component of the innate immune system. Macrophages recognize and absorb foreign pathogens through various mechanisms, including both non-specific mass endocytosis and binding to specific receptors on the cell surface; these receptors bind to epitopes on the bacterial surface itself or to mammalian proteins (antibodies, complement proteins, or other opsonins) bound to the bacterial surface. After internalization of pathogens by macrophages, the pathogens are encapsulated in membrane-bound compartments referred to as phagosomes. Phagosomes fuse with lysosomes to form phagolysosomes. Phagolysosomes contain enzymes, reactive oxygen species, and other toxic molecules that degrade pathogens. Macrophages also internalize and degrade infected cells and cellular debris from the site of active infection, helping to prevent further spread of infection and limit areas of tissue damage. Macrophages also play a role in innate and adaptive immunity by recruiting other immune cells to the site of infection. In humans, mature macrophages can express one or more of CD14, CD11b, CD68, CD163, F4 / 80, CD16, CD54, CD49e, CD38, Egr2, CD71, TLR2, and TLR4.

[0103] The term "manipulated" as used herein refers to cells that are stably transduced with heterologous polynucleotides or applied to gene editing to introduce, delete, or modify intracellular polynucleotides, or cells that are transiently transduced with polynucleotides in a manner that causes a stable phenotypic change in the cells.

[0104] The term "stem cell" as used herein is used to describe cells having an undifferentiated phenotype capable of differentiating into, for example, hematopoietic stem cells, myeloid stem cells, macrophage stem cells, myeloid cells, monocytes, and / or macrophages.

[0105] The term "pluripotency" as used herein means that a stem cell is capable of forming substantially all differentiated cell types of an organism, at least in a culture. For example, embryonic stem cells are a type of pluripotent stem cell capable of forming cells from each of the three germ layers (ectoderm, mesoderm, and endoderm).

[0106] The terms "induced pluripotent stem cell" and "iPSC" as used herein refer to cells derived from somatic cells that have been reprogrammed into a pluripotent state and are capable of proliferation, selective differentiation, and maturation. iPSCs are stem cells produced from differentiated adult, neonatal, or fetal cells that have been induced or modified—that is, reprogrammed—to become cells capable of differentiating into tissues of all three germ layers or the dermis (mesoderm, endoderm, and ectoderm). Produced iPSCs do not refer to cells found in nature.

[0107] The term "hematopoietic stem cell" as used herein refers to stem cells capable of generating both mature myeloid and lymphoid cell types, including macrophages, natural killer cells, T cells, and B cells. Hematopoietic stem cells are typically characterized as CD34+.

[0108] The term "progenitor" refers to a cell that has partially differentiated into a desired cell type. Progenitor cells maintain a certain degree of pluripotency and can differentiate into multiple cell types.

[0109] The term "hematopoietic progenitor cell" as used herein refers to an intermediate cell type capable of differentiating into blood cell lineages, wherein hematopoietic progenitor cells can differentiate into common myeloid progenitor cells or common lymphoid progenitor cells. Hematopoietic progenitor cells are typically characterized as CD34+ and CD45+. CD38 is also considered a marker for hematopoietic progenitor cells. CD45 is considered a hematopoietic lineage marker.

[0110] The terms "myeloid progenitor cell" or "macrophage progenitor cell" as used herein refer to cells that are precursors to myeloid cells, e.g., monocytes and macrophages. Myeloid progenitor cells are the first stage of differentiation of hematopoietic stem cells following the myeloid lineage of differentiation. The term "myeloid progenitor" as used herein refers to a cell capable of hematopoietic transition into a hematopoietic cell-type. Myeloid progenitor cells may be characterized as being CD45+, CD34+, CD150+, and FcγR+.

[0111] As used herein, "differentiate" or "differentiated" is used to refer to the processes and conditions under which undifferentiated or immature (e.g., unspecialized) cells acquire the characteristics of mature (specialized) cells, thereby acquiring specific forms and functions. Stem cells (unspecialized) are often exposed to various conditions (e.g., growth factors and morphogenetic factors) to induce specific lineage determination or differentiation of said stem cells.

[0112] "Expand" or "expand" as used herein refers to an increase in the number and / or purity of a cell type within a cell population through mitosis of cells with limited proliferative capacity, e.g., macrophages.

[0113] "Active," "to activate," or "activate" as used herein refers to the stimulation of activating receptors on macrophages to induce cell division, cytokine secretion, and / or the release of cytolytic granules to regulate or assist immune responses.

[0114] II. Cells engineered with synthetic cytokine receptors and a method for differentiating them into myeloid cells

[0115] Myeloid cells derived from stem or progenitor cells containing synthetic cytokine receptors are provided herein. In some embodiments, the synthetic cytokine receptor is any as described in Section II.B. In some embodiments, the synthetic cytokine receptor contains an intracellular domain from a common gamma chain intracellular signaling domain (e.g., interleukin-2 receptor subunit gamma, IL-2RG) and an intracellular domain from an interleukin-2 receptor subunit beta (IL-2RB). In some embodiments, the synthetic cytokine receptor also contains an extracellular domain that can be bound by a non-physiological ligand (e.g., rapamycin or an analog). In this way, the binding of a non-physiological ligand to the extracellular domain of a synthetic cytokine receptor activates cytokine receptor-mediated signaling, including JAK / STAT signaling, which is an important pathway for the differentiation of stem cells, e.g., iPSCs or other pluripotent stem cells, into downstream cell lineages, e.g., macrophages. Therefore, in the presence of a non-physiological ligand (e.g., rapamycin), the synthetic cytokine receptor may bind during cell differentiation, thereby eliminating the need for endogenous receptors or exogenous growth factors. In some embodiments, this increases the control of JAK / STAT signaling and reduces its variability during cell differentiation, thereby allowing for the efficient generation of induced myeloid cells (iMCs), e.g., induced macrophages (iMACs).

[0116] As described above, stem or progenitor cells capable of differentiating into myeloid cells using a synthetic cytokine receptor complex activated by a non-physiological ligand, and differentiated cells produced from such stem or progenitor cells for use in medical treatment are provided herein. The differentiated cells may be iMCs, but are not limited thereto. As a non-limiting example of the compositions and methods described herein, macrophages may be produced from pluripotent stem cells, such as induced pluripotent stem cells, engineered to express a synthetic cytokine receptor capable of inducing differentiation in addition to or instead of exogenous cytokines by being activated by a non-physiological ligand (e.g., rapamycin) as described. In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR) that uses rapamycin or rapalog to induce differentiation in addition to or instead of exogenous cytokines. An advantage of the embodiment may include the ability to generate effector cells expressing a synthetic cytokine receptor complex activated by a non-physiological ligand from a cell source (e.g., induced pluripotent stem cells), so that the proliferation of the patient's effector cells can be controlled by administering or discontinuing the non-physiological ligand. Another advantage of the embodiment includes, but is not limited to, the ability to eliminate the need for cell sorting after differentiation and to generate a uniform population of effector cells from source cells. Additionally, an additional advantage of effector cells expressing a synthetic cytokine receptor complex activated by a non-physiological ligand (e.g., macrophages) includes the activation of effector cells (e.g., macrophages) against tumor cells without the use of exogenous stimuli (e.g., TGF-β and / or IFN-γ).

[0117] A. Stem cells and engineered stem cells

[0118] In some embodiments, cells engineered with synthetic cytokine receptors, such as the synthetic cytokine receptor described in Section II.B, are provided herein. In some embodiments, the cells are stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0119] The method provided herein for producing myeloid cells from genetically engineered iPSCs may include an in vitro culture process, wherein the myeloid cells are differentiated from non-terminal differentiated cells. In some embodiments, the non-terminal differentiated cells are stem cells. In some embodiments, the non-terminal differentiated cells are iPSC cells. In some embodiments, the non-terminal differentiated cells are progenitor cells. In some embodiments, the non-terminal differentiated cells (e.g., stem cells, e.g., iPSCs) express synthetic cytokine receptors. In one aspect, the present disclosure provides a method for producing macrophages, comprising providing a stem or progenitor cell and differentiating the cell into a macrophage by controlled activation of the synthetic cytokine receptor or without activation of the synthetic cytokine receptor. In some embodiments, differentiation is performed by activation of the synthetic cytokine receptor with one or more additional cytokines. In some embodiments, differentiation may also be performed with cytokines without activation of the synthetic cytokine receptor.

[0120] In some embodiments, the stem cells are pluripotent stem cells. Various sources of pluripotent stem cells, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), may be used in the method. Various sources of pluripotent stem cells, including embryonic stem (ES) cells and induced pluripotent stem cells (iPSCs), may be used in the method. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs) artificially derived from non-pluripotent cells. In some aspects, non-pluripotent cells are cells having less self-renewal and differentiation efficacy than pluripotent stem cells. iPSCs may be generated by a process known as reprogramming, where non-pluripotent cells are effectively "redifferentiated" into an embryonic stem cell-like state by manipulating them to express genes such as OCT4, SOX2, and KLF4. Takahashi and Yamanaka Cell (2006) 126: 663-76.

[0121] In some embodiments, the source cells may be human embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs). In immunotherapy, the source cells are allogeneic or autogenic, meaning they originate from a donor or a subject, respectively. For example, allogeneic cells may be used if the subject being treated with the composition of the present disclosure has received high-dose chemotherapy or radiation therapy to destroy the subject's immune system.

[0122] In some embodiments, myeloid cells may be generated from induced pluripotent stem cells (iPSCs). iPSCs are a type of pluripotent stem cell derived from adult somatic cells that have been genetically reprogrammed into an embryonic stem cell-like state through the forced expression of genes and factors important for maintaining the defining characteristics of embryonic stem cells. iPSCs may be generated from tissues containing somatic cells, including but not limited to skin, tooth tissue, peripheral blood, and urine. To generate iPSCs, somatic cells may be reprogrammed through methods including but not limited to the transient expression of reprogramming factors, a virus-free method, an adenovirus, a plasmid, a minicircle vector, an episome vector, a Sendai virus, synthetic mRNA, self-replicating RNA, a retrovirus, a lentivirus, PhiC31 integrase, exciseable transposons, CRISPR-based gene editing, or recombinant proteins.

[0123] While the cell therapy industry has demonstrated the potential of using genetically engineered patient-derived cells to treat specific disease indications, many challenges remain with the use of patient-derived materials, including limited scalability and expansion capabilities, manufacturing complexity, high costs, patient diversity, and patient accessibility. In contrast, iPSCs are pluripotent stem cells, a cell type capable of theoretically differentiating into any other cell type, including macrophages applicable to cancer treatment. Using iPSCs can provide scalable and simplified manufacturing for targeted cell-attack (e.g., cancer-attack) cell therapies, such as macrophage-based ones, thereby reducing costs and improving patient access to cell therapy. iPSCs possess unlimited scalability, meaning they can reproduce and proliferate indefinitely, potentially generating a nearly endless supply of differentiated immune cells for therapies, such as cancer therapy. iPSCs are also processable with precise multigenome editing, allowing for the introduction of multiple genetic modifications to enhance disease targeting capabilities and ultimately improve the safety of immune cells. iPSCs can be similarly engineered with the goal of improving both early expansion and duration of engraftment by protecting against allogeneic rejection by the patient's own immune system. Furthermore, while patient-derived or donor-derived blood material is inconsistent, iPSCs provide consistent starting material derived from single-cell clones, which can allow for genomic consistency and integrity of the final cell product.

[0124] In some embodiments, the PSCs (e.g., iPSCs) are autologous to the subject to be treated, that is, the PSCs are derived from the same subject to which the differentiated cells are administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from the patient to be treated are reprogrammed to become iPSCs before differentiation into macrophages as described herein. In some embodiments, fibroblasts may be reprogrammed into iPSCs by transforming the fibroblasts with genes (OCT4, SOX2, NANOG, LIN28, and KLF4) cloned into plasmids (see, e.g., reference [Yu, et al., Science DOI: 10.1126 / science.1172482]). In some embodiments, non-pluripotent fibroblasts derived from a patient are reprogrammed to become iPSCs before differentiation into macrophages by, for example, the use of a non-integrating Sendai virus to reprogram the cells (e.g., the use of the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit). In some embodiments, the resulting differentiated cells are then administered to the patient derived from autologous cell therapy.

[0125] In some embodiments, the PSCs (e.g., iPSCs) are allogeneic to the subject to be treated, that is, the PSCs originate from a different individual from the subject to whom the differentiated cells are administered. In some embodiments, non-pluripotent cells (e.g., fibroblasts) derived from another individual (e.g., an individual without the disease or pathology to be treated, e.g., a healthy subject) are reprogrammed to become iPSCs before differentiation into macrophages. In some embodiments, reprogramming is achieved at least partially by the use of a non-integrating Sendai virus to reprogram the cells (e.g., the use of the CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit). In some embodiments, the resulting differentiated cells are then administered to an individual that is not the same as the individual from which the differentiated cells originated (e.g., allogeneic cell therapy or allogeneic cell transplantation). In these embodiments, the PSCs described herein (e.g., allogeneic cells) may be genetically engineered to be low immunogenic. Methods for reducing immunogenicity are known and include ablating the expression of polymorphic HLA-A / -B / -C and HLA class II molecules. An exemplary method for reducing one or more HLA molecules includes destroying the beta-2-microglobulin (B2M) gene as described herein.

[0126] In some embodiments, the present disclosure provides engineered stem cells that transiently or stably express a synthetic cytokine receptor complex. In some embodiments, the present disclosure provides engineered stem cells that stably express a synthetic cytokine receptor complex. In some embodiments, the present disclosure provides engineered stem cells that stably express a synthetic cytokine receptor complex and a chimeric antigen receptor (CAR).

[0127] In some embodiments, the engineered iPSC further comprises a destroyed B2M and / or SIRPA locus. In some embodiments, the genome further comprises a destroyed FKBP12 locus. In some embodiments, the genome further comprises a destroyed AAVS1 locus. In some embodiments, the engineered synthetic cytokine receptor is incorporated into the destroyed B2M locus, for example by HDR or other methods. In some embodiments, the CAR is incorporated into the destroyed AAVS1 locus, for example by HDR or other methods. In some embodiments, the gene encoding FKBP12 in the cell is further destroyed, for example, so that its expression is reduced or the gene encoding FKBP12 is knocked out. In some embodiments, the locus of the gene is destroyed by a gene editing technology, for example, a CRISPR-Cas system. In some embodiments, the destroyed locus inactivates the gene in the cell. In some embodiments, the destroyed locus involves the knockout of the gene in the cell. In some embodiments, the disrupted locus comprises an indel of the endogenous gene or a deletion of a continuous stretch of the genomic DNA of the endogenous gene. In some embodiments, the indel is a frameshift mutation or a deletion of a continuous stretch of the genomic DNA of the gene. In some embodiments, the indel is present in both alleles of the gene (indel / indel). Gene editing and manipulation methods are known and include the methods described in Section III. Any of these methods may be used to produce engineered stem cells further comprising a synthetic cytokine receptor complex as described herein. In some cases, the engineered stem cells further comprise a polynucleotide encoding a chimeric antigen receptor (CAR), thereby producing engineered stem cells that express the CAR. Exemplary CARs and methods for manipulating cells having the CAR are described in Sections III and IV.

[0128] In some cases, the engineered stem cells additionally contain a polynucleotide encoding an FRB, thereby generating engineered stem cells that express a cytosolic FRB. The FRB may have a sequence as described in Section II.C. Methods of engineering cells, such as using exogenous FRBs, are known and include any of those described in Section III.

[0129] In some embodiments, the engineered stem cells are iPSCs. In some embodiments, the engineered iPSCs are differentiated sequentially into hematopoietic progenitor cells (HPCs) and then into myeloid cell types (e.g., iMCs), such as macrophages (e.g., iMACs) or neutrophils (e.g., iNEUs), by a provided method involving binding of a non-physiological ligand to an engineered synthetic cytokine receptor. In some embodiments, the differentiation pathway may include intermediate differentiation into myeloid progenitor cells. In some embodiments, the engineered iPSCs are differentiated into hematopoietic progenitor cells (HPCs); the HPCs are differentiated into myeloid progenitor cells; and then the myeloid progenitor cells are differentiated sequentially into myeloid cells, such as macrophages (referred to as "iMAC" cells). In one variant, macrophages may be derived from HPCs by differentiating the HPCs into myeloid progenitor cells; and then the myeloid progenitor cells into iMAC cells. In additional variants, macrophages can be derived by differentiating myeloid progenitor cells into iMAC cells.

[0130] In some embodiments, hematopoietic stem cells may be engineered to express synthetic cytokine receptors. In some embodiments, myeloid progenitor cells may be engineered to express synthetic cytokine receptors. In some embodiments, macrophages may be engineered to express synthetic cytokine receptors.

[0131] In any of the provided embodiments, the synthetic cytokine receptor may be any as described herein that can be activated by a non-physiological ligand (e.g., rapamycin). In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR) that can be activated by rapamycin or rapalog. In the provided embodiments, activation of the synthetic cytokine receptor induces differentiation in addition to or instead of exogenous cytokines.

[0132] In some embodiments, a non-physiological ligand may induce differentiation in addition to or instead of an exogenous cytokine. In some embodiments, the non-physiological ligand may induce differentiation during one or more of mesodermogenesis, hematopoietic differentiation, myeloid progenitor cell differentiation, myeloid cell differentiation, and macrophage differentiation.

[0133] In some embodiments, manipulating cells to express synthetic cytokine receptors and activating the receptors with non-physiological ligands allows for the generation of a pure myeloid cell population (e.g., macrophages) that can differentiate and / or expand without the use of cell sorting.

[0134] B. Synthetic cytokine receptor complex

[0135] The synthetic cytokine receptor of the present disclosure comprises a synthetic gamma chain and a synthetic beta chain, each comprising a dimerization domain. The dimerization domain is controllably dimerized in the presence of a non-physiological ligand, thereby activating the signaling of the synthetic cytokine receptor.

[0136] In some embodiments, the synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. In some embodiments, the synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from the interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, the interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, or the interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. In certain embodiments, the beta chain intracellular domain is the IL-2RB intracellular domain. In some of these embodiments, the first and / or second transmembrane domain is a transmembrane domain sequence that is independently heterogeneous to the sequence from which the gamma chain or beta chain intracellular domain is derived. In some embodiments, the first and second transmembrane domains are different. In some of these embodiments, the first and / or second transmembrane domain is independently a transmembrane sequence from the same protein from which the gamma chain or beta chain intracellular domain is derived. In some embodiments, the first transmembrane domain is an IL-2RG transmembrane domain. In some embodiments, the second transmembrane domain is a beta chain transmembrane domain selected from the interleukin-2 receptor subunit beta (IL-2RB) transmembrane domain, the interleukin-7 receptor subunit beta (IL-7RB) transmembrane domain, or the interleukin-21 receptor subunit beta (IL-21RB) transmembrane domain. In some embodiments, the synthetic gamma chain polypeptide comprises a first dimerization domain, an interleukin-2 receptor subunit gamma (IL-2RG) transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain. The dimerization domain can be extracellular (N-terminus for the transmembrane domain) or intracellular (C-terminus for the transmembrane domain and N- or C-terminus for the IL-2G intracellular domain).

[0137] The synthetic beta-chain polypeptide comprises a second dimerization domain, an interleukin-2 receptor subunit beta (IL-2RB) transmembrane domain, and an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain. The dimerization domain may be extracellular (N-terminus for the transmembrane domain) or intracellular (C-terminus for the transmembrane domain and N- or C-terminus for the IL-2RB intracellular domain).

[0138] In some embodiments, the synthetic gamma-chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. In some embodiments, the synthetic beta-chain polypeptide is encoded by a nucleic acid sequence encoding a signal peptide. A person skilled in the art will easily become familiar with signal peptides that can provide a signal for transporting an initial protein in a cell. Any of the various signal peptides may be used. It should be understood that the signal peptide is cleaved, and a mature synthetic cytokine receptor (without the signal peptide) is generated and expressed on the cell surface.

[0139] In some embodiments, the signal peptide is the CD8a signal sequence represented as sequence identification number: 12: MALPVTALLLPLALLLHAARP.

[0140] In some embodiments, the signal peptide is the signal sequence represented as sequence identification number: 29: MPLGLLWLGLALLGALHAQA.

[0141] In some embodiments, a non-physiological ligand activates a synthetic cytokine receptor in macrophages to induce expansion and / or activation of engineered macrophages. In some embodiments, the non-physiological ligand is rapamycin or rapalog, and this synthetic cytokine receptor is referred to as a rapamycin-activated cytokine receptor (RACR).

[0142] In some embodiments, a non-physiological ligand activates synthetic cytokine receptors in macrophages to induce macrophage expansion. In some embodiments, activation of synthetic cytokine receptors results in an increased number of macrophages compared to uninduced cells by at least about 10 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, or at least about 4000 times. In some embodiments, activation of synthetic cytokine receptors results in a number of macrophages increased by at least about 5,000 times, at least about 6,000 times, at least about 7,000 times, at least about 8,000 times, at least about 9,000 times, at least about 10,000 times, at least about 50,000 times, at least about 100,000 times, at least about 250,000 times, at least about 500,000 times, at least about 750,000 times, or at least about 1,000,000 times compared to uninduced cells.

[0143] In some embodiments, macrophages increase by about 10 to about 100 times, about 50 to about 200 times, about 100 to about 300 times, about 200 to about 400 times, about 300 to about 500 times, about 400 to about 1000 times, about 500 to about 1500 times, about 1000 to about 2000 times, about 1500 to about 2500 times, about 2000 to about 3000 times, about 2500 to about 3500 times, about 3000 to about 4000 times, or any value between these ranges. In some embodiments, macrophages increase by about 4,000 to about 6,000 times, about 5,000 to about 7,000 times, about 6,000 to about 8,000 times, about 7,000 to about 9,000 times, about 8,000 to about 10,000 times, about 9,000 to about 50,000 times, about 10,000 to about 100,000 times, about 50,000 to about 250,000 times, about 100,000 to about 500,000 times, about 250,000 to about 750,000 times, about 500,000 to about 1,000,000 times, or any value between these ranges.

[0144] In some embodiments, a non-physiological ligand induces differentiation by activating a synthetic cytokine receptor in stem cells. In some embodiments, the non-physiological ligand is rapamycin or rapalog, and this synthetic cytokine receptor is referred to as a rapamycin-activated cytokine receptor (RACR).

[0145] In some embodiments, a non-physiological ligand activates a synthetic cytokine receptor in stem cells to induce expansion of hematopoietic progenitors differentiated from stem cells. In some embodiments, activation of the synthetic cytokine receptor results in an increased number of hematopoietic progenitors compared to non-engineered cells, at least about 10 times, at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, at least about 1000 times, at least about 1500 times, at least about 2000 times, at least about 2500 times, at least about 3000 times, at least about 3500 times, or at least about 4000 times.

[0146] In some embodiments, the hematopoietic progenitors increase by about 10 to about 100 times, about 50 to about 200 times, about 100 to about 300 times, about 200 to about 400 times, about 300 to about 500 times, about 400 to about 1000 times, about 500 to about 1500 times, about 1000 to about 2000 times, about 1500 to about 2500 times, about 2000 to about 3000 times, about 2500 to about 3500 times, about 3000 to about 4000 times, or any value between these ranges.

[0147] 1. Intracellular domain

[0148] In some embodiments, the intracellular signaling domain of the first transmembrane receptor protein comprises an interleukin-2 receptor subunit gamma (IL2Rg) domain. In some embodiments, the IL2Rg domain comprises the sequence presented in Sequence Identification No. 1. In some embodiments, the IL2Rg common gamma chain intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with Sequence Identification No. 1.

[0149] The sequence of the IL2RG common gamma chain intracellular domain is presented at sequence identification number: 1:

[0150] ERTMPRIPTLKNLEDLVTEYHGNFSAWSGVSKGLAESLQPDYSERLCLVSEIPPKGGALGEGPGASPCNQHSPYWAPPCYTLKPET.

[0151] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-2RB intracellular domain, and a second dimerization domain.

[0152] In some embodiments, the synthetic beta chain comprises an interleukin-2 receptor subunit beta (IL2RB) intracellular domain. IL2RB is also known as IL15RB or CD122. Therefore, as referred to herein, IL2RB may also mean IL15RB. That is, the terms are used interchangeably in this disclosure. In some embodiments, the IL2RB intracellular domain comprises the sequence presented in SEQ ID NO: 2. In some embodiments, the IL2RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 2.

[0153] The sequence of the IL2RB intracellular domain is presented at sequence identification number: 2:

[0154] NCRNTGPWLKKVLKCNTPDPSKFFSQLSSEHGGDVQKWLSSPFPSSSFSPGGLAPEISPLEVLERDKVTQLLLQQDKVPEPASLSSNHSLTSCFTNQGYFFFHLPDALEIEACQVYFTYDPYSEEDPDEGVAGAPTGSSPQPL QPLSGEDDAYCTFPSRDDLLLFSPSLLGGPSPPSTAPGGSGAGEERMPPSLQERVPRDWDPQPLGPPTPGVPDLVDFQPPPELVLREAGEEVPDAGPREGVSFPWSRPPGQGEFRALNARLPLNTDAYLSLQELQGQDPTHLV

[0155] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-7RB intracellular domain, and a second dimerization domain.

[0156] In some embodiments, the synthetic beta chain comprises an interleukin-7 receptor subunit beta (IL7RB) intracellular domain. In some embodiments, the IL7RB intracellular domain comprises the sequence presented in Sequence Identification No. 3. In some embodiments, the IL7RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with Sequence Identification No. 3.

[0157] The sequence of the IL7RB intracellular domain is presented at sequence identification number: 3:

[0158] KKRIKPIVWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQARDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLNPVAQGQPILTSLGSNQEEAYVTMSSFYQNQ

[0159] In some embodiments, the synthetic cytokine receptor comprises a first transmembrane receptor protein comprising an IL-2RG intracellular domain, a first dimerization domain, a second transmembrane receptor protein comprising an IL-21RB intracellular domain, and a second dimerization domain.

[0160] In some embodiments, the synthetic beta chain comprises an interleukin-21 receptor subunit beta (IL21RB) intracellular domain. In some embodiments, the IL21RB intracellular domain comprises the sequence presented in SEQ ID NO: 4. In some embodiments, the IL21RB intracellular domain has at least 80% amino acid identity, at least 85% amino acid identity, at least 90% amino acid identity, at least 95% amino acid identity, or 100% amino acid identity with SEQ ID NO: 4.

