Engineered cells and uses thereof

Abstract

The present disclosure provides engineered induced pluripotent stem cells (iPSCs) expressing B2M-HLA fusion constructs. The present disclosure also provides iPSC-derived macrophages which overexpress IL-12. The present disclosure further provides engineered immune cells having persistent cytokine expression.

Description

WSGR Docket No.61057-724.601 ENGINEERED CELLS AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 636,360 filed on April 19, 2024, the entirety of which is hereby incorporated by reference herein. BACKGROUND

[0002] The immunologically cold microenvironment of solid tumors limits the ability of immune cells to infiltrate and attack cancer cells. Engineered myeloid cells capable of targeting tumors are needed which can infiltrate the tumor microenvironment and which can avoid immune detection. SUMMARY

[0003] Accordingly, the present disclosure addresses the unmet need.

[0004] An aspect of the present disclosure is a nucleic acid molecule comprising a sequence encoding a fusion protein that comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, and an HLA- G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the nucleic acid molecule has a GC content of at most 70%, 65%, 64%, 63%, or 62%. In some embodiments, the HLA polypeptide comprises HLA-E polypeptide. In some embodiments, the HLA-E polypeptide comprises the sequence of SEQ ID NO: 5.

[0005] An aspect of the present disclosure is a method of engineering a cell, the method comprising inserting into the genome of the cell a sequence encoding a fusion protein, thereby producing a gene-edited cell, wherein the fusion protein comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA- E, an HLA-F, and an HLA-G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, and wherein the coding sequence for the linker in the sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%. In some embodiments, the HLA polypeptide comprises HLA-E. In some embodiments, the gene- edited cell expresses the fusion protein upon interferon-γ (IFNγ) stimulation. In some embodiments, the cell is an iPSC, and wherein the method comprises producing a population of gene-edited iPSCs. In some embodiments, at least 50%, 60%, 70%, 80%, or 90% of the iPSCs in the population express the fusion protein upon IFNγ stimulation. In some embodiments, the percentage of the iPSCs in the population that expresses the fusion protein upon IFNγ stimulation -1-WSGR Docket No.61057-724.601 is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold higher than the percentage of cells in a corresponding population of iPSCs that are otherwise the same but comprise a coding sequence for a fusion protein comprising a linker with higher than 70%, 80%, or 90% GC content.

[0006] An aspect of the present disclosure is a cell comprising an exogenous sequence encoding a fusion protein that comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, and an HLA-G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the exogenous sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%. In some embodiments, the HLA polypeptide comprises HLA-E polypeptide. In some embodiments, the cell is an induced pluripotent stem cell (iPSC). In some embodiments, the cell is a macrophage or a lymphocyte. In some embodiments, the cell expresses the HLA polypeptide upon interferon-γ (IFNγ) stimulation.

[0007] An aspect of the present disclosure is a method of manufacturing a therapeutic macrophage, the method comprising contacting the macrophage with a nucleic acid molecule to result in overexpression of an IL-12p70 dimer by the macrophage, thereby producing the therapeutic macrophage overexpressing the IL12p70 dimer. In some embodiments, the macrophage is differentiated from an induced pluripotent stem cell (iPSC). In some embodiments, the macrophage is differentiated from the iPSC in vitro. In some embodiments, the method further comprises differentiating a stem cell into the macrophage in vitro. In some embodiments, the stem cell is a hematopoietic stem cell (HSC). In some embodiments, the stem cell is an induced pluripotent stem cell (iPSC). In some embodiments, the method further comprises reprogramming a somatic cell into the iPSC. In some embodiments, the reprogramming comprises introducing into the somatic cell one or more synthetic RNA molecules encoding one or more reprogramming factors, resulting in the somatic cell expressing the one or more reprogramming factors. In some embodiments, the IL-12p70 dimer comprises IL-12α and IL-12β, and wherein the IL-12α and IL- 12β are fused via a peptide linker. In some embodiments, the peptide linker comprises a 2A sequence or a 2A-like sequence. In some embodiments, PBMC-T cells, when co-cultured with the therapeutic macrophage, induce at least 1.2-fold, 1.5-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold greater cell lysis of cancer cells that are in contact with the PBMC-T cells as compared to when co-cultured with the corresponding macrophage. In some embodiments, T cells that express a chimeric antigen receptor (CAR), when co-cultured with the therapeutic macrophage, induce at least 1.2-fold, 1.5-fold, 1.8-fold, 1.9-fold, or 2-fold greater cell lysis of cancer cells that are in contact with the T cells as compared to when the T cells are contacted to the cancer cells alone without co-culture with the macrophage that is otherwise the -2-WSGR Docket No.61057-724.601 same but lacks overexpression of the IL-12p70 dimer. In some embodiments, T cells that express a chimeric antigen receptor (CAR), when co-cultured with the therapeutic macrophage, induce at least 5%, 8%, 10%, 12%, 15%, 16%, 17%, 18%, or 20% greater cell lysis of cancer cells that are in contact with the T cells as compared to when co-cultured