[0161] The sequence of the IL21RB intracellular domain is presented at sequence identification number: 4:

[0162] SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCE DDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS

[0163] 2. Cross-country domain

[0164] The transmembrane (TM) domain is a sequence of a synthetic cytokine receptor that crosses a membrane. The transmembrane domain may contain a hydrophobic alpha helix. In some embodiments, the transmembrane domain is derived from a human protein.

[0165] The sequence of the transmembrane domain is represented as sequence identification number: 8: VVISVGSMGLIISLLCVYFWL.

[0166] The sequence of the TM domain is represented as sequence identification number: 9: VAVAGCVFLLISVLLLSGL.

[0167] The sequence of the TM domain is represented as sequence identification number: 10: PILLTISILSFFSVALLVILACVLW.

[0168] The sequence of the TM domain is represented as sequence identification number: 11: GWNPHLLLLLLLVIVFIPAFW.

[0169] The sequence of the TM domain is represented as sequence identification number: 36: IPWLGHLLVGLSGAFGFIILVYLLI.

[0170] In some embodiments, the TM domain and the intracellular signaling domain are derived from the same cytokine receptor. In some embodiments, the synthetic gamma-chain polypeptide contains an IL-2RG TM domain and an IL-2RG intracellular domain. In some embodiments, the synthetic beta-chain polypeptide contains an IL-2RB TM domain and an IL-2RB intracellular domain. In some embodiments, the synthetic beta-chain polypeptide contains an IL-7RB TM domain and an IL-7RB intracellular domain. In some embodiments, the synthetic beta-chain polypeptide contains an IL-21RB TM domain and an IL-21RB intracellular domain.

[0171] In some embodiments, one or more additional consecutive amino acids of an ectodomain directly adjacent to the TM domain of the cytokine receptor may also be included as part of the polypeptide sequence of the chain of the synthetic cytokine receptor. In some embodiments, 1 to 20 consecutive amino acids of an ectodomain adjacent to the TM domain of the cytokine receptor are included as part of the polypeptide sequence of the chain of the synthetic cytokine receptor. The portion of the ectodomain may be a consecutive sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids directly adjacent to the TM sequence (e.g., N-terminal).

[0172] In some embodiments, the synthetic gamma-chain polypeptide contains an IL-2RG™ domain containing the sequence presented in SEQ ID NO: 8 and an IL-2RG intracellular domain containing the sequence presented in SEQ ID NO: 1. In some embodiments, the synthetic gamma-chain polypeptide contains an IL-2RG™ domain containing the sequence presented in SEQ ID NO: 31 and an IL-2RG intracellular domain containing the sequence presented in SEQ ID NO: 1.

[0173] In some embodiments, the synthetic beta-chain polypeptide contains an IL-2RB™ domain containing the sequence presented in SEQ ID NO: 36 and an IL-2RB intracellular domain containing the sequence presented in SEQ ID NO: 2. In some embodiments, the synthetic beta-chain polypeptide contains an IL-2RB™ domain containing the sequence presented in SEQ ID NO: 35 and an IL-2RB intracellular domain containing the sequence presented in SEQ ID NO: 2.

[0174] 3. Dimeric domain

[0175] The dimerization domain may be a heterodimerization domain comprising, but not limited to, an FK506-binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain of size 12 kD, which is known in the art to dimerize in the presence of rapamycin or rapalog. In some embodiments, the FRB domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. In some embodiments, the FKBP domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5. In some embodiments, the FKBP domain may include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence identification number: 49. In some embodiments, the FKBP domain may include a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to sequence identification number: 30.

[0176] In some embodiments, the sequence of an exemplary FKBP domain is presented in sequence identification number: 5:

[0177] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

[0178] In some embodiments, the sequence of an exemplary FKBP domain is presented in sequence identification number: 49:

[0179] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKL

[0180] In some embodiments, the sequence of an exemplary FKBP domain is presented in sequence identification number: 30:

[0181] GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLGE

[0182] In some embodiments, the sequence of an exemplary FRB domain is presented in sequence identification number: 6:

[0183] ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRISK

[0184] In some embodiments, the sequence of the variant FRB domain (FRB mutant domain) is presented in sequence identification number: 7:

[0185] ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK

[0186] In some embodiments, the first dimerization domain is presented in sequence identification number: 5, and the second dimerization domain is presented in sequence identification number: 6.

[0187] In some embodiments, the first dimerization domain is presented in sequence identification number: 49, and the second dimerization domain is presented in sequence identification number: 6.

[0188] In some embodiments, the first dimerization domain is presented in sequence identification number: 30, and the second dimerization domain is presented in sequence identification number: 6.

[0189] In some embodiments, the first dimerization domain is presented in sequence identification number: 5, and the second dimerization domain is presented in sequence identification number: 7.

[0190] In some embodiments, the first dimerization domain is presented in sequence identification number: 49, and the second dimerization domain is presented in sequence identification number: 7.

[0191] In some embodiments, the first dimerization domain is presented in sequence identification number: 30, and the second dimerization domain is presented in sequence identification number: 7.

[0192] Alternatively, the first dimerization domain and the second dimerization domain may be an FK506-binding protein (FKBP) and a calcineurin domain of size 12 kD, which are known in the art to dimerize in the presence of FK506 or an analog thereof.

[0193] In some embodiments, the dimerization domain is a homomerization domain selected from the following:

[0194] i) FK506-binding protein (FKBP) of size 12 kD;

[0195] ii) cyclophyllial a (CypA); or

[0196] iii) Kiraze B (CyrB);

[0197] The corresponding non-physiological ligands are as follows:

[0198] i) FK1012, AP1510, AP1903 or AP20187;

[0199] ii) cyclosporine-A (CsA); or

[0200] iii) Cumermycin or its analogues.

[0201] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the FKBP domain and the cyclophyllin domain.

[0202] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are the FKBP domain and the bacterial dihydrofolate reductase (DHFR) domain.

[0203] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are a calcineurin domain and a cyclophyllin domain.

[0204] In some embodiments, the first and second dimerization domains of the transmembrane receptor protein are PYR1-like 1 (PYL1) and abscisic acid insensitivity 1 (ABI1).

[0205] In some embodiments, the synthetic cytokine receptor consists of a synthetic gamma-chain polypeptide containing an FKBP12 dimerization domain and an IL-2RG intracellular domain, and a synthetic beta-chain polypeptide containing an FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic gamma-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 28. In some embodiments, the synthetic beta-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 33.

[0206] In some embodiments, the synthetic cytokine receptor consists of the synthetic gamma chain polypeptide presented in sequence identification number: 28 and the synthetic beta chain polypeptide presented in sequence identification number: 33.

[0207] In some embodiments, the synthetic cytokine receptor consists of a synthetic gamma-chain polypeptide containing an FKBP12 dimerization domain and an IL-2RG intracellular domain, and a synthetic beta-chain polypeptide containing an FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic gamma-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 28. In some embodiments, the synthetic beta-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 55.

[0208] In some embodiments, the synthetic cytokine receptor consists of the synthetic gamma chain polypeptide presented in sequence identification number: 28 and the synthetic beta chain polypeptide presented in sequence identification number: 55.

[0209] In some embodiments, the synthetic cytokine receptor consists of a synthetic gamma-chain polypeptide containing an FKBP12 dimerization domain and an IL-2RG intracellular domain, and a synthetic beta-chain polypeptide containing an FRB dimerization domain and an IL-2RB intracellular domain. In some embodiments, the synthetic gamma-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 56. In some embodiments, the synthetic beta-chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in SEQ ID NO: 57.

[0210] In some embodiments, the synthetic cytokine receptor consists of the synthetic gamma chain polypeptide presented in sequence identification number: 56 and the synthetic beta chain polypeptide presented in sequence identification number: 57.

[0211] In some embodiments, the synthetic cytokine receptor may be bound by a non-physiological ligand rapamycin or a rapamycin analog. In some embodiments, the synthetic cytokine receptor responds to a non-physiological ligand rapamycin or a rapamycin analog, wherein the binding of the non-physiological ligand to the dimerization domain of the synthetic cytokine receptor induces cytokine receptor-mediated signaling in the cell, for example, through the JAK / STAT pathway.

[0212] 4. Non-physiological ligands

[0213] In various embodiments of the compositions and methods of the present disclosure, the system comprises a non-physiological ligand. Exemplary small molecules useful as ligands include, without limitation: rapamycin, fluorescein, fluorescein isothiocyanate (FITC), 4-[(6-methylpyrazine-2-yl)oxy]benzoic acid (aMPOB), folate, rhodamine, acetazolamide, and CA9 ligand.

[0214] In some embodiments, synthetic cytokine receptors are activated by a ligand. In some embodiments, the ligand is a non-physiological ligand.

[0215] In some embodiments, the non-physiological ligand is a rapalog.

[0216] In some embodiments, the non-physiological ligand is rapamycin.

[0217] In some embodiments, the non-physiological ligand is AP21967.

[0218] In some embodiments, the non-physiological ligand is FK506.

[0219] In some embodiments, the non-physiological ligand is FK1012. In some embodiments, the non-physiological ligand is AP1510. In some embodiments, the non-physiological ligand is AP1903. In some embodiments, the non-physiological ligand is AP20187. In some embodiments, the non-physiological ligand is cyclosporine-A (CsA). In some embodiments, the non-physiological ligand is coumermycin.

[0220] In some embodiments, the synthetic cytokine receptor complex is activated by folate, fluorescein, aMPOB, acetazolamide, CA9 ligand, tacrolimus, rapamycin, rapalog (rapamycin analog), CD28 ligand, poly(his) tag, Strep-tag, FLAG-tag, VS-tag, Myc-tag, HA-tag, NE-tag, biotin, digoxigenin, dinitrophenol, or a derivative thereof.

[0221] In some embodiments, the non-physiological ligand may be an inorganic or organic compound with less than 1,000 daltons.

[0222] In some embodiments, the ligand may be rapamycin or a rapamycin analog (rapalog). In some embodiments, the rapalog comprises a variant of rapamycin having one or more of the following modifications relative to rapamycin: demethylation, removal, or replacement of a methoxy at C7, C42, and / or C29; removal, derivatization, or replacement of a hydroxyl at C13, C43, and / or C28; reduction, removal, or derivatization of a ketone at C14, C24, and / or C30; replacement of a 6-membered pipecholate ring with a 5-membered prolyl ring; and alternative substitution on the cyclohexyl ring or replacement of the cyclohexyl ring with a substituted cyclopentyl ring.

[0223] Therefore, in some embodiments, rapalog is everolimus, novolimus, pimecrolimus, lidaphorolimus, tacrolimus, temsirolimus, umirolimus, zotarolimus, temsirolimus (CCI-779), C20-metallylapamycin, C16-(S)-3-methylindolapamycin, C16-(S)-3-methylindolapamycin (C16-iRap), AP21967 (A / C heteromerizing agent, Takara Bio®), sodium mycophenolic acid, benidipine hydrochloride, rapamine, AP23573 (lidaphorolimus), AP1903 (limiducid), or metabolites, derivatives and / or combinations thereof.

[0224] In some embodiments, the ligand comprises FK1012 (a semisynthetic dimer of FK506), tacrolimus (FK506), FKCsA (a complex of FK506 and cyclosporine), rapamycin, cumermycin, gibberellin, HaXS dimerizer (a chemical dimerizer of HaloTag and SNAP-tag), TmP-HTag (a trimethoprim halosene protein dimerizer), or ABT-737 or its functional derivative.

[0225] In some embodiments, the non-physiological ligand is 0 nM to 1000 nM, e.g., 0.05 nM, 0.1 nM, 0.5 nM, 1.0 nM, 5.0 nM, 10.0 nM, 15.0 nM, 20.0 nM, 25.0 nM, 30.0 nM, 35.0 nM, 40.0 nM, 45.0 nM, 50.0 nM, 55.0 nM, 60.0 nM, 65.0 nM, 70.0 nM, 75.0 nM, 80.0 nM, 90.0 nM, 95.0 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 It exists or is provided in an amount of nM, 800 nM, 900 nM, or 1000 nM, or in an amount within a range defined by any two of the amounts mentioned above.

[0226] In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is AP21967 and is present or provided at 100 nM.

[0227] In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is rapamycin and is present or provided at 50 nM.

[0228] In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 1 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 10 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 20 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 50 nM. In some embodiments, the non-physiological ligand is a rapalog and is present or provided at 100 nM.

[0229] In some embodiments, a non-physiological ligand is present or provided at 1 nM.

[0230] In some embodiments, a non-physiological ligand is present or provided at 10 nM.

[0231] In some embodiments, a non-physiological ligand is present or provided at 100 nM.

[0232] In some embodiments, a non-physiological ligand is present or provided at 1000 nM.

[0233] C. Cytosol FRB

[0234] In some embodiments, engineered cells, such as stem cells or macrophages, may be contacted with free cytosolic FRBs. As described in more detail elsewhere in this invention, after rapamycin normally binds to FBP12, the FKBP12-rapamycin complex binds to the FRB subunit of mTOR and blocks mTOR signaling. Therefore, contacting cells with rapamycin may inhibit or reduce cell growth and expansion in some cases. In some embodiments, cells may be made "rapamycin resistant" by providing free cytosolic FRBs to cells to complex with rapamycin and thereby eliminate or reduce rapamycin-mediated growth inhibition of source cells or iMACs.

[0235] In some embodiments, soluble FRBs may be microinjected into stem cells or macrophages to remove or reduce rapamycin-mediated growth inhibition. In some embodiments, stem cells or macrophages may be transduced into a vector containing soluble FRBs to remove or reduce rapamycin-mediated growth inhibition. In some embodiments, soluble FRBs may be added to cell culture media to remove or reduce rapamycin-mediated growth inhibition.

[0236] In an embodiment where a soluble FRB is microinjected into a stem cell or macrophage, the soluble FRB is injected at a concentration of 4 mg / mL, 4.5 mg / mL, 5 mg / mL, 5.5 mg / mL, or 6 mg / mL. In an embodiment where a soluble FRB is microinjected into a stem cell or macrophage, the soluble FRB is injected at a concentration of 1 μM.

[0237] In some embodiments, a nucleic acid molecule encoding FRB is introduced into a cell, for example, by the introduction of a vector construct encoding FRB. In some embodiments, the construct is designed to insert the nucleic acid encoding FRB into an endogenous locus within the cell. Methods of gene insertion or melting are known and include any of the methods described in Section III. In some embodiments, the insertion of the FRB-coding construct is, for example, by homology-directed repair using a CRISPR-Cas system. In some embodiments, engineered cells expressing FRB in an endogenous locus may express free cytosolic FRB in the cell.

[0238] The FRB domain is an approximately 100-amino acid domain derived from mTOR protein kinase. It is a freely diffusing soluble protein that can be expressed in the cytosol. Advantageously, the FRB domain reduces the inhibitory effect of rapamycin on mTOR in engineered cells and promotes the consistent activation of engineered cells, thereby providing the cells with a proliferative advantage over natural cells.

[0239] In some embodiments, the synthetic cytokine receptor complex comprises a complex comprising a cytosol polypeptide that binds to a ligand or a ligand.

[0240] In some embodiments, the cytosolic polypeptide comprises an FRB domain. In some embodiments, the cytosolic polypeptide comprises an FRB domain, and the ligand is rapamycin. The cytosolic FRB domain may comprise a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7. The FRB domain may be a naked FRB domain essentially composed of a polypeptide having a polypeptide sequence that is at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6 or SEQ ID NO: 7.

[0241] In some embodiments, the cell is in contact with an FRB domain protein having the sequence presented in sequence identification number: 6.

[0242] In some embodiments, the cell is in contact with an FRB domain protein having the sequence presented in sequence identification number: 7.

[0243] Advantageously, cytosolic FRBs confer resistance to the immunosuppressive effects of non-physiological ligands (e.g., rapamycin or rapalog).

[0244] D. Differentiation of Stem Cells

[0245] A method for differentiating pluripotent stem cells, such as iPSCs, engineered with synthetic cytokine receptors into myeloid cells is provided herein. A method for generating myeloid cells derived from pluripotent stem cells, such as iPSCs, engineered with synthetic cytokine receptors is provided herein. In some embodiments, iPSC differentiation is by a pathway including differentiation into hematopoietic progenitor (HP) cells and myeloid progenitor cells. In some embodiments, iPSC differentiation is by a pathway including differentiation into hematopoietic progenitor (HP) cells.

[0246] While iPSC-derived cell therapies are promising, the current process for inducing immune cells from iPSCs is complex, variable, and costly. Current protocols for inducing downstream cell types from iPSCs utilize sequential steps of "coxing" into the differentiation pathway of iPSCs by binding to endogenous receptors with exogenous factors. In some embodiments, this process may require long culture times and expensive protein materials, and can be highly variable as it depends on the constantly changing expression patterns of endogenous genes and receptors.

[0247] In some embodiments provided herein, iPSC differentiation is oriented at least partially by signaling induced from synthetic cytokine receptors. In embodiments of the provided method, binding of a synthetic cytokine receptor (e.g., RACR) by a homologous non-physiological ligand (e.g., rapamycin) can deliver cytokine signals to the cell to induce the JAK / STAT pathway and drive differentiation. The provided embodiments in which iPSCs are engineered with synthetic cytokine receptors promote differentiation through highly controlled synthetic receptors, which have the potential to reduce the variability of cell differentiation as well as the manufacturing cost of these cells by replacing expensive growth factors and cytokines with small molecule binders (e.g., rapamycin). In some aspects, the requirement for additional growth factors or cytokines to drive differentiation at one or more different stages of the process is reduced or eliminated, thereby providing oriented and consistent differentiation.

[0248] In some embodiments, the provided method comprises culturing pluripotent stem cells (e.g., iPSCs) under conditions for differentiating cells engineered with synthetic cytokine receptors into a myeloid cell population. In some embodiments, the method may comprise one or more incubations of adding different molecules to the culture medium. In some embodiments, the method comprises incubation in the presence of a non-physiological ligand (e.g., rapamycin or rapalog). In some embodiments, the method may comprise replacing the medium to supplement or add any one or more molecules to the culture medium.

[0249] In some embodiments, the provided method comprises culturing pluripotent stem cells (e.g., iPSCs) under conditions for differentiating cells engineered with synthetic cytokine receptors into a myeloid cell population or its progenitors (e.g., hematopoietic progenitors (HP) or myeloid progenitor cells).

[0250] In some embodiments, the provided method comprises culturing pluripotent stem cells (e.g., iPSCs) under conditions for differentiating cells engineered with a synthetic cytokine receptor into a progenitor cell population. In some embodiments, the provided method comprises a) culturing pluripotent stem cells (e.g., iPSCs) under conditions for differentiating cells engineered with a synthetic cytokine receptor into a progenitor cell population, and b) culturing the cells produced in a) by incubation under conditions for generating myeloid cells.

[0251] Examples of methods for differentiating stem cells (e.g., iPSCs) into pluripotent hematopoietic progenitor cells are provided in U.S. Patent No. 9,624,470, U.S. Patent Application No. 2020 / 0080059, as well as in the literature [Mesquitta et al., Sci. Rep. 9:6622 (2019)] (the full text of which is incorporated herein by reference). In some embodiments, culture in a) is performed by a first incubation under conditions for producing embryoid bodies (EB), followed by one or more additional incubations in the presence of a non-physiological ligand. In some embodiments, during the additional incubations, the culture optionally comprises one or more myeloid cell differentiation factors selected from one or more of IL-3, M-CSF, and GM-CSF.

[0252] In some embodiments, the first incubation is performed in a first medium. In some embodiments, one or more additional incubations include a second incubation in a second medium. In some embodiments, one or more additional incubations include a second incubation in a second medium and a third incubation in a third medium.

[0253] In some embodiments, the culture in b) is carried out by one or more additional incubations under conditions for producing myeloid cells. In some embodiments, the culture in b) is carried out by a fourth incubation in a fourth medium to produce myeloid cells. In some embodiments, the fourth incubation is carried out in the presence of a non-physiological ligand.

[0254] In some embodiments, culture of the cell population may be performed under serum-free conditions. Examples of commercially available serum-free media suitable for cell attachment and / or induction include mTeSR™1, STEMdiff APEL 2 medium or TeSR™2 from Stem Cell Technologies (Vancouver, Canada), primate ES / iPS cell medium from ReproCELL (Boston, Massachusetts), StemPro®-34 from Invitrogen (Carlsbad, California), StemPro® hESC SFM from Invitrogen, and X-VIVO™ from Lonza (Basel, Switzerland).

[0255] In some embodiments, the medium comprises one or more of the following: nutrients, extracts, growth factors, hormones, cytokines, and medium additives. Exemplary nutrients and extracts may include, for example, DMEM / F-12 (Dulbeco Modified Eagle Medium / Nutrient Mixture F-12), which is a basic medium widely used to support the growth of many different mammalian cells; KOSR (Knockout Serum Replacement); L-glut; and NEAA (Non-essential Amino Acids). Medium additives may include, but are not limited to, MTG, ITS, ME, and antioxidants (e.g., ascorbic acid). In some embodiments, the medium of the present invention comprises one or more of the following cytokines or growth factors: basic fibroblast growth factor (bFGF, also known as FGF2), transforming growth factor beta (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF or VEGF-165), various interleukins (e.g., IL-3), various colony-stimulating factors (e.g., granulocyte / macrophage colony-stimulating factor (GM-CSF) and / or macrophage colony-stimulating factor (M-CSF)), various interferons (e.g., IFNγ) and other cytokines, such as stem cell factor (SCF).

[0256] These cytokines may be commercially available and may be natural or recombinant. In some embodiments, growth factors, mitogens, and cytokines are phase and / or cell type specific at concentrations determined empirically or guided by established cytokine technology. Examples of exogenous cell culture medium additives and supplements and cell selection kit components are provided in WO 2020 / 124256, the full text of which is incorporated herein by reference.

[0257] In some embodiments, the medium of the present invention comprises one or more of the following: a PI3K inhibitor, an AHR antagonist, a pyrimido-[4,5-b]-indole derivative, and a ROCK inhibitor. In some embodiments, the PI3K inhibitor is LY294002. In some embodiments, the AHR antagonist is StemRegenin-1. In some embodiments, the pyrimido-[4,5-b]-indole derivative is UM729. In some embodiments, the ROCK inhibitor is Y27632.

[0258] In some embodiments, the first medium comprises one or more of BMP4, FGF2, VEGF-165, and a Rock inhibitor, optionally wherein the Rock inhibitor is Y27632. In some embodiments, the second medium comprises one or more of BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF, and M-CSF. In some embodiments, the third medium comprises one or more of UM729, StemRegenin-1, BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF, and M-CSF. In some embodiments, the fourth medium comprises one or more of SCF, GM-CSF, M-CSF, IL-3, an AHR antagonist, a pyrimido-[4,5-b]-indole derivative (e.g., UM729), and StemRegenin-1. In some embodiments, the first, second, third, and fourth media further comprise a non-physiological ligand.

[0259] In some embodiments, culture in the first medium is carried out for 1 to 4 days. In some embodiments, culture is carried out for 1 day or about 1 day, 2 days, 3 days or about 3 days, or 4 days or about 4 days.

[0260] In some embodiments, culture in the second medium is carried out for 3 to 6 days. In some embodiments, culture is carried out for 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, or 6 days or about 6 days.

[0261] In some embodiments, culture in the third medium is carried out for 3 to 6 days. In some embodiments, culture is carried out for 3 days or about 3 days, 4 days or about 4 days, 5 days or about 5 days, or 6 days or about 6 days.

[0262] In some embodiments, culture in the fourth medium is carried out for 9 to 15 days. In some embodiments, culture is carried out for 9 days or about 9 days, 10 days or about 10 days, 11 days or about 11 days, 12 days or about 12 days, 13 days or about 13 days, 14 days or about 14 days, or 15 days or about 15 days.

[0263] In some embodiments, the concentration of BMP4 in the medium is about 0.5 ng / mL - 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL. In some cases, the concentration of BMP4 is at least 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL or 50 ng / mL. In some cases, the concentration of BMP4 is about 10 ng / mL.

[0264] In some embodiments, the concentration of FGF2 in the medium is about 0.5 ng / mL - 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 5 ng / mL - 10 The values ​​are ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, and 10 ng / mL - 50 ng / mL (each including boundary values). In some embodiments, the concentration of FGF2 in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, or 50 ng / mL. In some embodiments, the concentration of FGF2 in the medium is about 10 ng / mL. In some embodiments, the concentration of FGF2 in the medium is about 50 ng / mL.

[0265] In some embodiments, the concentration of VEGF in the medium is about 0.5 ng / mL - 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 0.5 ng / mL - 100 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 100 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 5 ng / mL - 100 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, 10 ng / mL - 100 ng / mL (each with cut-off values). In some embodiments, the concentration of VEGF in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL or 100 ng / mL. In some cases, the concentration of VEGF is about 50 ng / mL.

[0266] In some embodiments, the concentration of Y27632 in the medium is about 0.5 μM - 2.5 μM, 0.5 μM - 5 μM, 0.5 μM - 10 μM, 0.5 μM - 15 μM, 0.5 μM - 20 μM, 0.5 μM - 30 μM, 0.5 μM - 50 μM, 2.5 μM - 5 μM, 2.5 μM - 10 μM, 2.5 μM - 15 μM, 2.5 μM - 20 μM, 2.5 μM - 30 μM, 2.5 μM - 50 μM, 5 μM - 10 μM, 5 μM - 15 μM, 5 μM - 20 μM, 5 μM - 30 μM, 5 μM - 50 μM, 10 μM - The values ​​are 15 μM, 10 μM - 20 μM, 10 μM - 30 μM, and 10 μM - 50 μM (each including boundary values). In some embodiments, the concentration of Y27632 in the medium is at least about 0.5 μM, 2.5 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM. In some embodiments, the concentration of Y27632 in the medium is about 10 μM.

[0267] In some embodiments, the concentration of UM729 in the medium is about 0.5 μM - 1 μM, 0.5 μM - 5 μM, 0.5 μM - 10 μM, 0.5 μM - 15 μM, 0.5 μM - 20 μM, 0.5 μM - 30 μM, 0.5 μM - 50 μM, 1 μM - 5 μM, 1 μM - 10 μM, 1 μM - 15 μM, 1 μM - 20 μM, 1 μM - 30 μM, 1 μM - 50 μM, 5 μM - 10 μM, 5 μM - 15 μM, 5 μM - 20 μM, 5 μM - 30 μM, 5 μM - 50 μM, 10 μM - 15 μM, 10 μM - 20 The values ​​are μM, 10 μM to 30 μM, and 10 μM to 50 μM (each including boundary values). In some embodiments, the concentration of UM729 in the medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM. In some embodiments, the concentration of UM729 in the medium is about 1 μM.

[0268] In some embodiments, the concentration of M-CSF in the medium is about 0.5 ng / mL - 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 0.5 ng / mL - 100 ng / mL, 0.5 ng / mL - 200 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 100 ng / mL, 2.5 ng / mL - 200 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 5 ng / mL - 100 ng / mL, 5 ng / mL - 200 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, 10 ng / mL - 100 ng / mL, 10 ng / mL - 200 ng / mL (each with cut-off values). In some cases, the concentration of M-CSF is at least 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL or 200 ng / mL. In some cases, the concentration of M-CSF is about 50 ng / mL. In some cases, the concentration of M-CSF in the urine is increased to 100 ng / mL.

[0269] In some embodiments, the concentration of GM-CSF in the medium is about 0.5 ng / mL - 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 0.5 ng / mL - 100 ng / mL, 0.5 ng / mL - 200 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 100 ng / mL, 2.5 ng / mL - 200 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 5 ng / mL - 100 ng / mL, 5 ng / mL - 200 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, 10 ng / mL - 100 ng / mL, 10 ng / mL - 200 ng / mL (each with cut-off values). In some cases, the concentration of GM-CSF is at least 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL or 200 ng / mL. In some cases, the concentration of GM-CSF is about 50 ng / mL. In some cases, the concentration of GM-CSF in the stomach is increased to 100 ng / mL.

[0270] In some embodiments, the concentration of PI3K inhibitor in the medium is about 0.5 μM - 1 μM, 0.5 μM - 5 μM, 0.5 μM - 10 μM, 0.5 μM - 15 μM, 0.5 μM - 20 μM, 0.5 μM - 30 μM, 0.5 μM - 50 μM, 1 μM - 5 μM, 1 μM. μM - 10 μM, 1 μM - 15 μM, 1 μM - 20 μM, 1 μM - 30 μM, 1 μM - 50 μM, 5 μM - 10 μM, 5 μM - 15 μM, 5 μM - 20 μM, 5 μM - 30 μM, 5 μM - 50 μM, 10 μM - 15 μM, 10 μM - 20 The concentrations are μM, 10 μM–30 μM, and 10 μM–50 μM (each including boundary values). In some embodiments, the concentration of the PI3K inhibitor in the medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM. In some embodiments, the concentration of the PI3K inhibitor in the medium is about 4 μM. In some embodiments, the PI3K inhibitor is LY294002.

[0271] In some embodiments, the concentration of the AHR antagonist in the medium is about 0.5 μM - 1 μM, 0.5 μM - 5 μM, 0.5 μM - 10 μM, 0.5 μM - 15 μM, 0.5 μM - 20 μM, 0.5 μM - 30 μM, 0.5 μM - 50 μM, 1 μM - 5 μM, 1 μM - 10 μM, 1 μM - 15 μM, 1 μM - 20 μM, 1 μM - 30 μM, 1 μM - 50 μM, 5 μM - 10 μM, 5 μM - 15 μM, 5 μM - 20 μM, 5 μM - 30 μM, 5 μM - 50 μM, 10 μM - 15 μM, 10 μM - 20 μM, The concentrations are 10 μM to 30 μM and 10 μM to 50 μM (each including boundary values). In some embodiments, the concentration of the AHR antagonist in the medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM. In some embodiments, the concentration of the AHR antagonist in the medium is about 1 μM. In some embodiments, the AHR antagonist is StemRegenin-1.

[0272] In some embodiments, the concentration of IL-3 in the medium is about 0.5 ng / mL - 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 0.5 ng / mL - 100 ng / mL, 0.5 ng / mL - 200 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 100 ng / mL, 2.5 ng / mL - 200 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 5 ng / mL - 100 ng / mL, 5 ng / mL - 200 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, 10 ng / mL - 100 ng / mL, 10 ng / mL - 200 ng / mL (each with cut-off values). In some cases, the concentration of IL-3 is at least 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL, 100 ng / mL or 200 ng / mL. In some cases, IL-3 concentration is 20 ng / mL. In some cases, the concentration of IL-3 is about 25 ng / mL. In some cases, the concentration of IL-3 in the stool is increased to 40 ng / mL.

[0273] In some embodiments, the concentration of SCF in the medium is about 0.5 ng / mL - 2.5 ng / mL, 0.5 ng / mL - 5 ng / mL, 0.5 ng / mL - 10 ng / mL, 0.5 ng / mL - 15 ng / mL, 0.5 ng / mL - 20 ng / mL, 0.5 ng / mL - 30 ng / mL, 0.5 ng / mL - 50 ng / mL, 0.5 ng / mL - 100 ng / mL, 2.5 ng / mL - 5 ng / mL, 2.5 ng / mL - 10 ng / mL, 2.5 ng / mL - 15 ng / mL, 2.5 ng / mL - 20 ng / mL, 2.5 ng / mL - 30 ng / mL, 2.5 ng / mL - 50 ng / mL, 2.5 ng / mL - 100 ng / mL, 5 ng / mL - 10 ng / mL, 5 ng / mL - 15 ng / mL, 5 ng / mL - 20 ng / mL, 5 ng / mL - 30 ng / mL, 5 ng / mL - 50 ng / mL, 5 ng / mL - 100 ng / mL, 10 ng / mL - 15 ng / mL, 10 ng / mL - 20 ng / mL, 10 ng / mL - 30 ng / mL, 10 ng / mL - 50 ng / mL, 10 ng / mL - 100 ng / mL (each with cut-off values). In some embodiments, the concentration of SCF in the medium is at least about 0.5 ng / mL, 2.5 ng / mL, 5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 30 ng / mL, 50 ng / mL or 100 ng / mL. In some cases, the concentration of SCF is about 20 ng / mL.

[0274] In some embodiments, the concentration of UM729 in the medium is about 0.5 μM - 1 μM, 0.5 μM - 5 μM, 0.5 μM - 10 μM, 0.5 μM - 15 μM, 0.5 μM - 20 μM, 0.5 μM - 30 μM, 0.5 μM - 50 μM, 1 μM - 5 μM, 1 μM - 10 μM, 1 μM - 15 μM, 1 μM - 20 μM, 1 μM - 30 μM, 1 μM - 50 μM, 5 μM - 10 μM, 5 μM - 15 μM, 5 μM - 20 μM, 5 μM - 30 μM, 5 μM - 50 μM, 10 μM - 15 μM, 10 μM - 20 The values ​​are μM, 10 μM to 30 μM, and 10 μM to 50 μM (each including boundary values). In some embodiments, the concentration of UM729 in the medium is at least about 0.5 μM, 1 μM, 5 μM, 10 μM, 15 μM, 20 μM, 30 μM, or 50 μM. In some embodiments, the concentration of UM729 in the medium is about 1 μM.

[0275] In some embodiments, one or more of the steps for producing myeloid cells may include adding a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or an analog) to the culture medium to induce differentiation.

[0276] In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is rapalog. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at concentrations of 2.5 nM to 200 nM, 2.5 nM to 150 nM, 2.5 nM to 100 nM, 2.5 nM to 50 nM, 2.5 nM to 20 nM, and 2.5 nM to 10 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is present in the medium at 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to It is added at concentrations of 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM.

[0277] In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 10 nM or about 10 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapamycin is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapalog is added to the medium at a concentration of 100 nM or about 100 nM.

[0278] In some embodiments, it was surprisingly discovered that low concentrations of non-physiological ligands (e.g., rapamycin or rapamycin analogs) can support differentiation and / or expansion. In some embodiments, non-physiological ligands (e.g., rapamycin or rapamycin analogs) are added to the medium at a concentration of 10 nM or less. In some embodiments, non-physiological ligands (e.g., rapamycin or rapamycin analogs) are added to the medium at a concentration of 2.5 nM to 10 nM, e.g., 3 nM to 7 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 3 nM or about 3 nM, 4 nM or about 4 nM, 5 nM or about 5 nM, 6 nM or about 6 nM, 7 nM or about 7 nM, 8 nM or about 8 nM, 9 nM or about 9 nM, or 10 nM or about 10 nM, or any value between any of the above. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 10 nM or about 10 nM. In some embodiments, rapamycin is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, rapamycin is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, rapamycin is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, rapalog is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, rapalog is added to the medium at a concentration of 6.2 nM or about 6.2 nM.

[0279] In some embodiments, the culture of the cell population may be transferred to a suitable vessel to promote cell aggregation. In some embodiments, pluripotent aggregates may be formed in a bioreactor by culturing engineered iPSCs in a suspension in a bioreactor. A conventional strategy utilizes the formation of embryoids as a common and important intermediate to initiate lineage-specific differentiation. An embryoid is an aggregate of stem cells induced to differentiate by changes in environmental stimuli (e.g., exposure to and / or removal of specific molecular / chemical factors; and / or exposure to / interaction with three-dimensional structures). The formation of embryoids induces the cells to differentiate into mesodermal specificity.

[0280] Hematopoietic cells can be generated from embryoids derived from pluripotent cells. Pluripotent cells may be allowed to form embryoids or aggregates as part of the differentiation process. The formation of "embryonic bodies" (EBs) or growing cell clusters to induce differentiation typically involves the in vitro aggregation of human pluripotent stem cells into EBs, allowing human pluripotent stem cells to spontaneously and randomly differentiate into multiple tissue types exhibiting endoderm, ectoderm, and mesoderm origins. Without specific culture conditions, it may take about two weeks for EBs to differentiate into any of the three germ layers, and the differentiation process proceeds in a random pattern.

[0281] In some embodiments, the culture of the cell population may be transferred to a suitable container to promote cell aggregation. The container may be a 2D or 3D container. Examples of suitable 2D containers for culturing source cells include any Petri dish or culture dish regularly used in a laboratory for culturing cells.

[0282] In some embodiments, the vessel is suitable for three-dimensional (3D) culture. Without being bound by specific theories or mechanisms, it is believed that 3D culture may be more effective than two-dimensional (2D) culture in providing scaffolds for cell differentiation. Suitable 3D culture systems may include, for example, hanging drop 3D culture, e.g., hanging drop plates; 3D microwell culture, e.g., ultra-low adhesion multiwell plates; 3D culture on hydrophobic surfaces; rotational culture; static 3D suspension culture; or bioreactors. Hanging drop plates are commercially available, such as the PERFECTA3D hanging drop plate available from Biospherix (Paris, NY). Ultra-low adhesion multiwell plates (also referred to in some cases as non-adhesive culture vessels) are also commercially available, such as the AGGREWELL™ ultra-low adhesion multiwell plate available from Stem Cell Technologies (Vancouver, Canada).

[0283] In some embodiments, the container is not treated to promote cell adhesion and growth. In some embodiments, the container is a standard tissue culture plate, but is not treated to promote cell adhesion and growth. In some embodiments, the cells do not adhere or are substantially not adhered during culture. In some embodiments, culture takes place in a suspension.

[0284] In some embodiments, the vessel is a multi-well plate. The multi-well plate may be a 96-well plate, a 24-well plate, or a 6-well plate.

[0285] In some embodiments, the vessel is a bioreactor. In some embodiments, the bioreactor is used for the process of generating and proliferating myeloid cells following the development of EB. The bioreactor allows for the optimization of cell culture conditions to achieve optimal hydrogen production yield and process robustness. Environmental conditions that may be adjusted or monitored in the bioreactor include gas composition (e.g., air, oxygen, nitrogen, carbon dioxide), gas flow rate, temperature, pH, dissolved oxygen level, and stirring speed / circulation speed within the cell culture. Any type of bioreactor known in the art may be used for cell culture for the differentiation and expansion of cultured myeloid cells and includes, but is not limited to, stirred-tank bioreactors, pneumatic bioreactors (e.g., bubble column or airlift bioreactors), membrane bioreactors, hollow-fiber bioreactors, wave bioreactors, vertical wheel bioreactors, gas permeable rapid expansion (G-Rex) bioreactors, or disposable bioreactors. In some embodiments, the bioreactor is a gas permeable rapid expansion (G-Rex) bioreactor. In some embodiments, the bioreactor is a vertical wheel bioreactor. In some embodiments, the bioreactor is a stirred-tank bioreactor. In some embodiments, the stirred-tank bioreactor is a Sartorius Ambr250 stirred-tank bioreactor.

[0286] The differentiation and expansion of myeloid cells can be scaled to any desired volume for various purposes. For example, for high to medium-throughput screening under various culture conditions, the process can be scaled to be performed in a microbioreactor (e.g., about 15 mL to about 500 mL) or a benchtop-scale bioreactor (e.g., in the range of about 0.5 L to about 15 L). Alternatively, the process can be scaled to a pilot-scale bioreactor (e.g., in the range of about 15 L to about 15,000 L) or a manufacturing-scale bioreactor (e.g., in the range of about 15,000 L to about 75,000 L or more). In some embodiments, the bioreactor is a vertical wheel bioreactor having a volume of about 10 mL to about 1,000 mL. In some embodiments, the bioreactor used to differentiate iPSC cells into HPs is a vertical wheel bioreactor having a volume of about 100 mL. In some embodiments, the bioreactor used to differentiate HP into iMAC is a vertical wheel bioreactor with a volume of about 500 mL.

[0287] Methods for dissociating cells are known to those skilled in the art. Any of the various methods may be used. In some embodiments, dissociation is performed using Gentle Cell Dissociation Reagent (GDCR; Stem Cell Technologies). In some embodiments, dissociation is performed using EDTA.

[0288] In some embodiments, one or more of the steps for producing HP cells may include adding a non-physiological ligand of a synthetic cytokine receptor (e.g., rapamycin or an analog) to a culture medium to induce differentiation. In some embodiments, the non-physiological ligand is rapamycin or a rapamycin analog. In some embodiments, the rapamycin analog is rapalog. In some embodiments, the non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at concentrations of 2.5 nM to 200 nM, 2.5 nM to 150 nM, 2.5 nM to 100 nM, 2.5 nM to 50 nM, 2.5 nM to 20 nM, and 2.5 nM to 10 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is present in the medium at 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to It is added at concentrations of 100 nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM.

[0289] In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 10 nM or about 10 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapamycin is added to the medium at a concentration of 100 nM or about 100 nM. In some embodiments, rapalog is added to the medium at a concentration of 100 nM or about 100 nM.

[0290] In some embodiments, it was surprisingly discovered that low concentrations of non-physiological ligands (e.g., rapamycin or rapamycin analogs) can support differentiation and / or expansion. In some embodiments, non-physiological ligands (e.g., rapamycin or rapamycin analogs) are added to the medium at a concentration of 10 nM or less. In some embodiments, non-physiological ligands (e.g., rapamycin or rapamycin analogs) are added to the medium at a concentration of 2.5 nM to 10 nM, e.g., 3 nM to 7 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 3 nM or about 3 nM, 4 nM or about 4 nM, 5 nM or about 5 nM, 6 nM or about 6 nM, 7 nM or about 7 nM, 8 nM or about 8 nM, 9 nM or about 9 nM, or 10 nM or about 10 nM, or any value between any of the above. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 10 nM or about 10 nM. In some embodiments, a non-physiological ligand (e.g., rapamycin or a rapamycin analog) is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, rapamycin is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, rapamycin is added to the medium at a concentration of 6.2 nM or about 6.2 nM. In some embodiments, rapalog is added to the medium at a concentration of 3.1 nM or about 3.1 nM. In some embodiments, rapalog is added to the medium at a concentration of 6.2 nM or about 6.2 nM.

[0291] In some embodiments, the provided method comprises the steps of: culturing engineered stem cells, e.g., engineered stem cells with a synthetic cytokine receptor and / or CAR, with a non-physiological ligand for a first period sufficient to produce HP; and bringing the HP into contact with a differentiation medium for a second period sufficient to produce iMAC.

[0292] In some embodiments, in addition to activation using a synthetic cytokine receptor or other conditions, may be used in a method for differentiating engineered stem cells into macrophages.

[0293] In some embodiments, the provided stem cells, e.g., iPSCs, engineered with synthetic cytokine receptors may be differentiated instead or alternatively through any other method known for differentiating macrophages. In some embodiments, one or more growth factors or cytokines commonly used in connection with the differentiation and / or activation of macrophages may be used in the provided method in addition to the non-physiological ligand binding of the synthetic cytokine receptor.

[0294] Various differentiation protocols for macrophages are known in the relevant technical field.

[0295] In some embodiments, stem cells are adapted to feeder-free culture. A “feeder-free” (FF) environment as used herein refers to an environment in which feeder or stromal cells are essentially absent or / or not pre-conditioned by the culture of feeder cells, such as culture conditions, cell cultures, or culture media. A “pre-conditioned” medium refers to a medium harvested after culturing feeder cells in the medium for a certain period, for example, at least one day. The pre-conditioned medium contains many mediating substances, including growth factors and cytokines secreted by feeder cells cultured in the medium.

[0296] E. Expansion and activation of iMCs (e.g., iMAC cells)

[0297] In some embodiments, after induced myeloid (iMC) cells (e.g., iMac cells) are transduced with a CAR and / or synthetic cytokine receptor, the cells are cultured under conditions that promote cell activation and expansion.

[0298] Culture conditions may be such that the cells can be administered to a patient without concern regarding reactivity to the components of the culture medium. For example, culture conditions may omit bovine serum products, such as bovine serum albumin. In one exemplary aspect, activation may be achieved by introducing a known activator into the culture medium. In one aspect, a cell population may be cultured under conditions that promote activation for about 1 day to about 4 days. In one embodiment, an appropriate level of activation may be determined by cell size, proliferation rate, or activation markers determined by flow cytometry. In some embodiments, any culture method disclosed herein may be used to promote the activation of myeloid cells.

[0299] In some embodiments, a method for preparing and / or expanding a population of engineered cells comprising synthetic cytokine receptors for non-physiological ligands is provided herein. In some embodiments, the method comprises: providing engineered cells comprising synthetic cytokine receptors by optionally introducing a polynucleotide encoding the synthetic cytokine receptor into source cells; and incubating the engineered cells in a medium comprising a non-physiological ligand.

[0300] In this embodiment, the cytokine receptor comprises a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an intracellular domain of the interleukin-2 receptor subunit gamma (IL-2RG); and a synthetic beta chain polypeptide comprising an intracellular domain selected from a second dimerization domain, a second transmembrane domain and an intracellular domain of the interleukin-2 receptor subunit beta (IL-2RB) intracellular domain; wherein a non-physiological ligand activates the synthetic cytokine receptor in the engineered cell to induce expansion and / or activation of the engineered cell.

[0301] In some embodiments, myeloid cells are engineered to express a synthetic cytokine receptor, such as RACR, and are activated using a rapalog in the medium without recombinant cytokines in the medium.

[0302] Generating myeloid cells (e.g., macrophages) from iPSCs using conventional approaches is generally inefficient and is a current bottleneck in the manufacture of effector immunotherapy. For example, in some cases, conventional approaches achieve only a ~1X yield of macrophages from iPSCs (1 macrophage per iPSC). In some embodiments, the provided method may result in an increased yield of macrophages from iPSCs exceeding 10x (10X), 20x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, 200x, 250x, 300x, or approximately the above multipliers. In some embodiments, the provided method may result in an increased yield of macrophages from iPSCs exceeding 350, 400, 450, 500, 550, 600, 650, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, or approximately exceeding the above multiples. In some embodiments, the provided method may result in an increased yield of more than 7,000-fold or about 7,000-fold of myeloid cells from iPSCs. This result represents a significant improvement compared to other approaches for differentiating and expanding iPSCs into myeloid cells. Furthermore, generating iMACs from HPs using the provided embodiments results in highly potent iMACs, as demonstrated by the apoptosis and / or phagocytosis of tumor target cells.

[0303] In some embodiments, the expansion step is performed in a feeder-free cell culture.

[0304] In some embodiments, a method is provided for administering an effective amount of a non-physiological ligand to a subject, wherein the non-physiological ligand induces the iPSC cells to differentiate into myeloid cells according to any of the aforementioned embodiments.

[0305] In some embodiments, a method for differentiating and expanding iMAC cells by culturing them in a suitable vessel is provided. The vessel may be a 2D or 3D vessel. Examples of suitable 2D vessels for culturing source cells include any Petri dish or culture dish regularly used in a laboratory for culturing cells. The culture vessel may be coated with a suitable culture medium, for example, an extracellular medium for the adhesion and / or differentiation of the cultured cells. In some embodiments, the vessel is treated to promote cell adhesion and growth. An example of a medium suitable for use in the method of the present invention is Matrigel™ Membrane Matrix (BD Biosciences, Franklin Lakes, NJ).

[0306] In some embodiments, the vessel is suitable for three-dimensional (3D) culture. Without being bound by specific theories or mechanisms, it is believed that 3D culture may be more effective than two-dimensional (2D) culture in providing scaffolds for cell differentiation. Suitable 3D culture systems may include, for example, hanging drop 3D culture, e.g., hanging drop plates; 3D microwell culture, e.g., ultra-low adhesion multiwell plates; 3D culture on hydrophobic surfaces; rotational culture; static 3D suspension culture; or bioreactors. Hanging drop plates are commercially available, such as the PERFECTA3D hanging drop plate available from Biospherics (Paris, NY). Ultra-low adhesion multiwell plates (also referred to in some cases as non-adhesive culture vessels) are also commercially available, such as the AgriWell™ ultra-low adhesion multiwell plate available from Stem Cell Technologies (Vancouver, Canada).

[0307] In some embodiments, the container is not treated to promote cell adhesion and growth. In some embodiments, the container is a standard tissue culture plate, but is not treated to promote cell adhesion and growth. In some embodiments, the cells do not adhere or are substantially not adhered during culture. In some embodiments, culture takes place in a suspension.

[0308] In some embodiments, the vessel is a multi-well plate. The multi-well plate may be a 96-well plate, a 24-well plate, or a 6-well plate.

[0309] In some embodiments, the vessel is a bioreactor. In some embodiments, the bioreactor is used for the iMC generation and proliferation process following the development of the EB. The bioreactor allows for the optimization of cell culture conditions to achieve optimal hydrogen production yield and process robustness. Environmental conditions that may be adjusted or monitored in the bioreactor include gas composition (e.g., air, oxygen, nitrogen, carbon dioxide), gas flow rate, temperature, pH, dissolved oxygen level, and stirring speed / circulation speed within the cell culture. Any type of bioreactor known in the art may be used for cell culture for the differentiation and expansion of cultured iMC cells and includes, but is not limited to, stirred-tank bioreactors, pneumatic bioreactors (e.g., bubble column or airlift bioreactors), membrane bioreactors, hollow-fiber bioreactors, wave bioreactors, vertical wheel bioreactors, gas permeable rapid expansion (G-Rex) bioreactors, or disposable bioreactors. In some embodiments, the bioreactor is a gas permeable rapid expansion (G-Rex) bioreactor. In some embodiments, the bioreactor is a vertical wheel bioreactor. In some embodiments, the bioreactor is a stirred-tank bioreactor. In some embodiments, the vertical wheel bioreactor is a PBS bioreactor. In some embodiments, the stirred-tank bioreactor is a Sartorius Ambr250 stirred-tank bioreactor.

[0310] The expansion of iMC cells can be scaled to any desired volume for various purposes. For example, for high to medium-throughput screening under various culture conditions, the process can be scaled to be carried out in a microbioreactor (e.g., about 15 mL to about 500 mL) or a benchtop-scale bioreactor (e.g., in the range of about 0.5 L to about 15 L). Alternatively, the process can be scaled to a pilot-scale bioreactor (e.g., in the range of about 15 L to about 15,000 L) or a manufacturing-scale bioreactor (e.g., in the range of about 15,000 L to about 75,000 L or more). In some embodiments, the bioreactor is a vertical wheel bioreactor having a volume of about 10 mL to about 1,000 mL. In some embodiments, the bioreactor is a vertical wheel bioreactor having a volume of about 100 mL. In some embodiments, the bioreactor is a vertical wheel bioreactor having a volume of about 500 mL.

[0311] In some embodiments, one or more containers may be used for iMC expansion. In some embodiments, the cells in a container are day 0-3, day 0-10, day 0-15, day 0-20, day 0-25, day 0-30, day 0-35, day 0-40, day 0-50, day 0-60, day 0-100, day 3-10, day 3-15, day 3-20, day 3-25, day 3-30, day 3-35, day 3-40, day 3-50, day 3-60, day 3-100, day 10-20, day 10-25, day 10-30, day 10-35, day 10-40, day 10-50, day 10-60, Days 10-100, Days 15-20, Days 15-25, Days 15-30, Days 15-35, Days 15-40, Days 15-50, Days 15-60, Days 15-100, Days 20-25, Days 20-30, Days 20-35, Days 20-40, Days 20-50, Days 20-60, Days 20-100, Days 25-30, Days 25-35, Days 25-40, Days 25-50, Days 25-60, Days 25-100, Days 30-35, Days 30-40, Days 30-50, Days 30-60, Cells may be cultured on days 30–100, 35–40, 35–50, 35–60, 35–100, 40–50, 40–60, and 40–100 (each including boundary values). In some embodiments, cells may be cultured in a vessel on days 0–35. In some embodiments, cells may be cultured in a vessel on days 3–35. In some embodiments, the vessel is a bioreactor. In some embodiments, one or more bioreactors may be used for iAC expansion. In some embodiments, one or more bioreactors are bioreactors of different types. In some embodiments, one or more bioreactors are bioreactors of different sizes.

[0312] F. Myeloid cells differentiated from engineered stem cells (e.g., iMAC cells)

[0313] In some embodiments, macrophages may be derived from iPSCs by sequentially differentiating the iPSCs into hematopoietic progenitor cells (HPCs); and then the HPCs into macrophages (also referred to as "iMAC" cells). In one variant, macrophages may be derived from HPCs by sequentially differentiating the HPCs into myeloid progenitor cells; and then the myeloid progenitor cells into iMAC cells. In an additional variant, macrophages may be derived by differentiating the myeloid progenitor cells into iMAC cells. Manipulation of cells to express synthetic cytokine receptors may be performed at the iPSC, HPC, myeloid progenitor cell, or iMAC cell stage of the differentiation process.

[0314] In some embodiments, hematopoietic stem cells are characterized as being CD34+ and / or CD45+; common myeloid progenitor cells are characterized as being CD34+, CD90-, and CD45RA+; and macrophages are characterized as being CD45+ and CD14+ and optionally express CD11b, CD68, CD163, F4 / 80, CD16, CD54, CD49e, CD38, Egr2, CD71, TLR2, and / or TLR4. In some embodiments, myeloid progenitor cells are characterized as being CD34+, CD90-, and CD45RA+. In some embodiments, myeloid progenitor cells further express CD123.

[0315] In some embodiments, the myeloid progenitor cells comprise one or more of common myeloid progenitor cells ("CMP") (CD34+, CD90-, CD123+, CD45RA-), megakaryocyte / erythrocyte progenitor cells ("MEP") (CD34+, CD90-, CD123-, CD45RA-), and granulocyte / monocyte progenitor cells ("GMP") (CD34+, CD90-, CD123+, CD45RA+). In another embodiment, the cell population is substantially free of lymphoid progenitor cells, e.g., common lymphoid progenitor cells ("CLP") (CD34+, CD7+, CD10+), T cells (CD2+, CD3+), and B cells (CD19+, CD20+, CD33-) (e.g., less than 5%). In another embodiment, the cell population comprises pluripotent progenitor cells ("MPP") (CD34+, CD90+). In another embodiment, the cell population includes a granulocyte-macrophage progenitor that can differentiate into monocytes (CD34+, CD14+) and then differentiate into macrophages (CD34-, CD11b+, CD68+). Additionally, they can differentiate into granulocytes (CD34+, CD15+), which then differentiate into neutrophils (CD34-, CD15+, CD66b+, CD16+), basophils (CD34-, CD15+, CD123+), and eosinophils (CD34-, CD15+, CD66b+, CD11b+).

[0316] In some embodiments, the myeloid cells derived from genetically engineered myeloid progenitor cells are macrophages, neutrophils, megakaryocytes, monocytes, basophils, eosinophils, and / or erythrocytes. In some embodiments, the myeloid cells are macrophages. In some embodiments, the myeloid cells derived from genetically engineered myeloid progenitor cells are macrophages. In some embodiments, the macrophages are CD34-, CD11b+, and CD68+. In some embodiments, the myeloid cells derived from genetically engineered myeloid progenitor cells are granulocytes. In some embodiments, the granulocytes are CD34+ and CD15+. In some embodiments, the myeloid cells derived from genetically engineered myeloid progenitor cells are neutrophils. In some embodiments, the neutrophils are CD34-, CD15+, CD66b+, and CD16+. In some embodiments, the myeloid cells derived from genetically engineered myeloid progenitor cells are basophils. In some embodiments, the basophils are CD34-, CD15+, and CD123+. In some embodiments, the myeloid cells derived from genetically engineered myeloid progenitor cells are eosinophils. In some embodiments, the eosinophils are CD34-, CD15+, CD66b+, and CD11b+.

[0317] In some embodiments, cells are "derived from induced pluripotent stem cells" or "iPSC-derived" cells if they have differentiated beyond the pluripotent stem cell stage and contain genetic modifications consistent with those used to produce induced pluripotent stem cells, for example, if they contain the presence of recombinant DNA containing the Oct4, Sox2, Klf4, and / or cMyc genes. Recombinant DNA is DNA composed of two DNA sequences that are not normally linked in nature. For example, linking a heterologous promoter to a gene to form an expression construct represents a recombinant DNA molecule.

[0318] Myeloid progenitor cells, as well as the final differentiated myeloid cells developed therefrom, can be produced by a process involving the steps of providing mature cells, reprogramming the mature cells to produce induced pluripotent stem cells, making the induced pluripotent stem cells less immunogenic (e.g., by knocking out HLA class I / II genes and upregulating CD47), differentiating the pluripotent stem cells into hematopoietic stem cells, differentiating the hematopoietic stem cells into myeloid progenitor cells, and differentiating the myeloid progenitor cells into mature cells of the myeloid lineage, such as granulocytes (progenitors into neutrophils, eosinophils, basophils), monocytes (progenitors into macrophages), megakaryocytes (progenitors into platelets), and erythroid progenitors (progenitors into erythroids). At each step, the cells may also have low immunogenicity.

[0319] In some embodiments, iMAC cells are CD45+ CD14 high It is characterized by being.

[0320] In some embodiments, macrophages express one or more of CD14, CD11b, CD68, CD163, F4 / 80, CD16, CD54, CD49e, CD38, Egr2, CD71, TLR2, and TLR4. In some embodiments, the engineered cell population is 40% to 60% CD14+, 50% to 70% CD14+, 60% to 80% CD14+, 70% to 90% CD14+, 80% to 100% CD14+, or any percentage within the range defined by any two of the above-mentioned values.

[0321] In some embodiments, the engineered macrophage population is CD14lo. In some embodiments, the engineered macrophage population is CD14high.

[0322] In some embodiments, the engineered macrophage population is 40% to 60% CD45+, 50% to 70% CD45+, 60% to 80% CD45+, 70% to 90% CD45+, 80% to 100% CD45+, or any percentage within the range defined by any two of the above-mentioned values.

[0323] In some embodiments, the engineered macrophage population is 60% to 80% CD45+ CD56+, 65% to 85% CD45+ CD56+, 70% to 90% CD45+ CD56+, 75% to 95% CD45+ CD56+, 80% to 99% CD45+ CD14+, or any percentage within the range defined by any two of the above-mentioned values.

[0324] In some embodiments, the engineered macrophage population is at least 40% CD45+, at least 50% CD45+, at least 60% CD45+, at least 70% CD45+, at least 80% CD45+, at least 90% CD45+, or 100% CD45+.

[0325] In some embodiments, iMAC expresses CAR in engineered stem cells as described in Section IID.

[0326] Tumor cells are highly sensitive targets for the assay of macrophage cytotoxic activity in vitro. In the assay, macrophages are co-incubated at different rates with target tumor cells known to be sensitive to macrophage-mediated tumor death. Target cells are pre-labeled with fluorescent dyes to distinguish them from effector cells (macrophages). After the incubation period, dead target cells are identified by nucleic acid staining that specifically penetrates dead cells. % death is calculated by comparing the total number of viable cells in each assay well to a non-effector control well. As used herein, the term "activity" refers to the measurement of the cytotoxic ability of macrophages against target cells.

[0327] In some embodiments, the engineered macrophages may be fresh or frozen. In some embodiments, the engineered macrophages are fresh. In some embodiments, the engineered macrophages are frozen. In some embodiments, when the frozen macrophages are thawed, they retain viability and cytotoxicity compared to fresh macrophages. In some embodiments, the frozen / thawed macrophages also perform tumor control to the same extent as fresh macrophages.

[0328] In some embodiments, cells may be frozen by a cryopreservation method. In some embodiments, iMCs are applied to cryopreservation after differentiation according to the provided method. In some embodiments, iMCs are applied to cryopreservation after manipulation. In some embodiments, manipulated iMCs produced according to the provided method are applied to cryopreservation. In some embodiments, the method comprises cryopreserving cells in the presence of a cryoprotectant to produce a cryopreserved composition. In some aspects, any of various known freezing solutions and parameters may be used. In some embodiments, the cryoprotectant is DMSO. In some embodiments, cells are frozen, for example, cryopreserved in a solution having a final concentration of 1% to 15%, 6% to 12%, 5% to 10%, or 6% to 8% DMSO. In some embodiments, the cryopreservation medium is 5% or about 5% to 10% or about 10% DMSO (v / v). In some embodiments, the cryopreservation medium contains one or more additional excipients, such as plasmalite A or human serum albumin (HSA). In some embodiments, the solution for cryopreservation may also contain human serum albumin (HSA). In certain embodiments, cells are frozen, for example, cryopreserved in a solution having a final concentration of 0.1% to 5%, 0.25% to 4%, 0.5% to 2%, or 1% to 2% HSA. In some embodiments, the cryopreservation medium contains a commercially available cryopreservation solution (CryoStor™ CS10 or CS5). CryoStor™ CS10 is a cryopreservation medium containing 10% dimethyl sulfoxide (DMSO). CryoStor™ CS5 is a cryopreservation medium containing 5% dimethyl sulfoxide (DMSO). In some embodiments, the cells are generally then frozen at -80° or about -80° at a rate of 1° or about 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.In some aspects, the engineered iMC is thawed prior to use, for example, in connection with the treatment method described herein. In some embodiments, after thawing the cells, the method comprises washing the cryopreserved composition under conditions to reduce or remove the cryoprotectant.

[0329] III. Gene Editing and Manipulation

[0330] In some embodiments, pluripotent stem cells (e.g., iPSCs) or iMCs may be modified by gene editing. In some embodiments, pluripotent stem cells (e.g., iPSCs) or iMCs may be modified by genetic manipulation, for example, by introducing an exogenous nucleic acid encoding a transgene, for example, a chimeric antigen receptor (CAR). In some embodiments, the gene-edited iPSCs as described may be used as source cells for differentiation into iMCs (e.g., iMACs).

[0331] Genome editing generally refers to a process of editing or altering the nucleotide sequence of a genome in a preferably accurate, desirable, and / or predetermined manner. Examples of compositions, systems, and methods of genome editing described herein use site-directed nucleases to cut or cleave DNA at precise target locations in the genome, thereby creating double-strand breaks (DSBs) in the DNA. These breaks may be repaired by endogenous DNA repair pathways, such as homology-directed repair (HDR) and / or non-homology-directed end-junction (NHEJ) repair (see, e.g., Cox et al., (2015) Nature Medicine 21 (2):121-31).

[0332] In some embodiments, the cells described herein (e.g., stem cells, macrophages) are genetically modified. In some embodiments, the modification involves knocking out one or more endogenous genes and / or soluble one or more exogenous genes of interest using DNA-targeting proteins and nucleases or RNA-guided nucleases. In some embodiments, the gene of interest is soluble in a specific locus of interest. In some embodiments, the gene of interest is a synthetic cytokine receptor complex. In some embodiments, the synthetic cytokine receptor complex is activated by rapamycin. In some embodiments, the synthetic cytokine receptor complex is a rapamycin-activated cytokine receptor (RACR). In some embodiments, the RACR is soluble in a locus of interest. In some embodiments, the gene of interest is a chimeric antigen receptor.

[0333] In some embodiments, the modification involves contacting the cell with a DNA-targeting protein and nuclease or an RNA-guided nuclease. In some embodiments, the DNA-targeting protein and nuclease or the RNA-guided nuclease comprises a zinc finger protein (ZFP), a clustered, regularly spaced short palindromic nucleic acid (CRISPR), or a TAL-effector nuclease (TALEN). In some embodiments, CRISPR-Cas9 is used. In some embodiments, CRISPR-Mad7 is used.

[0334] Rejection of cell therapies (e.g., CAR T cells) is due to a mismatch in human leukocyte antigens (HLA) between at least the donor and the recipient. One recently identified solution is to disrupt the expression of genes associated with this rejection, such as beta-2-microglobulin (B2M). Accordingly, in some embodiments, the cells described herein (e.g., iPSCs, macrophages) are genetically engineered to knock out the B2M locus.

[0335] In some embodiments, the cells described herein are genetically engineered to be rapamycin resistant. Rapamycin is a small molecule drug that inhibits the mTOR pathway, which is an essential pathway for cell growth and expansion. Therefore, contacting cells with rapamycin may, in some cases, inhibit or reduce cell growth and expansion. In some embodiments, to eliminate or reduce rapamycin-mediated growth inhibition of source cells or macrophages using the provided method, the provided cells are destroyed at the endogenous genes associated with rapamycin function, thereby making these cells "rapamycin resistant." The reference to "rapamycin resistant" cells is understood to refer to the ability of the endogenous mTOR pathway in cells that is not affected or impacted by the presence of rapamycin or rapamycin analogs. However, it is further understood that "rapamycin resistant" cells may nevertheless respond to rapamycin through a pathway that does not involve mTOR, for example, due to the activation of synthetic RACR as described herein.

[0336] In some embodiments, cells are genetically engineered to destroy genes associated with rapamycin recognition. In some embodiments, cells are genetically engineered to destroy the mTOR gene. In some embodiments, the mTOR gene is FKBP-12 (also known as FKBP-1A, FKBP1, FKBP12, PKC12, PKCI2, and PPIASE). FKBP12 is an essential binder of rapamycin and is required for its function. In some embodiments, cells are genetically engineered to destroy the FKBP12 gene. In some embodiments, cells are genetically engineered to induce rapamycin resistance by knocking out the FKB12 ​​gene. In some embodiments, the destruction of the endogenous FKBP12 gene in source stem cells (e.g., iPSCs) is achieved through genetic knockout using the CRISPR-Cas system. In normal cells without genetic destruction of FKBP12, FKBP12 is the primary binder of rapamycin, and subsequently, the FKBP12-rapamycin complex binds to the FRB subunit of mTOR and blocks mTOR signaling. For example, by destroying the expression of the FKBP12 gene by FKBP12 knockout, rapamycin does not function without first complexing with FKBP1A, so the results of the present invention demonstrate successful rapamycin inhibitory activity. Therefore, genetic destruction of FKBP12, for example by gene knockout, causes stem cells (e.g., iPSCs) to have high resistance to rapamycin-mediated mTOR inhibition, enabling vigorous growth of stem cells (e.g., iPSCs) even in the presence of high doses of rapamycin. In some embodiments, the ability to make cells resistant to rapamycin growth inhibition allows for the binding of RACR by rapamycin during cell differentiation without adverse effects. In addition, knockout of FKBP12 avoids competition between RACR and FKBP12 for rapamycin binding.Therefore, in some embodiments, the ability to confer resistance to rapamycin growth on cells by FKBP12 knockout also allows for the activation of RACR-containing cells in vivo and suppresses potential allogeneic anti-graft responses through the suppression of mTOR of the host immune system. In some embodiments, the gRNA used to knock out FKBP12 was a pool of gRNA molecules. In some embodiments, the gRNA comprises one or more gRNAs selected from gRNAs comprising the sequences presented in SEQ ID NO: 19, SEQ ID NO: 20, or SEQ ID NO: 21. In some embodiments, the one or more gRNAs is a pool of gRNAs comprising two or three gRNAs.

[0337] In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor in an endogenous gene. In some embodiments, the synthetic cytokine receptor is genetically engineered so that the expression of the endogenous gene is not destroyed. In some embodiments, the cells described herein are genetically engineered to include a nucleotide sequence encoding a synthetic cytokine receptor complex in a destroyed gene, such as a gene inactivated or knocked out in the cell.

[0338] In some embodiments, the cells described herein are genetically engineered to contain a nucleotide sequence encoding a synthetic cytokine receptor in a target endogenous gene. In some embodiments, the synthetic cytokine receptor is engineered into a safe-haber locus. In some embodiments, the target endogenous gene is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target endogenous gene is B2M.

[0339] In some embodiments, the gene of interest inserted into the endogenous locus is a synthetic cytokine receptor complex. In some embodiments, an endogenous promoter of a specific locus is used.

[0340] In some embodiments, the exogenous promoter is operably coupled to the gene encoding the synthetic cytokine receptor complex to drive expression. In some embodiments, the promoter is the EF1A promoter (also known as the EEF1A promoter). In some embodiments, the promoter is the MND promoter. In some embodiments, additional promoter(s) are included so that two or more promoters can drive the expression of the exogenous gene of interest. In some embodiments, the two or more promoters may be the same or different. In some embodiments, the promoter is a dual promoter in which the synthetic cytokine receptor is under the operable control of two promoters. In some embodiments, the dual promoter is a dual EF1α promoter.

[0341] For example, in some embodiments, the cell comprises a destroyed B2M gene and a nucleotide sequence encoding a synthetic cytokine receptor within the destroyed B2M gene.

[0342] In some embodiments, the cells described herein (e.g., iPSCs, macrophages) comprise (i) a destroyed B2M locus, and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) under the control of an endogenous B2M promoter and an EEF1A promoter.

[0343] In some embodiments, the cell described herein (e.g., iPSC, macrophage) comprises (i) a destroyed B2M locus, and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) inserted into the endogenous B2M gene under the control of the endogenous B2M promoter and the EEF1A promoter.

[0344] In some embodiments, a cell comprising (i) a destroyed B2M locus and (ii) a nucleotide sequence encoding a synthetic cytokine receptor complex (e.g., RACR) is produced by any of the methods described below.

[0345] In some embodiments, the cell described herein (e.g., iPSC, macrophage) further comprises (i) a destroyed AAVS1 locus, and (ii) a nucleotide sequence encoding a CAR (e.g., CD19-CAR) under the control of an endogenous AAVS1 promoter.

[0346] A. System for genome editing

[0347] In some embodiments, the system for editing cells described herein comprises a site-directed nuclease, such as a CRISPR / Cas system, and optionally gRNA. In some embodiments, the system comprises a engineered nuclease. In some embodiments, the system comprises a site-directed nuclease. In some embodiments, the site-directed nuclease comprises a CRISPR / Cas nuclease system. In some embodiments, the Cas nuclease is Cas9. In some embodiments, the nuclease is Mad7. In some embodiments, the guide RNA comprising the CRISPR / Cas system is a single guide RNA (sgRNA).

[0348] The sequences herein present exemplary gRNA targeting sequences. In some embodiments, the gRNA targeting sequence may contain one or more thymines in a complementary subsequence substituted with uracil. A person skilled in the art will understand that uracil and thymine may both be denoted by 't' instead of 'u' for uracil and 't' for thymine; and that in the context of ribonucleic acid, 't' is used to denote uracil unless otherwise indicated.

[0349] 1. Nuclease

[0350] a. CRISPR / Cas nuclease system

[0351] The naturally occurring CRISPR / Cas system is a genetic defense system that provides a form of acquired immunity in prokaryotes. CRISPR consists of clustered, regularly spaced, short palindromic repeats ( C lustered R egularly I nterspaced S hort P alindromic R CRISPR is an abbreviation for epieats and refers to a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) similar to foreign DNA previously exposed to the cell by viruses that have infected or attacked prokaryotes. These DNA fragments are used by prokaryotes to detect and destroy similar foreign DNA (e.g., from similar viruses during a subsequent attack) upon reintroduction. Transcription of the CRISPR locus results in the formation of RNA molecules containing spacer sequences that associate with Cas (CRISPR-associated) proteins capable of recognizing and cleaving foreign exogenous DNA. Numerous types and classes of CRISPR / Cas systems have been described (see, for example, the literature [Koonin et al., (2017) Curr Opin Microbiol 37:67-78]).

[0352] Engineered versions of the CRISPR / Cas system have been developed in numerous forms to mutate or edit the genomic DNA of cells from different species. A general approach using the CRISPR / Cas system involves xeno-expressing or introducing site-directed nucleases (e.g., Cas nucleases) into cells in combination with guide RNA (gRNA) to induce DNA cleavage events (e.g., the formation of single-strand or double-strand breaks (SSBs or DSBs)) in the backbone of the cell's genomic DNA at precise and targetable locations. The way in which DNA cleavage events are repaired by the cell provides an opportunity to edit the genome by adding, removing, or modifying (substituting) DNA nucleotide(s) or sequences (e.g., genes).

[0353] In some embodiments, the system for editing cells described herein comprises a nuclease capable of inducing DNA breakage within an endogenous target gene in the cell. In some embodiments, the DNA breakage comprises a double-strand break (DSB) induced by a nuclease capable of inducing a DSB by cleaving both strands of double-stranded DNA at a cleavage site. In some embodiments, the DNA breakage comprises a single-strand break (SSB) at a cleavage site of the sense strand or the antisense strand of the endogenous target gene. In some embodiments, the DNA breakage comprises an SSB at a cleavage site of the sense strand and an SSB at a cleavage site of the antisense strand, thereby causing a DSB. In some embodiments, the DSB is induced by a pair of recombinant nucleases, for example, nikcases, which can each induce a single-strand break (SSB) of the opposite DNA strand at different cleavage sites, for example, a cleavage site upstream of a gene variant on one strand and a cleavage site downstream of a gene variant on the other strand of the target gene. In some embodiments, the first of the nikase pair forms a complex with a first guide RNA, e.g., a first sgRNA, to target a cleavage to one strand, e.g., the sense strand, and the second of the nikase pair forms a complex with a second guide RNA, e.g., a second sgRNA, to target a cleavage to the other strand, e.g., the antisense strand. In some embodiments, the DSB is induced in the cell via an SSB on each of the opposite strands of the endogenous target gene, namely the sense strand and the antisense strand.

[0354] Generally, genes are located on double-stranded DNA containing a sense strand and an antisense strand that are complementary to each other. The sense strand is also referred to as the coding strand because its sequence is the DNA version of the RNA sequence to which it is transcribed. The antisense strand is also referred to as the template strand because its sequence is complementary to the RNA sequence to which it is transcribed.

[0355] i. Guide RNA (gRNA)

[0356] The engineered CRISPR / Cas system comprises at least two components: 1) a guide RNA (gRNA) molecule and 2) a Cas nuclease (which interact to form a gRNA / Cas nuclease complex). The gRNA comprises at least a user-defined targeting domain referred to as a "spacer," which includes a nucleotide sequence and a CRISPR repeat sequence. In the engineered CRISPR / Cas system, the gRNA / Cas nuclease complex is targeted to a specific target sequence of interest within a target nucleic acid (e.g., genomic DNA molecule) by generating a gRNA containing a spacer having a nucleotide sequence capable of binding to the specific target sequence in a complementary manner (see references [Jinek et al., Science, 337, 816-821 (2012)] and [Deltcheva et al., Nature, 471, 602-607 (2011)]). Therefore, the spacer provides the targeting function of the gRNA / Cas nuclease complex.

[0357] In naturally occurring type II-CRISPR / Cas systems, "gRNA" consists of two RNA strands: 1) CRISPR RNA (crRNA) containing a spacer and a CRISPR repeat sequence, and 2) trans-activated CRISPR RNA (tracrRNA). In type II-CRISPR / Cas systems, a portion of the crRNA containing the CRISPR repeat sequence and a portion of the tracrRNA hybridize to form a crRNA:tracrRNA duplex that interacts with a Cas nuclease (e.g., Cas9). The terms "split gRNA" or "modular gRNA" as used herein refer to a gRNA molecule comprising two RNA strands, wherein the first RNA strand incorporates the crRNA function(s) and / or structure, and the second RNA strand incorporates the tracrRNA function(s) and / or structure, wherein the first and second RNA strands are partially hybridized.

[0358] Accordingly, in some embodiments, the gRNA comprises two RNA molecules. In some embodiments, the gRNA comprises CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). In some embodiments, the gRNA is a split gRNA. In some embodiments, the gRNA is a modular gRNA. In some embodiments, the split gRNA comprises a first strand comprising a first region of spacer and complementarity from 5' to 3'; a second region of complementarity from 5' to 3'; and optionally a second strand comprising a tail domain.

[0359] In some embodiments, the crRNA comprises a spacer comprising a nucleotide sequence that is complementary and hybridizes to a sequence that is complementary to a target sequence on a target nucleic acid (e.g., a genomic DNA molecule). In some embodiments, the crRNA comprises a region that is complementary and hybridizes to a portion of the tracrRNA.

[0360] In some embodiments, the target nucleic acid (e.g., endogenous gene) is B2M. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 18, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence presented in SEQ ID NO: 18. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 18.

[0361] In some embodiments, the target nucleic acid (e.g., endogenous gene) is FKBP12. In some embodiments, the crRNA comprises the nucleotide sequence presented in any one of SEQ ID NOs: 19, 20, and 21, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence presented in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises the nucleotide sequence presented in any one of SEQ ID NOs: 19, 20, and 21. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 19, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence presented in SEQ ID NO: 19. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 19. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 20, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence presented in SEQ ID NO: 20. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 20. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 21, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence presented in SEQ ID NO: 21. In some embodiments, the crRNA comprises the nucleotide sequence presented in SEQ ID NO: 21.

[0362] In some embodiments, the target nucleic acid (e.g., endogenous gene) is AAVS1. In some embodiments, the gRNA comprises the nucleotide sequence presented in SEQ ID NO: 52, or a nucleotide sequence having at least 80%, 85%, 90%, or 95% sequence identity with the nucleotide sequence presented in SEQ ID NO: 52. In some embodiments, the gRNA comprises the nucleotide sequence presented in SEQ ID NO: 52.

[0363] In some embodiments, tracrRNA may comprise all or part of a wild-type tracrRNA sequence from a naturally occurring CRISPR / Cas system. In some embodiments, tracrRNA may comprise a truncated or modified variant of wild-type tracrRNA. The length of tracrRNA may depend on the CRISPR / Cas system used. In some embodiments, tracrRNA may comprise a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 nucleotides. In certain embodiments, tracrRNA is at least 26 nucleotides long. In additional embodiments, tracrRNA is at least 40 nucleotides long. In some embodiments, tracrRNA may include specific secondary structures, such as, for example, one or more hairpin or stem-loop structures, or one or more bulge structures.

[0364] a) Single guide RNA (sgRNA)

[0365] Engineered CRISPR / Cas nuclease systems often combine crRNA and tracrRNA into a single RNA molecule (referred herein to as "single guide RNA" (sgRNA)) by adding a linker between these components. Without being bound by theory, similar to duplexed crRNA and tracrRNA, sgRNA will form a complex with a Cas nuclease (e.g., Cas9), guide the Cas nuclease to a target sequence, and activate the Cas nuclease to cleave a target nucleic acid (e.g., genomic DNA). Thus, in some embodiments, the gRNA may comprise operably linked crRNA and tracrRNA. In some embodiments, the sgRNA may comprise a crRNA covalently linked to a tracrRNA. In some embodiments, the crRNA and tracrRNA are covalently linked via a linker. In some embodiments, the sgRNA may comprise a stem-loop structure through base pairing between the crRNA and tracrRNA. In some embodiments, the sgRNA comprises a 5' to 3' spacer, a first complementary region, a linking domain, a second complementary region, and optionally a tail domain.

[0366] sgRNA may be unmodified or modified. For example, modified sgRNA may contain one or more 2'-O-methylphosphorothioate nucleotides.

[0367] For example, guide RNA used in CRISPR / Cas systems, or other smaller RNAs, can be easily synthesized by chemical means as exemplified herein and described in the relevant art. While chemical synthesis procedures are continuously expanding, the purification of these RNAs by procedures, such as high-performance liquid chromatography (HPLC avoiding the use of gels like PAGE), tends to become more difficult as polynucleotide lengths increase far beyond about 100 nucleotides. One approach used to generate longer RNAs is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding the Cas9 endonuclease, are more easily generated enzymatically. Various types of RNA modifications may be introduced during or after the chemical synthesis and / or enzymatic generation of RNA, and modifications that improve stability and / or reduce the likelihood or extent of an innate immune response or enhance other properties may be introduced, for example, as described in the relevant art.

[0368] b) Spacer

[0369] In some embodiments, the gRNA includes a spacer sequence. The spacer sequence is a sequence that defines a target site on a target nucleic acid (e.g., DNA). The target nucleic acid is a double-stranded molecule: one strand contains a target sequence adjacent to the PAM sequence and is referred to as the "PAM strand," and the second strand is referred to as the "non-PAM strand" and is complementary to both the PAM strand and the target sequence. Both the gRNA spacer and the target sequence are complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is complementary to the non-PAM strand of the target nucleic acid. Therefore, in some embodiments, the spacer sequence corresponding to the target sequence adjacent to the PAM sequence is identical to the PAM strand. The gRNA spacer sequence hybridizes to the complementary strand (e.g., the non-PAM strand of the target nucleic acid / target site). In some embodiments, the spacer is sufficiently complementary to the complementary strand of the target sequence (e.g., the non-PAM strand) to target the Cas nuclease to the target nucleic acid. In some embodiments, the spacer is at least 80%, 85%, 90%, or 95% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer is 100% complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1, 2, 3, 4, 5, 6, or more nucleotides that are not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 1 nucleotide that is not complementary to the non-PAM strand of the target nucleic acid. In some embodiments, the spacer comprises 2 nucleotides that are not complementary to the non-PAM strand of the target nucleic acid.

[0370] In some embodiments, the 5' nucleotide of the gRNA includes the 5' nucleotide of the spacer. In some embodiments, the spacer is located at the 5' end of the crRNA. In some embodiments, the spacer is located at the 5' end of the sgRNA. In some embodiments, the spacer is about 15-50, about 20-45, about 25-40, or about 30-35 nucleotides long. In some embodiments, the spacer is about 19-22 nucleotides long. In some embodiments, the spacer is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the spacer is 19 nucleotides long. In some embodiments, the spacer is 20 nucleotides long. In some embodiments, the spacer is 21 nucleotides long.

[0371] In some embodiments, the nucleotide sequence of the spacer is designed or selected using a computer program. The computer program may use variables, such as predicted melting temperature, secondary structure formation, predicted annealing temperature, sequence identity, genomic context, chromatin accessibility, % GC, genomic frequency (e.g., identical or similar but diverse sequences at one or more points as a result of mismatch, insertion or deletion), methylation status, and / or the presence of SNPs.

[0372] In some embodiments, the spacer comprises at least one modified nucleotide(s) such as those described herein. The present disclosure provides a gRNA molecule comprising a spacer that may comprise the nucleobase uracil (U), whereas any DNA encoding the gRNA comprising the spacer that comprises the nucleobase uracil (U) will comprise the nucleobase thymine (T) at the corresponding position(s).

[0373] ii. Method for preparing gRNA

[0374] Methods for producing gRNA are known to those skilled in the art and include, but are not limited to, in vitro transcription (IVT), synthetic and / or chemical synthesis methods, or combinations thereof. Enzymatic (IVT), solid-phase, liquid-phase, combined synthesis methods, small-region synthesis, and ligation methods are utilized. In one embodiment, gRNA is produced using an IVT enzymatic synthesis method. Methods for producing polynucleotides by IVT are known in the art and are described in international application PCT / US2013 / 30062. Accordingly, the present disclosure also includes polynucleotides, e.g., DNA, constructs, and vectors, which are used to transcribe the gRNA described herein in vitro.

[0375] In some embodiments, a non-naturally modified nucleobase is introduced into a polynucleotide, e.g., gRNA, during or after synthesis. In certain embodiments, the modification is in the nucleoside linkage, purine or pyrimidine base, or sugar. In some embodiments, the modification is introduced to the ends of the polynucleotide using chemical synthesis or polymerase enzymes. Examples of modified nucleic acids and their synthesis are disclosed in PCT application number PCT / US2012 / 058519. The synthesis of modified polynucleotides is also described in the literature [Verma and Eckstein, Annual Review of Biochemistry, vol. 76, 99-134 (1998)].

[0376] In some embodiments, enzymatic or chemical ligation methods are used to conjugate polynucleotides or regions thereof having different functional moieties, such as targeting agents or delivery agents, fluorescent labels, liquids, nanoparticles, etc. Conjugates of polynucleotides and modified polynucleotides are reviewed in the literature [Goodchild, Bioconjugate Chemistry, vol. 1(3), 165-187 (1990)].

[0377] In some embodiments, the present disclosure provides a nucleic acid, e.g., a vector, encoding a gRNA as described herein. In some embodiments, the nucleic acid is a DNA molecule. In other embodiments, the nucleic acid is an RNA molecule. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a spacer flanked by all or part of a repeat sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid comprises a nucleotide sequence encoding a tracrRNA. In some embodiments, the crRNA and tracrRNA are encoded by two separate nucleic acids. In other embodiments, the crRNA and tracrRNA are encoded by a single nucleic acid. In some embodiments, the crRNA and tracrRNA are encoded by opposite strands of a single nucleic acid. In other embodiments, the crRNA and tracrRNA are encoded by the same strand of a single nucleic acid.

[0378] In some embodiments, the gRNA provided by this disclosure is chemically synthesized by any means described in the art (see, for example, WO / 2005 / 01248). While chemical synthesis procedures are continuously expanding, the purification of such RNA by procedures, such as high-performance liquid chromatography (HPLC avoiding the use of gels like PAGE), tends to become more difficult as the polynucleotide length increases far beyond about 100 nucleotides. One approach used to produce longer RNAs is to produce two or more molecules that are ligated together.

[0379] In some embodiments, more than one guide RNA may be used with a CRISPR / Cas nuclease system. Each guide RNA may contain a different targeting sequence so that the CRISPR / Cas system cleaves more than one target nucleic acid. In some embodiments, more than one guide RNA may have the same or different properties, such as activity or stability, within the Cas9 RNP complex. When more than one guide RNA is used, each guide RNA may be encoded in the same or different vectors. The promoters used to drive the expression of the more than one guide RNA may be the same or different.

[0380] Guide RNA can target any sequence of interest through the targeting sequence of crRNA (e.g., a spacer sequence). In some embodiments, the degree of complementarity between the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule are 100% complementary. In other embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain at least one mismatch. For example, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 1 to 6 discrepancies. In some embodiments, the targeting sequence of the guide RNA and the target sequence on the target nucleic acid molecule may contain 5 or 6 discrepancies.

[0381] The length of the targeting sequence may depend on the CRISPR-Cas system and components used. For example, different Cas9 proteins from different bacterial species have varying optimal targeting sequence lengths. Accordingly, the targeting sequence may include a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more than 50 nucleotides. In some embodiments, the targeting sequence may include a length of 18 to 24 nucleotides. In some embodiments, the targeting sequence may include a length of 19 to 21 nucleotides. In some embodiments, the targeting sequence may include a length of 20 nucleotides.

[0382] In some embodiments of the present disclosure, the CRISPR / Cas nuclease system comprises at least one guide RNA. In some embodiments, the guide RNA and the Cas protein may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex. The guide RNA may guide the Cas protein to a target sequence on a target nucleic acid molecule (e.g., a genomic DNA molecule), where the Cas protein cleaves the target nucleic acid. In some embodiments, the CRISPR / Cas complex is a Cpf1 / guide RNA complex. In some embodiments, the CRISPR complex is a type-II CRISPR / Cas9 complex. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the CRISPR / Cas9 complex is a Cas9 / guide RNA complex. In some embodiments, the CRISPR / Cas complex is an engineered class 2 type V CRISPR system. In some embodiments, the endonuclease is Mad7.

[0383] iii. Cas nuclease

[0384] In some embodiments, the present disclosure provides compositions and systems (e.g., engineered CRISPR / Cas systems) comprising a site-directed nuclease, wherein the site-directed nuclease is a Cas nuclease. The Cas nuclease may comprise at least one domain that interacts with a guide RNA (gRNA). Additionally, the Cas nuclease is directed to a target sequence by the guide RNA. Once directed to the target sequence, the guide RNA interacts with the target sequence as well as the Cas nuclease so that the Cas nuclease can cleave the target sequence. In some embodiments, the guide RNA provides specificity for cleaving the target sequence, and the Cas nuclease is universal and pairs with different guide RNAs to cleave different target sequences.

[0385] In some embodiments, the CRISPR / Cas system comprises components derived from Type I, Type II, or Type III systems. The updated classification scheme for CRISPR / Cas locus defines Class 1 and Class 2 CRISPR / Cas systems having Types I through V or VI (Makarova et al., (2015) Nat Rev Microbiol, 13(11):722-36; Shmakov et al., (2015) Mol Cell, 60:385-397). Class 2 CRISPR / Cas systems have a single protein effector. Cas proteins of Type II, V, and VI are single-protein, RNA-guided endonucleases (referred herein to as "Class 2 Cas nucleases"). Class 2 Cas nucleases include, for example, the Cas9, Cpf1, C2c1, C2c2, and C2c3 proteins. Cpf1 nuclease (Zetsche et al., (2015) Cell 163:1-13) is homologous to Cas9 and contains a RuvC-like nuclease domain.

[0386] In some embodiments, the Cas nuclease is derived from a type-II CRISPR / Cas system (e.g., the Cas9 protein from the CRISPR / Cas9 system). In some embodiments, the Cas nuclease is derived from a class 2 CRISPR / Cas system (a single-protein Cas nuclease, e.g., the Cas9 protein or the Cpf1 protein). Proteins of the Cas9 and Cpf1 families are enzymes having DNA endonuclease activity and can be directed to cleave a desired nucleic acid target by designing a suitable guide RNA as further described herein.

[0387] Type-II CRISPR / Cas system components are derived from Type-IIA, Type-IIB, or Type-IIC systems. Cas9 and its orthologs are included. Non-limiting exemplary species from which Cas9 nucleases or other components are derived include the following: Streptococcus pyogenes ( Streptococcus pyogenes ), Streptococcus thermophilus ( Streptococcus thermophilus ), Streptococcus species ( Streptococcus sp. ), Staphilococcus aureus( Staphylococcus aureus ), Listeria inocula( Listeria innocua ), Lactobacillus gasseri( Lactobacillus gasseri ), Francisela Novicida ( Francisella novicida ), Wallinella succinogenes( Wolinella succinogenes ), Suterella wazwottensis( Sutterella wadsworthensis ), Gamma Proteobacterium ( Gamma proteobacterium ), Neisseria meningitidis( Neisseria meningitidis ), Campylobacter jejuni( Campylobacter jejuni ), Pasteurella water tossida ( Pasteurella multocida ), Fibrobacter succinogenes( Fibrobacter succinogene ), Rhodospirilum rubrum( Rhodospirillum rubrum ), Nocardiopsis dasonvillei( Nocardiopsis dassonvillei ), Streptomyces pristinaespiralis ( Streptomyces pristinaespiralis ), Streptomyces viridochromogenes ( Streptomyces viridochromogenes), Streptomyces viridochromogenes ( Streptomyces viridochromogenes ), Streptosporangium roseum( Streptosporangium roseum ), Streptosporangium roseum( Streptosporangium roseum ), Allycyclobacillus acidocaldarius( Alicyclobacillus acidocaldarius ), Bacillus pseudomycoides( Bacillus pseudomycoides ), Bacillus selenityreducens( Bacillus selenitireducens ), Exiguobacterium sibiricum( Exiguobacterium sibiricum ), Lactobacillus delbrueckii( Lactobacillus delbrueckii ), Lactobacillus salivarius( Lactobacillus salivarius ), Lactobacillus buccneri( Lactobacillus buchneri ), Treponema denticolla ( Treponema denticola ), Microsila marinaa ( Microscilla marina ), Burgholderiales bacterium ( Burkholderiales bacterium ), Polaromonas naphthalenivorans( Polaromonas naphthalenivorans ), Polaromonas species ( Polaromonas sp. ), Crocospaera Wassoni ( Crocosphaera watsonii ), Cyanotheca species ( Cyanothece sp. ), Microcystis aeruginosa( Microcystis aeruginosa ), Synechococcus species ( Synechococcus sp. ), Acetohalobium arabaticum( Acetohalobium arabaticum ), Ammonifex degensii( Ammonifex degensii ), Caldicellulose sylphthor bexyi( Caldicelulosiruptor becscii ), Candidatus desulphorudis( Candidatus Desulforudis ), Clostridium botulinum ( Clostridium botulinum ), Clostridium difficile ( Clostridium difficile ), Pinegoldia Magna( Finegoldia magna ), Natranaerobius Thermophilus( Natranaerobius thermophilus ), Pelotomaculum thermopropionicum ( Pelotomaculum thermopropionicum ), Acidithiobacillus caldus( Acidithiobacillus caldus ), Acidithiobacillus ferrooxidans ( Acidithiobacillus ferrooxidans ), Allochromatium vinosum( Allochromatium vinosum ), Marinobacter species ( Marinobacter sp. ), Nitrosococcus halopillus( Nitrosococcus halophilus ), Nitrosococcus wassoni( Nitrosococcus watsoni), Pseudoalteromonas haloplanchthys( Pseudoalteromonas haloplanktis ), Ctedonobacter racemifer ( Ktedonobacter racemifer ), Metanohalobium bestigatum( Methanohalobium evestigatum ), Anabaena variabilis( Anabaena variabilis ), Nodularia spumigena ( Nodularia spumigena ), Northstock species ( Nostoc sp. ), Arthrospira Maxima ( Arthrospira maxima ), Arthrospira platensis( Arthrospira platensis ), Arthrospira species ( Arthrospira sp. ), Lingvia species ( Lyngbya sp. ), Microcoleus xtonoplastes( Microcoleus chthonoplastes ), Oscillatoria species ( Oscillatoria sp. ), Petroto ga Mobilis ( Petrotoga mobilis ), Thermosipho Africanus( Thermosipho africanus ), Streptococcus Pasteurianus( Streptococcus pasteurianus ), Neiceria Cinerea( Neisseria cinerea ), Campylobacter lari( Campylobacter lari ), Parvivaculum lavamentivorans( Parvibaculum lavamentivorans ), Corynebacterium diphtheria( Corynebacterium diphtheria ), or Akariochloris Marina ( Acaryochloris marina In some embodiments, the Cas9 protein is derived from Streptococcus pyogenes (SpCas9). In some embodiments, the Cas9 protein is derived from Streptococcus thermophilus (StCas9). In some embodiments, the Cas9 protein is derived from Neisseria meningitidis (NmCas9). In some embodiments, the Cas9 protein is derived from Staphylococcus aureus (SaCas9). In some embodiments, the Cas9 protein is derived from Campylobacter jejuni (CjCas9).

[0388] In some embodiments, the Cas nuclease may include more than one nuclease domain. For example, the Cas9 nuclease may include at least one RuvC-like nuclease domain (e.g., Cpf1) and at least one HNH-like nuclease domain (e.g., Cas9). In some embodiments, the Cas9 nuclease introduces a DSB into the target sequence. In some embodiments, the Cas9 nuclease is modified to contain only one functional nuclease domain. For example, the Cas9 nuclease is modified so that one of the nuclease domains is mutated or completely or partially deleted to reduce nucleic acid cleavage activity. In some embodiments, the Cas9 nuclease is modified so as not to contain a functional RuvC-like nuclease domain. In other embodiments, the Cas9 nuclease is modified so as not to contain a functional HNH-like nuclease domain. In some embodiments where only one of the nuclease domains is functional, the Cas9 nuclease is a nikase capable of introducing a single-strand break (“nik”) into a target sequence. In some embodiments, a conserved amino acid within the Cas9 nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, the Cas9 nuclease nikase includes amino acid substitutions in the RuvC-like nuclease domain. An exemplary amino acid substitution of the RuvC-like nuclease domain includes D10A (based on S. pyogenes Cas9 nuclease). In some embodiments, the nikase includes amino acid substitutions in the HNH-like nuclease domain. Exemplary amino acid substitutions of the HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on S. pyogenes Cas9 nuclease). In some embodiments, the nuclease system described herein comprises a nikase complementary to the sense and antisense strands of the target sequence and a pair of guide RNAs.Guide RNA instructs the nickcase to target and introduce the DSB by generating a nick on the opposite strand of the target sequence (i.e., double nicking). Chimeric Cas9 nucleases are used when one domain or region of a protein is replaced by a part of a different protein. For example, the Cas9 nuclease domain is replaced by a domain from a different nuclease, such as Fok1. Cas9 nuclease is a modified nuclease.

[0389] In some embodiments, the Cas nuclease is derived from a type-I CRISPR / Cas system. In some embodiments, the Cas nuclease is a component of a cascade complex of a type-I CRISPR / Cas system. For example, the Cas nuclease is a Cas3 nuclease. In some embodiments, the Cas nuclease is derived from a type-III CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type-IV CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type-V CRISPR / Cas system. In some embodiments, the Cas nuclease is derived from a type-VI CRISPR / Cas system.

[0390] In some embodiments, the Cas nuclease is a Mad endonuclease. The CRISPR / Mad system is closely related to type V (Cpf1-like) of the class-2 family of Cas enzymes. In some embodiments, the CRISPR-Mad system uses Eubacterium rectale Mad7 endonuclease or a variant thereof. The Mad7-crRNA complex cleaves target DNA by identifying PAM 5'-YTTN.

[0391] b. Engineered nuclease

[0392] In some embodiments, the cells described herein are genetically engineered with site-directed nucleases, wherein the site-directed nuclease is an engineered nuclease. Exemplary engineered nucleases are meganucleases (e.g., homogenetic endonucleases), ZFNs, TALENs, and megaTALs.

[0393] Naturally occurring meganucleases can recognize and cleave double-stranded DNA sequences of about 12 to 40 base pairs and are generally grouped into five families. In some embodiments, meganucleases are selected from the LAGLIDADG family, GIY-YIG family, HNH family, His-Cys box family, and PD-(D / E)XK family. In some embodiments, the DNA binding domain of the meganuclease is engineered to recognize and bind to sequences other than its homologous target sequences. In some embodiments, the DNA binding domain of the meganuclease is fused to a heterologous nuclease domain. In some embodiments, a meganuclease, such as a homing endonuclease, is fused to a TAL module to produce a hybrid protein, such as a "megaTAL" protein. The megaTAL protein has enhanced DNA targeting specificity by recognizing both the DNA binding domain of the meganuclease and the target sequence of the TAL module.

[0394] ZFN is a fusion protein comprising a zinc-finger DNA binding domain ("zinc finger" or "ZF") and a nuclease domain. Each naturally occurring ZF can bind to three consecutive base pairs (DNA triplets), and ZF repeats are combined to recognize a DNA target sequence and provide sufficient affinity. Therefore, engineered ZF repeats are combined to recognize longer DNA sequences, e.g., 9-, 12-, 15-, or 18-bp, etc. In some embodiments, ZFN comprises a ZF fused to a nuclease domain from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain includes a dimerization domain, for example, when the nuclease dimerizes to become active, and a pair of ZFNs comprising a ZF repeat and a nuclease domain are designed to target a target sequence comprising two half-target sequences recognized by each ZF repeat on opposite strands of a DNA molecule, with an interconnection sequence between them (sometimes referred to as a spacer in the literature). For example, the interconnection sequence is 5 to 7 bp in length. When both ZFNs of the pair bind, the nuclease domain dimerizes and can introduce a DSB into the interconnection sequence. In some embodiments, the dimerization domain of the nuclease domain includes a Knop-into-Hole motif to promote dimerization. For example, the ZFN includes a Knop-into-Hole motif in the dimerization domain of FokI.

[0395] The DNA binding domain of a TALEN typically comprises a variable number of 34 or 35-amino acid repeats ("modules" or "TAL modules"), each of which binds to a single DNA base pair, A, T, G, or C. Adjacent residues at positions 12 and 13 of each module ("repeat-variable double residues" or RVDs) specify the single DNA base pair to which the module binds. A module used to recognize G may also have an affinity for A, but TALENs have the advantage of a simple recognition code (one module for each of the four bases) that greatly simplifies the customization of the DNA-binding domain to recognize specific target sequences. In some embodiments, TALENs may include a nuclease domain from a restriction endonuclease. For example, the restriction endonuclease is FokI. In some embodiments, the nuclease domain can be dimerized to become active, and a pair of TALENs are designed to target a target sequence, which comprises two half-target sequences recognized by each DNA binding domain on opposite strands of a DNA molecule, with an interconnect sequence between them. For example, each half-target sequence is in the range of 10 to 20 bp, and the interconnect sequence is 12 to 19 bp in length. When both TALENs of the pair bind, the nuclease domain can dimerize and introduce a DSB into the interconnect sequence. In some embodiments, the dimerization domain of the nuclease domain may include a knop-into-hole motif to promote dimerization. For example, the TALEN may include a knop-into-hole motif in the dimerization domain of FokI.

[0396] c. Target area

[0397] In some embodiments, the site-directed nuclease described herein is directed toward a target nucleic acid molecule (e.g., an endogenous gene) and cleaves it (e.g., introduces a DSB). In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target nucleic acid is a gene associated with the rapamycin response. In some embodiments, the target nucleic acid is FKBP12. In some embodiments, the target nucleic acid is B2M.

[0398] The target nucleic acid molecule is any DNA molecule that is endogenous or exogenous to the cell. As used herein, the term “endogenous sequence” refers to a sequence that is natural to the cell. In some embodiments, the target nucleic acid molecule is a genomic DNA (gDNA) molecule or a chromosome from or within the cell. In some embodiments, the target sequence of the target nucleic acid molecule is a genomic sequence from or within the cell. In some embodiments, the target sequence may be located in the coding sequence of a gene, the intron sequence of a gene, the transcription control sequence of a gene, the translation control sequence of a gene, or a non-coding sequence between genes. In some embodiments, the gene may be a protein-coding gene. In other embodiments, the gene may be a non-coding RNA gene. In some embodiments, the target sequence may include all or part of a disease-associated gene.

[0399] In some embodiments, the target sequence may be located in a non-genetic functional region of the genome that controls aspects of chromatin organization, such as a scaffold region or a locus control region. In some embodiments, the target sequence may be a genetically safe harbor region, that is, a locus that facilitates safe genetic modification.

[0400] In some embodiments, the target sequence may be adjacent to a protospacer adjacent motif (PAM), which is a short sequence recognized by the CRISPR / Cas complex. In some embodiments, the PAM may be adjacent to or within 1, 2, 3, or 4 nucleotides at the 3' end of the target sequence. In some embodiments, the target sequence may contain the PAM. The length and sequence of the PAM may depend on the Cas protein used. For example, the PAM may be selected from a consensus or specific PAM sequence for a specific Cas nuclease or Cas ortholog, including those disclosed in Fig. 1 of the literature [Ran et al., (2015) Nature, 520:186-191 (2015)] (incorporated by reference herein). In some embodiments, the PAM may have a length of 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. Non-restrictive exemplary PAM sequences include the following: NGG (SpCas9 WT, SpCas9 nikase, dimeric dCas9-Fok1, SpCas9-HF1, SpCas9 K855A, eSpCas9 (1.0), eSpCas9 (1.1)), NGAN or NGNG (SpCas9 VQR variant), NGAG (SpCas9 EQR variant), NGCG (SpCas9 VRER variant), NAAG (SpCas9 QQR1 variant), NNGRRT or NNGRRN (SaCas9), NNNRRT (KKH SaCas9), NNNNRYAC (CjCas9), NNAGAAW (St1Cas9), NAAAAC (TdCas9), NGGNG (St3Cas9), NG (FnCas9), NAAAAN (TdCas9), NNAAAAW (StCas9), NNNNACA (CjCas9), GNNNCNNA (PmCas9), and NNNNGATT (NmCas9) (e.g., literature [Cong et al., (2013) Science 339:819-823]; [Kleinstiver et al., (2015) Nat Biotechnol 33:1293-1298]; [Kleinstiver et al., (2015) Nature 523:481-485]; [Kleinstiver et al., (2016) Nature 529:490-495]; [Tsai et al., (2014) Nat Biotechnol 32:569-576]; [Slaymaker et al., (2016) Science 351:84-88]; [Anders et al., (2016) Mol Cell 61:895-902]; [Kim et al., (2017) Nat Comm 8:14500]; [Fonfara et al., (2013) Nucleic Acids Res 42:2577-2590]; [See Garneau et al., (2010) Nature 468:67-71]; [Magadan et al., (2012) PLoS ONE 7:e40913]; [Esvelt et al., (2013) Nat Methods 10(11):1116-1121] (where N is defined as any nucleotide, W is defined as A or T, R is defined as a purine (A) or (G), and Y is defined as a pyrimidine (C) or (T). In some embodiments, the PAM sequence is NGG. In some embodiments, the PAM sequence is NGAN. In some embodiments, the PAM sequence is NGNG. In some embodiments, the PAM is NNGRRT. In some embodiments, the PAM sequence is NGGNG. In some embodiments, the PAM sequence may be NNAAAAW.

[0401] In some embodiments, the PAM sequence recognized by a nuclease, e.g., Cas9, varies depending on the specific nuclease and the bacterial species from which it originates. In some embodiments, the PAM sequence recognized by SpCas9 is the nucleotide sequence 5'-NGG-3', where "N" is any nucleotide. In some embodiments, the PAM sequence recognized by SaCas9 is the nucleotide sequence 5'-NGRRT-3' or the nucleotide sequence 5'-NGRRN-3', where "N" is any nucleotide and "R" is a purine (e.g., guanine or adenine). In some embodiments, the PAM sequence recognized by NmeCas9 is the nucleotide sequence 5'-NNNNGATT-3', where "N" is any nucleotide. In some embodiments, the PAM sequence recognized by CjCas9 is the nucleotide sequence 5'-NNNNRYAC-3', where "N" is any nucleotide, "R" is a purine (e.g., guanine or adenine), and "Y" is a pyrimidine (e.g., cytosine or thymine). In some embodiments, the PAM sequence recognized by StCas9 is the nucleotide sequence 5'-NNAGAAW-3', where "N" is any nucleotide and "W" is adenine or thymine.

[0402] In some embodiments, the recombinant nuclease is Cas9, and the PAM sequence is a nucleotide sequence: (a) 5'-NGG-3'; (b) 5'-NGRRT-3' or 5'-NGRRN-3'; (c) 5'-NNNNGATT-3'; (d) 5'-NNNNRYAC-3'; or (e) 5'-NNAGAAW-3'; where "N" is any nucleotide, "R" is a purine (e.g., guanine or adenine), "Y" is a pyrimidine (e.g., cytosine or thymine), and "W" is adenine or thymine. In some embodiments, the recombinant nuclease is Cas9, e.g., SpCas9, and the PAM sequence is 5'-NGG-3', where "N" is any nucleotide. In some embodiments, the recombinant nuclease is Cas9, e.g., SaCas9, and the PAM sequence is 5'-NGRRT-3' or 5'-NGRRN-3', where "N" is any nucleotide and "R" is a purine, e.g., guanine or adenine. In some embodiments, the recombinant nuclease is Cas9, e.g., NmeCas9, and the PAM sequence is 5'-NNNNGATT-3', where "N" is any nucleotide. In some embodiments, the recombinant nuclease is Cas9, e.g., CjCas9, and the PAM sequence is 5'-NNNNRYAC-3', where "N" is any nucleotide and "R" is a purine, e.g., guanine or adenine, and "Y" is a pyrimidine, e.g., cytosine or thymine. In some embodiments, the recombinant nuclease is Cas9, e.g., StCas9, and the PAM sequence is 5'-NNAGAAW-3', where "N" is any nucleotide and "W" is adenine or thymine.

[0403] d. Ribonucleoprotein

[0404] In some embodiments, the site-directed polypeptide (e.g., Cas nuclease) and the genome-targeted nucleic acid (e.g., gRNA or sgRNA) may be administered separately to a cell or a target, respectively. In some embodiments, the site-directed polypeptide may be pre-complexed with one or more guide RNAs or one or more sgRNAs. Such pre-complexed materials are known as ribonucleoprotein particles (RNPs). In some embodiments, the nuclease system comprises a ribonucleoprotein (RNP). In some embodiments, the nuclease system comprises a Cas9 RNP comprising a Cas9 protein purified by complexing with gRNA. In some embodiments, the nuclease system comprises a Mad7 RNP comprising a Mad7 protein purified by complexing with gRNA. The Cas9 and Mad7 proteins may be expressed and purified by any means known in the art. Ribonucleoproteins can be assembled in vitro and delivered directly to cells using standard electroporation or transfection techniques known in the relevant art.

[0405] B. Targeted gene insertion

[0406] In some embodiments, a synthetic cytokine receptor (e.g., RACR) is incorporated into a target nucleic acid molecule (e.g., an endogenous gene). In some embodiments, incorporation into the target endogenous gene may disrupt the expression of the target endogenous gene in the cell. In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target nucleic acid is B2M. In some embodiments, the nucleic acid encoding the engineered cytokine receptor is incorporated into a B2M locus that is disrupted, for example, by HDR or other methods. In some embodiments, HDR may be used to incorporate a donor template containing the nucleic acid encoding the synthetic cytokine receptor (e.g., RACR) into the target nucleic acid molecule (e.g., an endogenous gene). For example, a construct encoding a synthetic cytokine receptor by the HDR method further includes a first homologous arm and a second homologous arm that are homologous to the target gene locus for CRISPR-based homologous-directed repair.

[0407] In some embodiments, one or more additional genes may be infused into or inserted into the cell genome. In some embodiments, a gene encoding a chimeric antigen receptor (CAR), such as that described in Section IV, is inserted into the cell genome. In some embodiments, a gene encoding an FRB, such as that described in Section C, is inserted into the cell genome. In some embodiments, each of the one or more additional genes may be individually incorporated into an endogenous gene. In some embodiments, incorporation into a target endogenous gene may disrupt the expression of the target endogenous gene in the cell. In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target nucleic acid molecule is a safe harbor gene. In some embodiments, the safe harbor gene is AAVS1. In some embodiments, the target nucleic acid is B2M. In some embodiments, HDR may be used to incorporate a donor template containing a nucleic acid encoding an additional gene (e.g., CAR or FRB) into an endogenous gene. For example, by the HDR method, a construct encoding an additional gene (e.g., a nucleic acid encoding CAR or FRB) further comprises a first homologous arm and a second homologous arm that are homologous to a target gene locus for CRISPR-based homologous-directed repair. In some embodiments, a nucleic acid construct encoding a synthetic cytokine receptor is incorporated into the B2M locus, and a nucleic acid encoding FRB or CAR is incorporated into the AAVS1 locus. In some embodiments, a nucleic acid construct encoding a synthetic cytokine receptor is incorporated into the B2M locus, a nucleic acid encoding FRB is incorporated into one of the ACTB or EF1A locus, and a nucleic acid encoding CAR is incorporated into the other of the AAVS1 locus.

[0408] In some embodiments, transient BCL-XL overexpression is performed in cells deprived of a specific endogenous gene that is an essential gene (Li et al. (2018) Nucleic Acids Research, 46:10195-10215). For example, in some cases, editing an essential gene requires anti-apoptotic support to enable clonal selection, which can be achieved by providing transient overexpression of BCL-2 during editing. In some embodiments, transient BCL-XL overexpression can be achieved by introducing BCL-XL mRNA into cells.

[0409] In some embodiments, stem cells, e.g., iPSCs, are manipulated with targeted gene insertions or insertions. In some embodiments, progenitor cells, e.g., HPs, are manipulated with targeted gene insertions or insertions. In some embodiments, iMACs are manipulated with targeted gene insertions or insertions.

[0410] Methods for introducing exogenous genes, such as synthetic cytokine receptors, into target nucleic acid molecules (e.g., endogenous genes) are widely known in the relevant art (e.g., literature [Menke D. Genesis (2013) 51: 618]; [Capecchi, Science (1989) 244:1288-1292]; [Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814]; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; U.S. Patent Nos. 8,771,945, 8,586,526, 6,774,279 and UP Patent Application Publication Nos. 20030232410, 20050026157, See US20060014264; the contents thereof are incorporated by reference), targeted homologous recombination, site-specific recombinases, PB transposases, and genome editing by engineered nucleases. Agents for introducing synthetic cytokine receptors into target nucleic acid molecules may be designed from publicly available sources or obtained commercially from Transposagen, Addgene, and Sangamo Biosciences.

[0411] In some embodiments, gene editing technology may be used for the knockout or knockdown of a gene. In some embodiments, gene editing technology may be used for the knock-in or integration of DNA into a region of the genome. In some embodiments, gene editing technology mediates double-strand breaks (DSB) in relation to non-homologous end-linking (NHEJ) or homology-directed repair (HDR). In some embodiments, DNA base editing or prime-editing gene editing technology may be used. In some embodiments, programmable addition (PASTE) gene editing technology through site-specific targeting elements may be used.

[0412] Exemplary methods used to introduce synthetic cytokine receptors into target nucleic acid molecules include genome editing using endonucleases, meganucleases, zinc-finger nucleases, and transcription activator-like effector nucleases (TALENs).

[0413] In some embodiments, a method for introducing an exogenous gene, such as a gene encoding a synthetic cytokine receptor, into a target nucleic acid molecule involves genome editing using engineered endonucleases. In some embodiments, this approach involves a reverse genetic method in which a specific double-strand break is cut and generated at a desired location(s) in the genome using an artificially engineered nuclease, and then repaired by endogenous cellular processes, such as homology-directed repair (HDR) and non-homologous end-linking (NHEJ). NHEJ directly links the DNA ends at the double-strand break, whereas HDR utilizes a homologous sequence as a donor template to regenerate the missing DNA sequence at the break site. To introduce specific nucleotide modifications (e.g., mutations, such as amino acid substitutions) into genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases because most restriction enzymes target only a few base pairs of DNA, and since combinations of recognized base pairs are found at many locations in the genome, there is a very high probability that they will result in multiple cuts not restricted to the desired location. To overcome these challenges and generate site-specific single- or double-strand breaks, several distinct classes of nucleases have been discovered and biotechnologically engineered to date. These include meganucleases, zinc finger nucleases (ZFNs), transcription-activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs), such as type II and type V RGNs.

[0414] It will be apparent to those skilled in the art from reading the present disclosure that the disclosed cells, systems, and manufacturing methods can be produced using various editing mechanisms. Multiple different nuclease-based systems exist for providing editing to the genome of an organism, each of which may be used in a single editing system, a sequential editing system (e.g., using different nuclease-directed systems sequentially to provide two or more genome edits in a cell), and / or a recursive editing system (e.g., utilizing a single nuclease-directed system to introduce two or more genome edits in a cell). Therefore, those skilled in the art will recognize from reading the present disclosure that various enzyme-directed editing systems are useful for the disclosed embodiments.

[0415] In some embodiments, the targeted insertion may be by targeted-primed reverse transcription (TPRT) or "prime editing." In some embodiments, prime editing mediates the targeted insertion in human cells without requiring a DSB or donor DNA template. Prime editing is a genome editing method that uses a nucleic acid programmable DNA binding protein ("napDNAbp") that operates in association with a polymerase to write new genetic information directly to a specified DNA site (i.e., in the form of a fusion protein or otherwise provided as a trans with napDNAbp), wherein the prime editing system is programmed with a prime editing (PE) guide RNA ("PEgRNA") that both specify the target site and template the synthesis of the desired edit in the form of an alternative DNA strand through an extension (DNA or RNA) engineered into the guide RNA (e.g., the 5' or 3' end, or the inner part of the guide RNA). A replacement strand containing the desired edit (e.g., a single nucleotide substitution) shares the same sequence as the endogenous strand of the target site to be edited (excluding the fact that it contains the desired edit). Through DNA repair and / or replication mechanisms, the endogenous strand of the target site is replaced by the newly synthesized replacement strand containing the desired edit.

[0416] In some embodiments, targeted insertion is achieved through programmable addition via a site-specific targeting element (PASTE). In some aspects, PASTE is a platform where genomic insertion is directed via a CRISPR-Cas9 nicase fused to both reverse transcriptase and serine integrase. As described in the literature [Ioannidi et al. (doi.org / 10.1101 / 2021.11.01.466786)], PASTE does not produce double-strand breaks but allows for the integration of sequences as large as ~36 kb. In some embodiments, the serine integrase may be any known in the art. In some embodiments, the serine integrase has sufficient orthogonality so that PASTE can be used for multiplexed gene integration and can simultaneously integrate at least two different genes from at least two genomic loci. In some embodiments, PASTE has activity in non-dividing cells and has editing efficiency comparable to or better than homology-oriented repair or non-homology-terminal linkage-based integration with fewer detectable off-target events.

[0417] 1. Homology-Oriented Recovery (HDR)

[0418] In some aspects, the provided embodiments involve the targeted incorporation of a nucleic acid sequence, e.g., a donor template, into a target nucleic acid sequence, e.g., an endogenous gene. In some embodiments, the target nucleic acid molecule is a housekeeping gene. In some embodiments, the housekeeping gene is eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), or actin beta (ACTB). In some embodiments, the target nucleic acid is a safe harbor gene. In some embodiments, the safe harbor gene is AAVS1. In some embodiments, the target nucleic acid is B2M.

[0419] In some embodiments, the DNA repair mechanism may be induced by a nuclease after (i) two SSBs in which an SSB is present on each strand to induce a single-strand overhang; or (ii) a DSB occurring at the same cleavage site on both strands to induce a smooth end break.

[0420] In some embodiments, HDR is utilized for the targeted incorporation or insertion of nucleic acid sequence(s), e.g., donor templates, into one or more target nucleic acid molecules (e.g., endogenous gene(s)). In some embodiments, HDR may be used to incorporate a donor template comprising a synthetic cytokine receptor (e.g., RACR) and / or CAR into a target nucleic acid molecule (e.g., endogenous gene). For example, HDR may be used to incorporate a donor template encoding RACR into the B2M gene locus. For example, HDR may be used to further incorporate a donor template encoding CAR into the AAVS1 gene locus.

[0421] Agents capable of inducing DSBs, such as Cas nucleases (e.g., Cas9), TALENs, and ZFNs, promote genome editing by inducing DSBs at cleavage sites within target nucleic acid molecules, such as endogenous genes, such as B2M, as discussed in the previous section.

[0422] Agents capable of inducing SSB (sometimes referred to as nicks) include recombinant nucleases having nickase activity, such as those described in the previous section, e.g., Cas9. Examples of agents having nickase activity include Cas9 from Streptococcus pyogenes containing mutations selected from the group consisting of, e.g., D10A, H840A, H854A, and H863A.

[0423] Upon cleavage by one of these agents, a target endogenous gene containing an SSB or DSB, e.g., B2M, undergoes one of two major pathways for DNA damage repair: (1) error-prone non-homologous end junction (NHEJ), or (2) high-fidelity homology-directed repair (HDR) pathway.

[0424] In some embodiments, SSB or DSB obtains cells previously induced by one or more agent(s) comprising a nuclease, and introduces a donor template, e.g. ssODN, resulting in HDR and integration of the donor template into a target endogenous gene, e.g. B2M.

[0425] Generally, in the absence of a repair template, such as a donor template, such as ssODN, the NHEJ process religates the ends of the cut DNA strands, which frequently results in nucleotide deletions and insertions at the cut sites.

[0426] Alteration of the nucleic acid sequence in a target endogenous gene locus, e.g., the B2M gene locus, may be induced by HDR by incorporating an exogenously provided donor template encoding a synthetic cytokine receptor (e.g., RACR). The HDR pathway may occur via the canonical HDR pathway or an alternative HDR pathway. Unless otherwise specified, the terms “HDR” or “homology-oriented repair” as used herein include both canonical HDR and alternative HDR.

[0427] Canonical HDR, "canonical homology-directed repair," or "cHDR" are used interchangeably and refer to a process of repairing DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences, e.g., sister chromatids; or exogenous nucleic acids, e.g., donor templates). Canonical HDR typically acts when there is a significant resection in a DSB to form at least one single-stranded portion of DNA. In normal cells, Canonical HDR typically involves a series of steps, such as break recognition, break stabilization, resection, single-stranded DNA stabilization, DNA crossover intermediate formation, crossover intermediate degradation, and ligation. The Canonical HDR process requires RAD51 and BRCA2, and the homologous nucleic acid, e.g., the donor template, is typically double-stranded. In Canonical HDR, a double-stranded polynucleotide, e.g., a double-stranded donor template, is introduced, which contains a sequence homologous to the targeting sequence within the target endogenous gene locus and will either be directly incorporated into the targeting sequence or The sequence or a portion of the sequence of the donor template will be used as a template for insertion into a target endogenous gene, e.g., B2M. After resection at break, repair may proceed by different pathways, e.g., by the double Holiday junction model (also referred to as the double-strand break repair or DSBR pathway) or by the synthetic-dependent strand annealing (SDSA) pathway.

[0428] In the double Holiday junction model, strand intrusion occurs due to two single-strand overhangs of the targeting sequence to a homologous sequence of a double-stranded polynucleotide, e.g., a double-stranded donor template, which results in the formation of an intermediate having two Holiday junctions. The junction is displaced as new DNA is synthesized from the ends of the intrusive strands to fill the gap caused by excision. The ends of the newly synthesized DNA ligate to the excision ends, and the junction dissolves, resulting in insertion into the targeting sequence or a portion of the targeting sequence containing a gene variant. Crossing over with a polynucleotide, e.g., a donor template, may occur upon the dissolution of the junction.

[0429] In the SDSA pathway, only one single-stranded overhang penetrates a polynucleotide, e.g., a donor template, and new DNA is synthesized from the end of the penetrating strand to fill the gap caused by excision. Then, the newly synthesized DNA is annealed to the remaining single-stranded overhang, new DNA is synthesized to fill the gap, and the strands are ligated to produce a modified DNA duplex.

[0430] Alternative HDR, or "alternative homology-directed repair," or "alternative HDR" are used interchangeably and refer to a process of repairing DNA damage using homologous nucleic acids (e.g., endogenous homologous sequences, e.g., sister chromatids; or exogenous nucleic acids, e.g., donor templates) in some embodiments. Alternative HDR differs from canonical HDR in that the process utilizes a different pathway than canonical HDR and can be inhibited by the canonical HDR mediators RAD51 and BRCA2. Furthermore, alternative HDR is also distinguished by the involvement of single-stranded or nicked homologous nucleic acid templates, e.g., donor templates, whereas canonical HDR generally involves double-stranded homologous templates. In the alternative HDR pathway, a single-stranded template polynucleotide, e.g., a donor template, is introduced. A nick, single-strand break, or DSB at a cleavage site to modify a desired target site, e.g., a target endogenous gene, e.g., B2M, is mediated by a nuclease molecule, e.g., any nuclease as described herein, and a cleavage occurs at the break to expose a single-strand overhang. The incorporation of a template polynucleotide sequence, e.g., a donor template, to modify a target site of DNA typically occurs via the SDSA pathway as described herein.

[0431] In some embodiments, HDR is performed by introducing into a cell one or more agonist(s) capable of inducing DSB, such as any of those described herein, and a donor template, such as ssODN, such as any of those described herein. Introduction may be performed by any suitable delivery means, such as any of those described herein. The conditions under which HDR is allowed to occur may be any conditions suitable for performing HDR in a cell.

[0432] In some embodiments, HDR is performed by introducing into a cell one or more agonist(s) capable of inducing SSB in each strand, such as any of those described herein, and a donor template, such as ssODN, such as any of those described herein. Introduction may be performed by any suitable delivery means, such as any of those described herein. The conditions under which HDR is allowed to occur may be any conditions suitable for performing HDR in a cell.

[0433] a. Donor mold

[0434] In some embodiments, the provided method comprises the use of a donor template encoding a synthetic cytokine receptor, e.g., RACR and / or CAR, which is homologous to a portion(s) of a targeting sequence within a target gene, e.g., B2M and / or AAVS1. In some embodiments, the targeting sequence is contained within the sense strand. In some embodiments, the targeting sequence is contained within the antisense strand. Additionally, in some embodiments, a donor template is provided for use in the provided method as a template for HDR-mediated incorporation of a nucleic acid sequence encoding, e.g., RACR.

[0435] In some embodiments, the donor template is used with one or more agent(s) capable of inducing DNA breakage, e.g., SSB or DSB. In some embodiments, the donor template is used with one or more agent(s) capable of inducing DSB and guide RNA, e.g., sgRNA, to melt a nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) in a target endogenous gene locus (e.g., B2M). In some embodiments, the donor template is used with one or more agent(s) capable of inducing SSB; a first guide RNA, e.g., a first sgRNA; and a second guide RNA, e.g., a second sgRNA, to melt a nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR) in a target endogenous gene locus (e.g., B2M).

[0436] In some embodiments, the donor template comprises a nucleic acid sequence homologous to a cleavage site within the target gene, for example, B2M. In some embodiments, the donor template comprises a nucleic acid sequence homologous to a cleavage site within the sense strand of the target gene, for example, B2M. In some embodiments, the donor template comprises a nucleic acid sequence homologous to a cleavage site within the antisense strand of the target gene, for example, B2M. In some embodiments, the target gene, for example, B2M, comprises a sense strand and an antisense strand, and the sense strand comprises a targeting sequence. In some embodiments, the target gene, for example, B2M, comprises a sense strand and an antisense strand, and the antisense strand comprises a targeting sequence.

[0437] In some embodiments, a donor template, e.g., ssODN, comprises a nucleic acid sequence containing a PAM sequence homologous to the PAM sequence within the targeting sequence.

[0438] In some embodiments, the donor template is single-stranded. In some embodiments, the donor template is a single-stranded DNA oligonucleotide (ssODN). In some embodiments, the donor template is double-stranded.

[0439] In some embodiments, the ssODN comprises a 5' ssODN arm and a 3' ssODN arm. In some embodiments, the 5' ssODN arm is directly linked to the 3' ssODN arm. In some embodiments, the 5' ssODN arm is homologous to the sequence of a target gene, e.g., B2M, located immediately upstream of the cleavage site, and the 3' ssODN arm is homologous to the sequence of a target gene located immediately downstream of the cleavage site.

[0440] In some embodiments, the 5' ssODN arm and / or the 3' ssODN arm have a length of 250 to 750 nucleotides. In some embodiments, the 5' ssODN arm has a length of 250 to 750 nucleotides. In some embodiments, the 3' ssODN arm has a length of 250 to 750 nucleotides. In some embodiments, the 5' ssODN arm and the 3' ssODN arm each have a length of 250 to 750 nucleotides. In some embodiments, the 5' ssODN arm and / or the 3' ssODN arm have a length of about 500 nucleotides. In some embodiments, the 5' ssODN arm has a length of about 500 nucleotides. In some embodiments, the 3' ssODN arm has a length of about 500 nucleotides. In some embodiments, the 5' ssODN arm and the 3' ssODN arm each have a length of about 500 nucleotides.

[0441] In some embodiments, the target gene is B2M, and the donor template comprises a nucleic acid sequence homologous to a cleavage site within the B2M gene. In some embodiments, the donor template comprises a nucleic acid sequence homologous to a cleavage site within the sense strand of the B2M target gene. In some embodiments, the donor template comprises a nucleic acid sequence homologous to a cleavage site within the antisense strand of the B2M target gene. In some embodiments, the donor template is ssODN, and the 5' ssODN arm is homologous to the sequence of the B2M target gene immediately upstream of the cleavage site, and the 3' ssODN arm is homologous to the sequence of the B2M target gene immediately downstream of the cleavage site.

[0442] In some embodiments, the 5' ssODN arm comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence as presented in SEQ ID NO: 22. In some embodiments, the 5' ssODN arm comprises the nucleic acid sequence as presented in SEQ ID NO: 22.

[0443] In some embodiments, the 3' ssODN arm comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence as presented in SEQ ID NO: 23. In some embodiments, the 3' ssODN arm comprises the nucleic acid sequence as presented in SEQ ID NO: 23.

[0444] In some embodiments, the 5' ssODN arm comprises a nucleic acid sequence as presented in sequence identification number: 22, and the 3' ssODN arm comprises a nucleic acid sequence as presented in sequence identification number: 23.

[0445] In some embodiments, the target gene is AAVS1, and the donor template comprises a nucleic acid sequence homologous to a cleavage site within the AAVS1 gene. In some embodiments, the donor template comprises a nucleic acid sequence homologous to a cleavage site within the sense strand of the AAVS1 target gene. In some embodiments, the donor template comprises a nucleic acid sequence homologous to a cleavage site within the antisense strand of the AAVS1 target gene. In some embodiments, the donor template is an ssODN, the 5' ssODN arm is homologous to the sequence of the AAVS1 target gene immediately upstream of the cleavage site, and the 3' ssODN arm is homologous to the sequence of the AAVS1 target gene immediately downstream of the cleavage site.

[0446] In some embodiments, the 5' ssODN arm comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence as presented in SEQ ID NO: 53. In some embodiments, the 5' ssODN arm comprises the nucleic acid sequence as presented in SEQ ID NO: 53.

[0447] In some embodiments, the 3' ssODN arm comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence as presented in SEQ ID NO: 54. In some embodiments, the 3' ssODN arm comprises the nucleic acid sequence as presented in SEQ ID NO: 54.

[0448] In some embodiments, the 5' ssODN arm comprises a nucleic acid sequence as presented in sequence identification number: 53, and the 3' ssODN arm comprises a nucleic acid sequence as presented in sequence identification number: 54.

[0449] Additionally, isolated nucleic acids are provided herein for use in a method of incorporating a synthetic cytokine receptor (e.g., RACR or CAR) into a target gene (e.g., B2M or AAVS1), comprising the nucleic acid sequence of any donor template described herein, e.g., ssODN or a part thereof, e.g., a 5' ssODN arm or a 3' ssODN arm.

[0450] In some embodiments, the crRNA comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 18; and the 5' ssODN arm comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 22; The 3' ssODN arm comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 23. In some embodiments, the crRNA comprises the nucleic acid sequence presented in any one of Sequence Identification No.: 18; the 5' ssODN comprises the nucleic acid sequence presented in Sequence Identification No.: 22; and the 3' ssODN comprises the nucleic acid sequence presented in Sequence Identification No.: 23.

[0451] Additionally, isolated nucleic acids are provided herein for use in a method of incorporating a synthetic cytokine receptor (e.g., CAR) into a target gene (e.g., AAVS1), comprising the nucleic acid sequence of any donor template described herein, e.g., ssODN or a part thereof, e.g., a 5' ssODN arm or a 3' ssODN arm.

[0452] In some embodiments, the crRNA comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 52; and the 5' ssODN arm comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 53; The 3' ssODN arm comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 54. In some embodiments, the crRNA comprises the nucleic acid sequence presented in any one of Sequence Identification No.: 52; the 5' ssODN comprises the nucleic acid sequence presented in Sequence Identification No.: 53; and the 3' ssODN comprises the nucleic acid sequence presented in Sequence Identification No.: 54.

[0453] In some embodiments, a donor template, e.g., ssODN, comprises a nucleic acid sequence encoding a transgene sequence encoding a synthetic cytokine receptor. In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR) that responds to rapamycin or an analogue (e.g., rapalog). In some embodiments, the transgene sequence is a tandem cassette encoding both polypeptides of the synthetic cytokine receptor.

[0454] In some embodiments, a transgene encoding a synthetic cytokine receptor (e.g., RACR) may be inserted such that its expression is driven by an endogenous promoter at the integration site, e.g., a promoter that drives the expression of the endogenous B2M gene. In some embodiments where the polypeptide coding sequence lacks a promoter, the expression of the integrated transgene is subsequently ensured by transcription driven by an endogenous promoter or other control element within the region of interest. For example, a transgene encoding part of a synthetic cytokine receptor (e.g., RACR) may be inserted without a promoter, but may be inserted in-frame with the coding sequence of an endogenous locus (e.g., B2M locus) such that the expression of the integrated transgene is controlled by transcription by an endogenous promoter and / or other control element at the integration site. In some embodiments, a multicistron element, such as a ribosomal skipping element / self-cleaving element (e.g., 2A element or internal ribosomal entry site (IRES)), is positioned upstream of the transgene so that the multicistron element is positioned in-frame with one or more exons of the endogenous open reading frame in the endogenous locus (e.g., B2M locus) so that the expression of the transgene is operably linked to the endogenous promoter.

[0455] In some embodiments, each nucleic acid encoding a polypeptide of a synthetic cytokine receptor within a “tandem” cassette is controlled independently by a regulatory element or is controlled as a multicistron (e.g., bicistron) expression system. In other embodiments, each nucleic acid encoding a polypeptide of a synthetic cytokine receptor within a “tandem” cassette may be operably linked to a promoter that may be the same or different. In some embodiments, the nucleic acid molecule may contain a promoter that drives the expression of two or more different polypeptide chains. In some embodiments, this nucleic acid molecule may be a multicistron (bicistron or tricistron, see, e.g., U.S. Patent No. 6,060,273). In some embodiments, the transcription unit may be operated as a bicistron unit containing an IRES (internal ribosomal entry site), which allows for the co-expression of the gene product by a message from a single promoter. Alternatively, in some cases, a single promoter may direct the expression of RNA containing two polypeptides separated from each other by a sequence encoding a cleavable linker as described herein in a single open reading frame (ORF). Thus, the ORF codes for a single polypeptide that is processed into individual polypeptide chains during or after translation. In some embodiments, the promoter is selected from human elongation factor 1 alpha (EF1α) promoters (e.g., presented in SEQ ID NOs: 24, 25, or 26). In some embodiments, the promoter is an MND promoter (e.g., presented in SEQ ID NO: 27).

[0456] In some embodiments, a donor template, e.g., ssODN, comprises a nucleic acid sequence encoding a synthetic cytokine receptor (e.g., RACR). In some embodiments, the nucleic acid sequence encoding the synthetic cytokine receptor (e.g., RACR) is located between the 5' ssODN arm and the 3' ssODN arm. In some embodiments, the nucleic acid sequence encoding the synthetic cytokine receptor (e.g., RACR) comprises an EF1-alpha promoter (e.g., sequence identification number: 24, 25, or 26). In some embodiments, the nucleic acid sequence encoding the synthetic cytokine receptor (e.g., RACR) comprises an MND promoter (e.g., sequence identification number: 27). In some embodiments, the synthetic cytokine receptor is a rapamycin-activated cytokine receptor (RACR). The RACR may be any, for example, as described in Section II.B. In some embodiments, the nucleic acid molecule is a tandem cassette encoding the first polypeptide sequence of RACR and the second polypeptide sequence of RACR.

[0457] In some embodiments, the first nucleic acid sequence encoding RACR comprises a nucleic acid sequence encoding a RACR-gamma chain (e.g., Sequence ID No.: 28) and a nucleic acid sequence encoding a RACR-beta chain (e.g., Sequence ID No.: 33). In some embodiments, the first nucleic acid sequence codes for a RACR-gamma chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence presented in Sequence ID No.: 28. In some embodiments, the first nucleic acid sequence codes for the RACR-gamma chain sequence presented in Sequence ID No.: 28. In some embodiments, the nucleic acid sequence encoding the RACR-gamma chain further codes for a signal peptide at the N-terminus of the initial protein to facilitate protein transport when expressed. In some embodiments, the signal peptide has the sequence presented in SEQ ID NO: 29. In some embodiments, the second nucleic acid sequence codes for a RACR-beta chain having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the amino acid sequence presented in SEQ ID NO: 33. In some embodiments, the second nucleic acid sequence codes for the RACR-beta chain presented in SEQ ID NO: 33. In some embodiments, the nucleic acid sequence encoding the RACR-beta chain further codes for a signal peptide at the N-terminus of the initial protein to facilitate protein transport when expressed. In some embodiments, the signal peptide has the sequence presented in sequence identification number: 34.

[0458] In some embodiments, the first nucleic acid sequence encoding the RACR-gamma chain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 37. In some embodiments, the first nucleic acid sequence encoding the RACR-gamma chain has the sequence presented in Sequence Identification No.: 37. In some embodiments, the second nucleic acid sequence encoding the RACR-beta chain has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 38. In some embodiments, a second nucleic acid sequence encoding the RACR-beta chain is presented in sequence identification number: 38.

[0459] In some embodiments, the nucleic acid sequence encoding the RACR-gamma chain and the nucleic acid sequence encoding the RACR-beta chain are separated by a nucleic acid sequence encoding a cleavable linker. In some embodiments, an additional nucleic acid sequence encoding a cleavable linker is located downstream of the nucleic acid sequence encoding the RACR-beta chain.

[0460] In some embodiments, the linker is a protein quantification reporter linker (PQR; e.g., sequence identification number: 42) comprising any of those described in Canadian patent application number CA2970093 (the full text of which is incorporated herein by reference). In some embodiments, the PQR linker has the sequence presented in sequence identification number: 42. In some embodiments, the PQR linker is coded by the nucleotide sequence presented in sequence identification number: 41.

[0461] In some embodiments, the cleavable linker is a self-cleaving peptide, e.g., a 2A ribosomal skip element. In some cases, the cleavable linker, e.g., T2A, causes the ribosome to skip the synthesis of a peptide bond at the C-terminus of the 2A element (ribosomal skipping), which can cause separation between the end of the 2A sequence and the downstream of the next peptide (see, e.g., [de Felipe. Genetic Vaccines and Ther. 2:13 (2004)] and [deFelipe et al. Traffic 5:616-626 (2004)]). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein are, without limitation, 2A sequences from foot-and-mouth disease virus (F2A, e.g., sequence identification number: 43), equine rhinitis A virus (E2A, e.g., sequence identification number: 44), and Tosea asigna ( Thosea asigna It includes ) virus (T2A, e.g., sequence identification number: 45 or 46), and porcine Tescovirus-1 (P2A, e.g., sequence identification number: 47 or 48) as described in U.S. Patent Publication No. 20070116690.

[0462] In some embodiments, by means of a cleavable element located between the first nucleic acid sequence and the second nucleic acid sequence, the expression of the nucleic acid sequence encoding RACR yields a first peptide (i.e., RACR-gamma chain) and a separate second peptide (i.e., RACR-beta chain).

[0463] In some embodiments, the transgene sequence may also include a sequence required for a transcription termination and / or polyadenylation signal. In some aspects, an exemplary polyadenylation signal is selected from SV40, hGH, BGH, and rbGlob transcription termination sequences and / or polyadenylation signals. In some embodiments, the transgene includes an SV40 polyadenylation signal. In some embodiments, when present within the transgene, the transcription termination sequence and / or polyadenylation signal is typically the most prominent 3' sequence within the transgene and is linked to one of the homologous arms. In some embodiments, the transgene sequence includes the polyadenylation sequence presented in sequence identification number: 39.

[0464] In some embodiments, the ssODN comprises, in order: a 5' ssODN arm, an EF1-alpha promoter, a nucleic acid sequence encoding a RACR-gamma chain, a nucleic acid sequence encoding a cleavable linker (e.g., a PQR linker), a nucleic acid sequence encoding a RACR-beta chain, a poly-A sequence, and a 3' ssODN arm.

[0465] In some embodiments, ssODN comprises the sequence presented in sequence identification number: 40, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in sequence identification number: 40. In some embodiments, ssODN is presented in sequence identification number: 40.

[0466] In some embodiments, after the ssODN is incorporated into the target gene, the target gene is knocked out. In some embodiments, the target gene is human B2M and / or AAVS1, and after the ssODN is incorporated, B2M and / or AAVS1 is knocked out. In some embodiments, a nucleic acid sequence encoding a synthetic cytokine receptor is incorporated into the B2M locus. In some embodiments, a nucleic acid sequence encoding CAR is incorporated into the AAVS1 locus. In some embodiments, engineered iPSCs and iMACs have a modified B2M locus in which the endogenous B2M gene is genetically destroyed by the knockout of the B2M gene and knocked out by the targeted incorporation of the nucleic acid encoding the synthetic cytokine receptor. In some embodiments, engineered iPSCs and iMACs have a modified AAVS1 locus in which the endogenous AAVS1 gene is genetically destroyed by the knockout of the AAVS1 gene and knocked out by the targeted incorporation of the nucleic acid encoding CAR. In some embodiments, the synthetic cytokine receptor is a RACR encoded by a nucleic acid sequence containing, in order: an EF1-alpha promoter, a nucleic acid sequence encoding a RACR-gamma chain, a nucleic acid sequence encoding a cleavable linker (e.g., a PQR linker), a nucleic acid sequence encoding a RACR-beta chain, and a poly-A sequence. In some embodiments, the nucleic acid sequence encoding the RACR incorporated into the B2M locus has the sequence presented in Sequence Identification No.: 32, or a sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleic acid sequence presented in Sequence Identification No.: 32. In some embodiments, the nucleic acid encoding the RACR incorporated into the B2M locus is presented in Sequence Identification No.: 32.

[0467] IV. Chimeric Antigen Receptor

[0468] In some cases, the stem cells or macrophages of the present disclosure comprise a polynucleotide encoding a chimeric antigen receptor (CAR) to thereby produce stem cells or macrophages that express the CAR.

[0469] In some embodiments, the present disclosure considers a chimeric antigen receptor (CAR) system for use in the treatment of a subject having cancer. In some embodiments, the macrophages of the present disclosure comprise a CAR sequence (CAR-macrophage or CAR-iMAC cell).

[0470] In some embodiments, stem cells or macrophages are engineered to express a CAR construct by transfecting a cell population with an expression vector encoding the CAR construct. Exemplary examples of cell populations that may be transfected include HSCs, blood progenitor cells, myeloid progenitor cells, or macrophages. Suitable means for producing a transfected macrophage population expressing a selected CAR construct are widely known to those skilled in the art and include, to some extent, retroviruses, lentiviruses (virus-mediated CAR gene delivery systems), sleeping beauty, and piggyback (transposon / transposase systems including non-virus-mediated CAR gene delivery systems). In some embodiments, any transfection method considered in this disclosure may be used to generate CAR-expressing stem cells or macrophages.

[0471] In some embodiments, stem cells or macrophages are engineered to express a CAR construct by genetically manipulating a cell population (e.g., via CRISPR) to express the CAR construct. In some embodiments, a nucleic acid molecule encoding CAR is introduced into the cell, for example, by the introduction of a vector construct encoding CAR. In some embodiments, the construct is designed to insert the nucleic acid encoding CAR into an endogenous locus within the cell. Methods for gene insertion or melting are known and include any of the methods described in Section III. In some embodiments, the insertion of the CAR-coding construct is, for example, by homology-directed repair using a CRISPR-Cas system.

[0472] In some embodiments, the CAR construct comprises an extracellular binding portion, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain and / or an activating signaling domain. In some embodiments, the CAR construct comprises an intracellular signaling domain comprising an extracellular binding portion, a transmembrane domain, and a co-stimulatory signaling domain. In some embodiments, the CAR construct comprises an intracellular signaling domain comprising an extracellular binding portion, a transmembrane domain, and an activating signaling domain. In some embodiments, the CAR construct comprises an intracellular signaling domain comprising an extracellular binding portion, a transmembrane domain, and a co-stimulatory signaling domain and an activating signaling domain.

[0473] In some embodiments, the CAR may include additional elements, such as a signal peptide to ensure proper ex-transport of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integrated membrane protein, and a hinge domain that provides flexibility to the recognition region and allows strong binding to the targeted moiety.

[0474] A. Extracellular binding portion

[0475] Typically, CARs are generated by fusing a polynucleotide encoding VL, VH, or scFv, if necessary, to the 5' end of a polynucleotide encoding transmembrane and intracellular domains, and by transgenerating cells with the polynucleotide as well as the corresponding VH or VL. Numerous variants of CARs are widely known in the art, and this disclosure considers the use of any known variant. Additionally, VL / VH pairs and scFvs for numerous haptens are known in the art or can be routinely generated by conventional methods. Accordingly, this disclosure considers the use of any known hapten-binding domain.

[0476] In any embodiment described herein, the binding portion of the CAR may be, for example, a single-chain fragment variable region (scFv) of an antibody, Fab, Fv, Fc, or (Fab')2 fragment, etc. The use of an unmodified (i.e., full-size) antibody, such as IgG, IgM, IgA, IgD, or IgE, in or as a CAR is excluded from the scope of the invention.

[0477] In some embodiments, the binding portion of the CAR may be directed toward any antigen that is desired to be targeted, for example, due to overexpression in cells or association with a disease or pathological condition such as cancer.

[0478] In some embodiments, the binding portion of the CAR is specific to tumor antigens. The selection of the antigen-binding domain will depend on the specific type of cancer to be treated. Tumor antigens are widely known in the relevant technical field, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), EGFRvIII, IL-11Ra, IL-13Ra, EGFR, FAP, B7H3, Kit, CA LX, CS-1, MUC1, BCMA, bcr-abl, HER2, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), ALK, CD19, CD123, cyclin B1, lectin-reactive AFP, Fos-associated antigen 1, ADRB3, thyroglobulin, EphA2, RAGE-1, RU1, RU2, SSX2, AKAP-4, LCK, OY-TES1, PAXS, SART3, CLL-1, fucosyl GM1, GloboH, MN-CA IX, EPCAM, EVT6-AML, TGS5, human Telomerase reverse transcriptase, polysialic acid, PLAC1, RU1, RU2 (AS), intestinal carboxyesterase, lewisY, sLe, LY6K, mut hsp70-2, M-CSF, MYCN, RhoC, TRP-2, CYPIBI, BORIS, prostase, prostate-specific antigen (PSA), PAX3, PAP, NY-ESO-1, LAGE-la, LMP2, NCAM, p53, p53 mutant, Ras mutant, gplOO, prosteine, OR51E2, PANX3, PSMA, PSCA, Her2 / neu, hTERT, HMWMAA, HAVCR1, VEGFR2, PDGFR-beta, survivin and telomerase, legumine, HPV E6, E7, sperm protein 17, SSEA-4, tyrosinase, TARP, WT1, Prostate-Carcinoma Tumor Antigen-1 (PCTA-1), ML-IAP, MAGE, MAGE-A1, MAD-CT-1, MAD-CT-2, MelanA / MART 1, XAGE1, ELF2M, ERG (TMPRSS2 ETS fusion gene), NA17, Neutrophil Elastase, Sarcoma Translocation Breakpoint, NY-BR-1,Includes ephnnB2, CD20, CD22, CD24, CD30, CD33, CD38, CD44v6, CD97, CD171, CD179a, androgen receptor, FAP, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, GD2, o-acetyl-GD2, GD3, GM3, GPRCSD, GPR20, CXORF61, folate receptor (FRa), folate receptor beta, ROR1, Flt3, TAG72, TN Ag, Tie 2, TEM1, TEM7R, CLDN6, TSHR, UPK2 and mesothelin. Non-limiting examples of tumor antigens include the following: differentiation antigens, e.g., tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens, e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi 5; overexpressed embryonic antigens, e.g., CEA; overexpressed oncogenes and mutated tumor-suppressor genes, e.g., p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; e.g., BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, e.g., Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include the following: TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, IL13Ra2, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50,MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG1 6, TA-90\Mac-2 binding protein\cyclophyllin C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, MUC16, LMP1, EBMA-1, BARF-1, CS1, CD319, HER1, B7H6, L1CAM, IL6, and MET.,

[0479] In some embodiments, the CAR comprises an extracellular domain comprising an FMC63 scFv binding domain for CD19 binding. In some embodiments, the CAR is a second-generation CAR composed of FMC63 mouse anti-human CD19 scFv linked to a 4-1BB co-stimulatory domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain for CD19, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain including an FMC63 scFv binding domain for CD19 binding, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulation domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain including an FMC63 scFv binding domain for CD19 binding, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulation domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises an extracellular domain including an FMC63 scFv binding domain for CD19 binding, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulation domain, and a CD3zeta signaling domain.

[0480] In some embodiments, the CAR is a second-generation CAR composed of FMC63 mouse anti-human CD19 scFv linked to a CD28 co-stimulatory domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR composed of FMC63 mouse anti-human CD19 scFv linked to a CD8 transmembrane domain, a 4-1BB co-stimulatory domain and a CD3zeta intracellular signaling domain.

[0481] In some embodiments, the antigen is BCMA. CAR T-therapy targeting BCMA has been approved by the FDA and includes Avecma and Carvicti. CARs targeting BCMA are described, for example, in U.S. Publication No. 2020 / 0246381; U.S. Patent No. 10,918,665; and U.S. Publication No. 2019 / 0161553, each of which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain to BCMA, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain to BCMA, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, CAR includes a binding domain to BCMA, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulation domain, and a CD3 zeta signaling domain.

[0482] In some embodiments, the antigen is G protein-coupled receptor class C group 5 member D (GPRC5D). CARs targeting GRC5D are described, for example, in U.S. Publications Nos. 2018 / 0118803 and 2021 / 10393689, each of which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain to GRC5D, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain to GRC5D, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain to GRC5D, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3zeta signaling domain.

[0483] In some embodiments, the antigen is Fc receptor-like 5 (FcRL5). A CAR targeting FcRL5 is described, for example, in U.S. Publication No. US 2017 / 0275362, which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain to FcRL5, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain to FcRL5, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain to FcRL5, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3zeta signaling domain.

[0484] In some embodiments, the antigen is receptor tyrosine kinase-like orphan receptor 1 (ROR1). A CAR targeting ROR1 is described, for example, in U.S. Publication No. 2022 / 0096651, which is incorporated herein by reference. In some embodiments, the CAR comprises a binding domain to ROR1, a CD8a hinge, a CD8a transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain to ROR1, an IgG4 hinge, a CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3zeta signaling domain. In some embodiments, the CAR comprises a binding domain to ROR1, a CD28 hinge, a CD28 transmembrane domain, a CD28 co-stimulatory domain, and a CD3zeta signaling domain.

[0485] In some embodiments, the CAR is a second-generation CAR comprising an anti-BCMA scFv linked to a 4-1BB co-stimulation domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR comprising an anti-GPRC5D scFv linked to a 4-1BB co-stimulation domain and a CD3zeta intracellular signaling domain. In some embodiments, the CAR is a second-generation CAR comprising an anti-ROR1 scFv linked to a 4-1BB co-stimulation domain and a CD3zeta intracellular signaling domain.

[0486] A person skilled in the art will easily become familiar with CARs for various tumor antigens. Any one of these CARs may be used as a CAR. Numerous CARs have been approved by the FDA and include, but are not limited to, anti-CD19 and anti-BCMA CAR T cells, such as tisagenlecleucel (Kymriah), acicaptagen ciloleucel (Yescarta), brexucaptagen otoleucel (Decartus), lysocaptagen marareucel (Brayangi), or idecaptagen vicleucel (Avekma). Creating similar constructs for the specific targeting of a desired tumor antigen is within the level of a person skilled in the art.

[0487] In some embodiments, the binding portion of the CAR may be directed toward a universal antigen to target a wide variety of tumors without the need to manufacture a separate CAR construct. The targeted moiety recognized by the CAR may also be maintained constant. In some embodiments, a ligand may be administered to the subject to allow interaction with the target cell and interaction with the binding portion of the CAR. It is only the ligand portion of the small conjugate molecule that needs to be modified to allow the system to target cancer cells of different identit...

Claims

Claim 1 A engineered cell comprising a myeloid progenitor cell having a synthetic cytokine receptor for a non-physiological ligand, wherein the engineered cell comprising the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Claim 2 In paragraph 1, the engineered cell in which the myeloid progenitor cell is a granulocyte / monocyte progenitor cell ("GMP"). Claim 3 A engineered cell according to claim 1 or 2, characterized in that the myeloid progenitor cells have surface phenotypes of CD34+, CD90-, and CD45RA+. Claim 4 A engineered cell according to claim 1 or 2, characterized in that the myeloid progenitor cells have surface phenotypes of CD34+, CD90-, CD123+, and CD45RA+. Claim 5 A engineered cell comprising a myeloid cell having a synthetic cytokine receptor for a non-physiological ligand, wherein the engineered cell comprising the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Claim 6 In paragraph 5, engineered cells in which the myeloid cells are macrophages, neutrophils, megakaryocytes, monocytes, basophils, eosinophils, and / or red blood cells. Claim 7 A engineered cell comprising a macrophage having a synthetic cytokine receptor for a non-physiological ligand, wherein the engineered cell comprising the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Claim 8 In claim 6 or 7, the engineered cell in which the macrophage is a mature macrophage expressing CD14. Claim 9 A engineered cell, which is a neutrophil, comprising a synthetic cytokine receptor for a non-physiological ligand, wherein the engineered cell wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain; and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain, and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain. Claim 10 In any one of paragraphs 1 to 9, the engineered cell is one that has been differentiated from a stem cell. Claim 11 In paragraph 10, the engineered cell is a pluripotent stem cell. Claim 12 In paragraph 10 or 11, the engineered cell in which the stem cell is an induced pluripotent stem cell. Claim 13 In any one of claims 1 to 6 and 10 to 12, the engineered cell is an induced myeloid cell (iMC) differentiated from a stem cell engineered with a synthetic cytokine receptor. Claim 14 In any one of claims 7, 8 and 10 through 12, the engineered cell is an induced macrophage (iMAC) differentiated from a stem cell engineered with a synthetic cytokine receptor. Claim 15 In any one of paragraphs 9 to 12, the engineered cell is an induced neutrophil (iNEU) differentiated from a stem cell engineered with a synthetic cytokine receptor. Claim 16 A engineered cell according to any one of claims 1 to 15, wherein the synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an IL-2RG intracellular domain in the order from N-terminus to C-terminus; and the synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain in the order from N-terminus to C-terminus. Claim 17 A manipulated macrophage according to any one of claims 1 to 16, wherein the first dimerization domain and the second dimerization domain are extracellular domains. Claim 18 A engineered cell according to any one of claims 1 to 17, wherein the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 1, or a polypeptide sequence as presented in SEQ ID NO:

1. Claim 19 A manipulated cell according to any one of claims 1 to 18, wherein the first transmembrane domain comprises an IL-2RG transmembrane domain. Claim 20 In claim 19, a engineered cell in which the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 8 or 31, or a polypeptide sequence as presented in SEQ ID NO: 8 or 31. Claim 21 A engineered cell according to any one of claims 1 to 20, wherein the beta-chain intracellular domain is an IL-2RB intracellular domain. Claim 22 In claim 21, a engineered cell in which the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 2, or a polypeptide sequence as presented in SEQ ID NO:

2. Claim 23 A engineered cell according to any one of claims 1 to 20, wherein the beta-chain intracellular domain is the IL-7RB intracellular domain. Claim 24 In claim 23, a engineered cell in which the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 3, or a polypeptide sequence as presented in SEQ ID NO:

3. Claim 25 A engineered cell according to any one of claims 1 to 20, wherein the beta-chain intracellular domain is the IL-21RB intracellular domain. Claim 26 In claim 25, a engineered cell in which the intracellular domain of IL-21RB comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 4, or a polypeptide sequence as presented in SEQ ID NO:

4. Claim 27 A engineered cell according to any one of claims 1 to 26, wherein the second transmembrane domain comprises a transmembrane domain from the same polypeptide as the beta-chain intracellular domain. Claim 28 A engineered cell according to any one of claims 1 to 27, wherein the second transmembrane domain is a polypeptide sequence that is at least 95% identical to SEQ ID NO: 35 or 36, or a transmembrane domain of IL-2RB comprising a polypeptide sequence as presented in SEQ ID NO: 35 or 36. Claim 29 A engineered cell according to any one of claims 1 to 22, 27 and 28, wherein the first transmembrane domain of the synthetic gamma chain polypeptide is an IL-2RG transmembrane domain comprising the sequence presented in SEQ ID NO: 8 or 31, and the IL-2RG intracellular domain comprises the sequence presented in SEQ ID NO: 1; and the second transmembrane domain of the synthetic beta chain polypeptide is an IL-2RB transmembrane domain comprising the sequence presented in SEQ ID NO: 35 or 36, and the beta chain intracellular domain comprises the IL-2RB intracellular domain comprising the sequence presented in SEQ ID NO:

2. Claim 30 A engineered cell according to any one of claims 1 to 29, wherein the first transmembrane domain and the IL-2RG intracellular domain of the synthetic gamma chain polypeptide comprise the sequences presented in Sequence Identification No. 31 and Sequence Identification No. 1; and the second transmembrane domain and the beta chain intracellular domain of the synthetic beta chain polypeptide comprise the sequences presented in Sequence Identification No. 35 and Sequence Identification No.

2. Claim 31 In any one of claims 1 to 30, the first dimerization domain and the second dimerization domain are heteromerization domains selected from the FKBP12-rapamycin binding (FRB) domain and the FK506-binding protein (FKBP) of size 12 kD; and the engineered cell in which the non-physiological ligand is rapamycin or rapalog. Claim 32 A manipulated cell according to any one of claims 1 to 31, wherein the first dimerization domain is FKBP and the second dimerization domain is FRB. Claim 33 A manipulated cell according to any one of claims 1 to 31, wherein the first dimerization domain is FRB and the second dimerization domain is FKBP. Claim 34 A engineered cell according to any one of claims 31 to 33, wherein the FRB domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 6 or sequence identification number:

7. Claim 35 A engineered cell according to any one of claims 31 to 34, wherein the FRB domain comprises the polypeptide sequence presented in sequence identification number: 6 or sequence identification number:

7. Claim 36 A engineered cell according to any one of claims 31 to 35, wherein the FKBP domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 5 or sequence identification number:

30. Claim 37 A engineered cell according to any one of claims 31 to 36, wherein the FKBP domain comprises the polypeptide sequence presented in sequence identification number: 5 or sequence identification number:

30. Claim 38 A manipulated cell according to any one of claims 1 to 37, wherein the synthetic gamma chain polypeptide has an amino acid sequence identical to the amino acid sequence presented in sequence identification number: 28 or sequence identification number: 28 by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and the synthetic beta chain polypeptide has an amino acid sequence identical to the amino acid sequence presented in sequence identification number: 55 or sequence identification number: 55 by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Claim 39 A engineered cell according to any one of claims 1 to 38, wherein the synthetic gamma chain polypeptide has an amino acid sequence identical to the amino acid sequence presented in sequence identification number: 56 or sequence identification number: 56 by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and the synthetic beta chain polypeptide has an amino acid sequence identical to the amino acid sequence presented in sequence identification number: 57 or sequence identification number: 57 by at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. Claim 40 In any one of claims 1 to 30, wherein the first dimerization domain and the second dimerization domain are homomerization domains selected from: i) FK506-binding protein (FKBP) of size 12 kD; ii) cyclopyli A (CypA); or iii) gyrase B (CyrB); and engineered cells in which the non-physiological ligand is each of the following: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine-A (CsA) or its analogs; or iii) cumermycin or its analogs. Claim 41 In any one of claims 1 to 40, the engineered cells are resistant to rapamycin-mediated mTOR inhibition. Claim 42 In any one of claims 1 to 41, the engineered cell expresses a cytosolic polypeptide that binds to a non-physiological ligand, optionally wherein the cytosolic polypeptide is a cytosolic FRB domain. Claim 43 A engineered cell according to any one of claims 1 to 42, wherein the non-physiological ligand is rapamycin or rapalog, and the engineered cell expresses a cytosolic FRB domain or a variant thereof. Claim 44 A engineered cell according to claim 42 or 43, wherein the cytosolic FRB domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 6 or sequence identification number:

7. Claim 45 A engineered cell according to claim 42 or 43, wherein the cytosolic FRB domain comprises a polypeptide sequence that is at least 98% identical to sequence identification number: 6 or sequence identification number:

7. Claim 46 A manipulated cell according to any one of claims 1 to 45, wherein the manipulated cell comprises a destroyed FKBP12 gene that reduces the expression of FKBP12 within the cell. Claim 47 A manipulated cell according to any one of claims 1 to 46, wherein the manipulated cell comprises a knockout of the FKBP12 gene. Claim 48 A manipulated cell according to any one of claims 1 to 47, wherein the manipulated cell comprises a nucleotide sequence encoding a synthetic cytokine receptor inserted into the genome of the cell. Claim 49 In paragraph 48, a engineered cell in which a nucleotide sequence encoding a synthetic cytokine receptor is inserted into a non-target locus within the cell's genome. Claim 50 In paragraph 48, a engineered cell in which a nucleotide sequence encoding a synthetic cytokine receptor is inserted into an endogenous gene of the cell. Claim 51 In paragraph 50, a manipulated cell in which the insertion reduces the expression of an endogenous gene within the locus. Claim 52 A manipulated cell according to paragraph 50 or 51, wherein the insertion knocks out an endogenous gene within the locus. Claim 53 A manipulated cell in which the insertion is by homology-directed repair in any one of paragraphs 50 to 52. Claim 54 A engineered cell in which the endogenous gene is a housekeeping gene, a blood-lineage specific locus, or an immune-related gene in any one of paragraphs 50 to 53. Claim 55 In paragraph 54, engineered cells in which the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB). Claim 56 In paragraph 54, an engineered cell in which the immune-related gene is selected from the beta-2-microglobulin (B2M) gene and the signal regulatory protein alpha (SIRPA) gene. Claim 57 A manipulated cell according to any one of claims 1 to 56, wherein the manipulated cell comprises a B2M knockout. Claim 58 A engineered cell according to any one of claims 1 to 57, wherein the engineered cell comprises a B2M knockout and an FKBP12 knockout. Claim 59 A engineered cell comprising a chimeric antigen receptor (CAR) in any one of claims 1 to 58. Claim 60 A engineered cell according to any one of claims 1 to 59, wherein the binding of a non-physiological ligand to a synthetic cytokine receptor activates the synthetic cytokine receptor in the engineered cell, thereby inducing expansion and / or activation of the engineered cell in a cell population. Claim 61 A group comprising engineered myeloid progenitor cells according to any one of paragraphs 1 through 4, 10 through 12 and 16 through 60. Claim 62 A group comprising engineered myeloid cells according to any one of paragraphs 5, 6, 10 through 13 and 16 through 60. Claim 63 A group comprising a manipulated macrophage according to any one of paragraphs 7, 8, 10 through 12, 14 and 16 through 60. Claim 64 A group comprising neutrophils engineered according to any one of paragraphs 9 through 12 and paragraphs 15 through 60. Claim 65 A method for generating genetically engineered myeloid cells differentiated from stem cells, comprising: a) culturing a population of stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a hematopoietic progenitor (HP) population, wherein the synthetic cytokine receptor comprises: a synthetic gamma-chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular interleukin-2 receptor subunit gamma (IL-2RG) domain, and a synthetic beta-chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta-chain intracellular domain selected from an intracellular interleukin-2 receptor subunit beta (IL-2RB) domain, an intracellular interleukin-7 receptor subunit beta (IL-7RB) domain, and / or an intracellular interleukin-21 receptor subunit beta (IL-21RB) domain; and b) a step of culturing the cells produced in a) by incubation under conditions for generating myeloid cells, wherein at least part of one or both of steps a) and b) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor. Claim 66 In paragraph 65, the method in which the myeloid cells are macrophages or neutrophils. Claim 67 A method for generating genetically engineered macrophages differentiated from stem cells, comprising: a) culturing a population of stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a hematopoietic progenitor (HP) population, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain and an interleukin-2 receptor subunit gamma (IL-2RG) intracellular domain, and a synthetic beta chain polypeptide comprising a second dimerization domain, a second transmembrane domain, and a beta chain intracellular domain selected from an interleukin-2 receptor subunit beta (IL-2RB) intracellular domain, an interleukin-7 receptor subunit beta (IL-7RB) intracellular domain and / or an interleukin-21 receptor subunit beta (IL-21RB) intracellular domain; and b) a step of culturing the cells produced in a) by incubation under conditions for generating macrophages, wherein at least part of one or both of steps a) and b) is performed in the presence of a non-physiological ligand of a synthetic cytokine receptor. Claim 68 A method according to any one of claims 65 to 67, wherein the culture in a) is performed by one or more additional incubations in the presence of a non-physiological ligand and one or more myeloid cell differentiation factors optionally selected from one or more of IL-3, M-CSF and GM-CSF, following a first incubation under conditions for producing an embryoid (EB). Claim 69 In paragraph 68, a method in which one or more myeloid cell differentiation factors are IL-3, M-CSF, and GM-CSF. Claim 70 A method according to claim 68 or 69, wherein one or more additional incubations comprise a second incubation in a second medium comprising one or more of IL-3, GM-CSF and M-CSF, and a third incubation in a third medium comprising one or more of IL-3, GM-CSF and M-CSF, wherein one or both of the second medium and the third medium comprise a non-physiological ligand. Claim 71 A method according to any one of claims 65 to 70, wherein step a) comprises the following: (i) performing a first incubation comprising culturing a population of stem cells engineered with a synthetic cytokine receptor under conditions for forming a first aggregate in a first medium; (ii) contacting the aggregate with a dissociating agent to form a dissociated cell population; (iii) performing a second incubation comprising culturing the dissociated cell population under conditions for forming a second aggregate in a second medium, optionally wherein the second medium comprises one or more of IL-3, GM-CSF, and M-CSF; and (iv) performing a third incubation comprising culturing the cell population of (iii) in a third medium, optionally wherein the third medium comprises one or more of IL-3, GM-CSF, and M-CSF. Claim 72 A method according to any one of claims 68 to 71, wherein the first incubation is performed in a first medium comprising one or more of BMP4, FGF2, VEGF-165 and Rock inhibitor. Claim 73 A method according to any one of claims 68 to 72, wherein the first incubation is performed in a first medium comprising BMP4, FGF2, VEGF-165 and a Rock inhibitor. Claim 74 A method according to paragraph 72 or 73 in which the Rock inhibitor is Y27632. Claim 75 A method according to any one of claims 70 to 74, wherein the second medium further comprises a non-physiological ligand of a synthetic cytokine receptor. Claim 76 A method according to any one of claims 70 to 74, wherein the second medium does not contain a non-physiological ligand of a synthetic cytokine receptor. Claim 77 A method according to any one of claims 70 to 76, wherein culture in the first medium is carried out for 1 to 3 days. Claim 78 A method according to any one of claims 70 to 77, wherein the second medium comprises one or more of BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF and M-CSF. Claim 79 A method according to any one of claims 70 to 77, wherein the second medium comprises BMP4, FGF2, VEGF, LY294002, IL-3, and M-CSF. Claim 80 A method according to any one of claims 70 to 79, wherein the second medium further comprises a non-physiological ligand of a synthetic cytokine receptor. Claim 81 A method according to any one of claims 70 to 79, wherein the second medium does not contain a non-physiological ligand of a synthetic cytokine receptor. Claim 82 A method according to any one of claims 70 to 81, wherein culture in the second medium is carried out for 3 to 6 days. Claim 83 A method according to any one of claims 70 to 82, wherein the third medium comprises one or more of UM729, StemRegenin-1, BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF, and M-CSF. Claim 84 A method according to any one of claims 70 to 82, wherein the third medium comprises UM729, StemRegenin-1, BMP4, FGF2, VEGF, LY294002, IL-3, GM-CSF and M-CSF. Claim 85 A method according to any one of claims 70 to 84, wherein the third medium further comprises a non-physiological ligand of a synthetic cytokine receptor. Claim 86 A method according to any one of claims 70 to 84, wherein the third medium does not contain a non-physiological ligand of a synthetic cytokine receptor. Claim 87 A method according to any one of claims 70 to 86, wherein culture in the third medium is carried out for 6 to 12 days. Claim 88 A method in which, in any one of paragraphs 67 to 87, the culture in a) produces myeloid progenitor cells. Claim 89 In paragraph 88, the method in which the myeloid progenitor cells are granulocyte / monocyte progenitor cells ("GMP"). Claim 90 A method according to claim 88 or 89, characterized in that the myeloid progenitor cells have surface phenotypes of CD34+, CD90-, and CD45RA+. Claim 91 A method according to claim 88 or 89, characterized in that the myeloid progenitor cells have surface phenotypes of CD34+, CD90-, CD123+, and CD45RA+. Claim 92 A method according to any one of claims 65 to 91, wherein the culture in b) is carried out in a medium comprising one or more of UM729, SCF, StemRegenin1, IL-3, GM-CSF and M-CSF. Claim 93 A method according to any one of claims 65 to 91, wherein the culture in b) is carried out in a medium containing UM729, SCF, StemRegenin1, IL-3, GM-CSF and M-CSF. Claim 94 A method according to any one of claims 65 to 93, wherein the culture in b) is carried out in a medium further comprising a non-physiological ligand of a synthetic cytokine receptor. Claim 95 A method according to any one of claims 65 to 93, wherein the culture in b) is carried out in a medium that does not contain a non-physiological ligand of a synthetic cytokine receptor. Claim 96 A method according to any one of claims 67 to 95, wherein the culture in b) is carried out for 12 to 24 days. Claim 97 A method for generating myeloid cells genetically engineered to express a synthetic cytokine receptor, comprising: culturing a population of hematopoietic progenitor (HP) cells derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a myeloid cell population, wherein at least part of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a transmembrane domain, and an intracellular domain of an interleukin-2 receptor subunit gamma (IL-2RG); and a synthetic beta chain comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain of a beta chain selected from an intracellular domain of an interleukin-2 receptor subunit beta (IL-2RB), an intracellular domain of an interleukin-7 receptor subunit beta (IL-7RB), and / or an intracellular domain of an interleukin-21 receptor subunit beta (IL-21RB). polypeptide Claim 98 A method for generating myeloid cells genetically engineered to express a synthetic cytokine receptor, comprising: culturing a myeloid progenitor cell population derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a myeloid cell population, wherein at least part of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of an interleukin-2 receptor subunit gamma (IL-2RG); and a synthetic beta chain comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain of a beta chain selected from an intracellular domain of an interleukin-2 receptor subunit beta (IL-2RB), an intracellular domain of an interleukin-7 receptor subunit beta (IL-7RB), and / or an intracellular domain of an interleukin-21 receptor subunit beta (IL-21RB). polypeptide Claim 99 In paragraph 97 or 98, the method in which the myeloid cells are macrophages or neutrophils. Claim 100 A method for generating macrophages genetically engineered to express a synthetic cytokine receptor, comprising: culturing a population of hematopoietic progenitor (HP) cells derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating the cells into a macrophage population, wherein at least a portion of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of an interleukin-2 receptor subunit gamma (IL-2RG); and a synthetic beta chain intracellular domain selected from a second dimerization domain, a second transmembrane domain, and an intracellular domain of an interleukin-2 receptor subunit beta (IL-2RB), an intracellular domain of an interleukin-7 receptor subunit beta (IL-7RB), and / or an intracellular domain of an interleukin-21 receptor subunit beta (IL-21RB). Chain polypeptide. Claim 101 A method for generating macrophages genetically engineered to express a synthetic cytokine receptor, comprising: culturing a myeloid progenitor cell population derived from stem cells engineered with a synthetic cytokine receptor under conditions for differentiating cells into a macrophage population, wherein at least part of the culture is performed in the presence of a non-physiological ligand of the synthetic cytokine receptor, wherein the synthetic cytokine receptor comprises: a synthetic gamma chain polypeptide comprising a first dimerization domain, a first transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit gamma (IL-2RG); and a synthetic beta chain comprising a second dimerization domain, a second transmembrane domain, and an intracellular domain of the interleukin-2 receptor subunit beta (IL-2RB), an intracellular domain of the interleukin-7 receptor subunit beta (IL-7RB), and / or an intracellular domain of the interleukin-21 receptor subunit beta (IL-21RB). polypeptide Claim 102 A method according to any one of claims 97 to 101, wherein the culture is carried out in a medium comprising one or more of UM729, SCF, StemRegenin1, IL-3, GM-CSF and M-CSF. Claim 103 A method according to any one of claims 97 to 101, wherein the culture is carried out in a medium comprising UM729, SCF, StemRegenin1, IL-3, GM-CSF and M-CSF. Claim 104 A method according to any one of claims 97 to 103, wherein the culture is carried out for 12 to 24 days. Claim 105 A method according to any one of claims 67 to 96, wherein the culture of one or both of steps a) and b) is carried out in a bioreactor. Claim 106 A method according to any one of claims 97 to 104, wherein the culture is performed in a bioreactor. Claim 107 A method according to claim 105 or 106 in which the bioreactor is a vertical wheel bioreactor. Claim 108 A method according to any one of claims 105 to 107, wherein the bioreactor is a vertical wheel bioreactor having a volume of about 10 mL to about 1000 mL. Claim 109 A method according to claim 105, wherein the culture in a) is carried out in a bioreactor, wherein the bioreactor is a vertical wheel bioreactor having a volume of about 100 mL. Claim 110 A method according to claim 105 or 109, wherein the culture in b) is performed in a bioreactor, and the bioreactor is a vertical wheel bioreactor having a volume of about 500 mL. Claim 111 In paragraph 106, the method wherein the bioreactor is a vertical wheel bioreactor having a volume of about 500 mL. Claim 112 A method in which, in any one of paragraphs 65 to 111, the stem cell is a pluripotent stem cell. Claim 113 In paragraph 112, the method in which the pluripotent stem cells are induced pluripotent stem cells. Claim 114 A method according to any one of claims 65 to 112, wherein the synthetic gamma chain polypeptide comprises a first dimerization domain, a first transmembrane domain, and an IL-2RG intracellular domain in the order from N-terminus to C-terminus, and the synthetic beta chain polypeptide comprises a second dimerization domain, a second transmembrane domain, and an intracellular domain in the order from N-terminus to C-terminus. Claim 115 A method according to any one of claims 65 to 114, wherein the first dimerization domain and the second dimerization domain are extracellular domains. Claim 116 A method according to any one of claims 65 to 115, wherein the IL-2RG intracellular domain comprises a polypeptide sequence that is at least 95% identical to SEQ ID NO: 1, or a polypeptide sequence as presented in SEQ ID NO:

1. Claim 117 A method according to any one of claims 65 to 116, wherein the first transmembrane domain comprises an IL-2RG transmembrane domain. Claim 118 A method according to claim 117, wherein the IL-2RG transmembrane domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 8 or 31, or a polypeptide sequence as presented in sequence identification number: 8 or 31. Claim 119 A method according to any one of claims 65 to 118, wherein the beta-chain intracellular domain comprises an IL-2RB intracellular domain. Claim 120 A method according to claim 119, wherein the IL-2RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 2, or a polypeptide sequence as presented in sequence identification number:

2. Claim 121 A method according to any one of claims 65 to 118, wherein the beta chain intracellular domain comprises an IL-7RB intracellular domain. Claim 122 A method according to claim 121, wherein the IL-7RB intracellular domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 3, or a polypeptide sequence as presented in sequence identification number:

3. Claim 123 A method according to any one of claims 65 to 118, wherein the beta-chain intracellular domain comprises an IL-21RB intracellular domain. Claim 124 A method according to claim 123, wherein the intracellular domain of IL-21RB comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 4, or a polypeptide sequence as presented in sequence identification number:

4. Claim 125 A method according to any one of claims 65 to 124, wherein the second transmembrane domain comprises a transmembrane domain from the same polypeptide as the intracellular domain. Claim 126 A method according to any one of claims 65 to 125, wherein the second transmembrane domain is a polypeptide sequence that is at least 95% identical to sequence identification number: 35 or 36, or a transmembrane domain of IL-2RB comprising a polypeptide sequence as presented in sequence identification number: 35 or 36. Claim 127 A method according to any one of claims 65 to 120, 125 and 126, wherein the first transmembrane domain of the synthetic gamma chain polypeptide is an IL-2RG transmembrane domain comprising the sequence presented in sequence identification number: 8 or 31, and the IL-2RG intracellular domain comprises the sequence presented in sequence identification number: 1; and the second transmembrane domain of the synthetic beta chain polypeptide is an IL-2RB transmembrane domain comprising the sequence presented in sequence identification number: 35 or 36, and the beta chain intracellular domain comprises the sequence presented in sequence identification number:

2. Claim 128 A method according to any one of claims 65 to 127, wherein the first transmembrane domain and the IL-2RG intracellular domain of the synthetic gamma chain polypeptide comprise the sequences presented in Sequence Identification No. 31 and Sequence Identification No. 1; and the second transmembrane domain and the beta chain intracellular domain of the synthetic beta chain polypeptide comprise the sequences presented in Sequence Identification No. 35 and Sequence Identification No.

2. Claim 129 In any one of claims 65 to 128, the first dimerization domain and the second dimerization domain are heteromerization domains selected from FKBP12-rapamycin binding (FRB) domains and FK506-binding protein (FKBP) of size 12 kD; and / or the non-physiological ligand is rapamycin or rapalog. Claim 130 A method according to any one of claims 65 to 129, wherein the first dimerization domain is FKBP and the second dimerization domain is FRB. Claim 131 A method according to any one of claims 65 to 129, wherein the first dimerization domain is FRB and the second dimerization domain is FKBP. Claim 132 A method according to any one of claims 129 to 131, wherein the FRB domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 6 or sequence identification number:

7. Claim 133 A method according to any one of claims 129 to 132, wherein the FRB domain comprises the polypeptide sequence presented in sequence identification number: 6 or sequence identification number:

7. Claim 134 A engineered cell according to any one of claims 129 to 133, wherein the FKBP domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 5 or sequence identification number:

30. Claim 135 A method according to any one of claims 129 to 134, wherein the FKBP domain comprises the polypeptide sequence presented in sequence identification number: 5 or sequence identification number:

30. Claim 136 A method according to any one of claims 65 to 135, wherein the synthetic gamma chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number: 28, and the synthetic beta chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number:

55. Claim 137 A method according to any one of claims 65 to 136, wherein the synthetic gamma chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number: 56, and the synthetic beta chain polypeptide has an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence presented in sequence identification number:

57. Claim 138 A method according to any one of claims 65 to 128, wherein the first dimerization domain and the second dimerization domain are homomerization domains selected from: i) FK506-binding protein (FKBP) of size 12 kD; ii) cyclopyli A (CypA); or iii) gyrase B (CyrB); and the non-physiological ligand is each of the following: i) FK1012, AP1510, AP1903 or AP20187 or their analogs; ii) cyclosporine-A (CsA) or its analogs; or iii) cumermycin or its analogs. Claim 139 A method according to any one of claims 65 to 138, wherein cells of a cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) are resistant to rapamycin-mediated mTOR inhibition. Claim 140 A method according to any one of claims 65 to 139, wherein cells of a cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) express a cytosolic polypeptide that binds to a non-physiological ligand, optionally wherein the cytosolic polypeptide is a cytosolic FRB domain. Claim 141 A method according to any one of claims 65 to 140, wherein the non-physiological ligand is rapamycin or rapalog, and the population of cells expresses a cytosolic FRB domain or a variant thereof. Claim 142 A method according to claim 140 or 141, wherein the cytosolic FRB domain comprises a polypeptide sequence that is at least 95% identical to sequence identification number: 6 or sequence identification number:

7. Claim 143 A method according to claim 140 or 141, wherein the cytosolic FRB domain comprises a polypeptide sequence that is at least 98% identical to sequence identification number: 6 or sequence identification number:

7. Claim 144 A method according to any one of claims 65 to 143, wherein the cells of a cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) contain a destroyed FKBP12 gene that reduces the expression of FKBP12 within the cells. Claim 145 A method according to any one of claims 65 to 144, wherein the cells of a cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) contain a knockout of the FKBP12 gene. Claim 146 A method according to any one of claims 65 to 145, wherein the synthetic cytokine receptor is incorporated into the endogenous gene of a cell of a cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) by targeted integration of the nucleotide sequence encoding the synthetic cytokine receptor into the endogenous gene. Claim 147 In paragraph 146, a method in which the targeted integration is by a non-homologous end linkage (NHEJ). Claim 148 In paragraph 146, a method in which targeted integration is achieved by homology-oriented recovery. Claim 149 A method according to any one of claims 146 to 148, wherein the insertion reduces the expression of an endogenous gene within the locus. Claim 150 A method according to any one of claims 146 to 149, wherein the insertion knocks out an endogenous gene within the locus. Claim 151 A method according to any one of paragraphs 146 to 150, wherein the insertion is by homology-oriented recovery. Claim 152 A method according to any one of claims 146 to 151, wherein the endogenous gene is a housekeeping gene, a blood-lineage specific locus, or an immune-related gene. Claim 153 A method according to claim 152 in which the housekeeping gene is selected from eukaryotic translation elongation factor 1 alpha (EEF1A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin C (UBC), and actin beta (ACTB). Claim 154 In paragraph 152, the method wherein the immune-related gene is selected from the beta-2-microglobulin (B2M) gene and the signal regulatory protein alpha (SIRPA) gene. Claim 155 A method according to any one of claims 65 to 154, wherein the cells of the cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) contain B2M knockout. Claim 156 A method according to any one of claims 65 to 155, wherein the cells of a cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) comprise B2M knockout and FKBP12 knockout. Claim 157 A method according to any one of claims 65 to 156, wherein the cells of a cell population (stem cell population, hematopoietic progenitor cell population or myeloid progenitor cell population) comprise a chimeric antigen receptor (CAR). Claim 158 A method according to any one of claims 66 to 96 and 99 to 157, wherein the macrophage is a mature macrophage expressing CD14. Claim 159 A method according to any one of claims 65 to 158, wherein the non-physiological ligand is rapamycin or a rapamycin analog. Claim 160 In paragraph 159, a method in which a rapamycin analog is rapalog. Claim 161 In any one of claims 65 to 160, a non-physiological ligand is present in the medium at 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 A method of adding at concentrations of nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM. Claim 162 A method according to any one of claims 65 to 161, wherein a non-physiological ligand is added to the medium at a concentration of 10 nM or about 10 nM. Claim 163 A method according to any one of claims 65 to 162, wherein a non-physiological ligand is added to the medium at a concentration of 100 nM or about 100 nM. Claim 164 A myeloid cell population produced by any one of the methods of paragraphs 65, 66, 68 through 99 and 102 through 163. Claim 165 A population of macrophages produced by any one of the methods of paragraphs 65 through 96 and paragraphs 99 through 163. Claim 166 In paragraph 165, a macrophage population that expresses CD14. Claim 167 A pharmaceutical composition comprising a manipulated myeloid cell population of claim 62 or 164. Claim 168 A pharmaceutical composition comprising a population of engineered macrophages according to any one of claims 63, 165 and 166. Claim 169 A pharmaceutical composition comprising pharmaceutically acceptable excipients in claim 167 or 168. Claim 170 A pharmaceutical composition comprising a cryoprotective agent in any one of claims 167 to 169. Claim 171 A method for expanding myeloid cells comprising contacting a myeloid cell population of claim 62 or 164 or a pharmaceutical composition of any one of claims 167, 169 and 170 with a non-physiological ligand of a synthetic cytokine receptor. Claim 172 A method for expanding macrophages comprising contacting a population of macrophages according to any one of claims 63, 165 and 166 or a pharmaceutical composition according to any one of claims 168, 169 and 170 with a non-physiological ligand of a synthetic cytokine receptor. Claim 173 A method performed in vitro or in vitro in accordance with Article 171 or Article 172. Claim 174 A method according to any one of claims 171 to 173, wherein the non-physiological ligand is rapamycin or a rapamycin analog. Claim 175 In paragraph 174, a method in which a rapamycin analog is rapalog. Claim 176 In any one of claims 171 to 175, the non-physiological ligand is 5 nM to 200 nM, 5 nM to 150 nM, 5 nM to 100 nM, 5 nM to 50 nM, 5 nM to 20 nM, 5 nM to 10 nM, 10 nM to 200 nM, 10 nM to 150 nM, 10 nM to 100 nM, 10 nM to 50 nM, 10 nM to 20 nM, 20 nM to 200 nM, 20 nM to 150 nM, 20 nM to 100 nM, 20 nM to 50 nM, 50 nM to 200 nM, 50 nM to 150 nM, 50 nM to 100 A method of contacting at concentrations of nM, 100 nM to 200 nM, 100 nM to 150 nM, and 150 nM to 200 nM. Claim 177 A method according to any one of claims 171 to 176, wherein a non-physiological ligand is contacted at a concentration of 10 nM or about 10 nM. Claim 178 A method according to any one of claims 171 to 177, wherein a non-physiological ligand is contacted at a concentration of 100 nM or about 100 nM. Claim 179 A method according to any one of claims 171 to 178, wherein the method is performed in vivo on a subject and a non-physiological ligand is administered to the subject. Claim 180 A method for treating a disease or pathological condition in a subject, comprising administering an effective amount of a myeloid cell population of claim 62 or 164 or a pharmaceutical composition of any one of claims 167, 169 and 170 to the subject. Claim 181 A method for treating a disease or pathological condition in a subject, comprising administering to the subject an effective amount of a macrophage population of any one of claims 63, 165 and 166 or a pharmaceutical composition of any one of claims 168, 169 and 170 together with a non-physiological ligand of a synthetic cytokine receptor. Claim 182 In paragraph 180 or 181, a method in which the disease or pathological condition is cancer. Claim 183 A method according to any one of claims 171 to 182, wherein the cell expresses a CAR directed toward an antigen expressed by the cell of the disease or pathological condition. Claim 184 In paragraph 183, a method in which the CAR targets a tumor antigen. Claim 185 A method comprising administering a non-physiological ligand of a synthetic cytokine receptor to a subject in any one of claims 171 to 184. Claim 186 A method according to any one of claims 171 to 185, wherein the non-physiological ligand is rapamycin or a rapamycin analog. Claim 187 In paragraph 186, a method in which a rapamycin analog is rapalog. Claim 188 A method according to any one of claims 171 to 187, wherein a non-physiological ligand is administered at a dose of 1 mg to 100 mg, optionally 10 to 100 mg, optionally 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, or about 10 mg, 20 mg, 25 mg, 30 mg, 40 mg, 50 mg, or any value between any of the above. Claim 189 A method according to any one of claims 171 to 188, wherein multiple doses of a non-physiological ligand are administered to a subject. Claim 190 A method according to claim 189 in which multiple doses are administered to a subject intermittently or at regular intervals for an arbitrarily predetermined period following administration of a macrophage population or a composition thereof. Claim 191 A method according to any one of claims 171 to 190, wherein 2 to 8 doses of a non-physiological ligand are administered to a subject. Claim 192 A method according to any one of claims 171 to 190, wherein a single dose of a non-physiological ligand is administered to a subject. Claim 193 A kit comprising a engineered cell of any one of claims 1 to 60, an engineered myeloid cell population of claim 62 or 164, a macrophage cell population of any one of claims 63, 165 and 166, or a pharmaceutical composition of any one of claims 168 to 170, and instructions for administering the same to a subject requiring it. Claim 194 A kit according to claim 193, further comprising a container containing a non-physiological ligand and instructions for administering the non-physiological ligand to a subject after administration of a cell population. Claim 195 A kit in which the subject has cancer, as in paragraph 193 or 194.

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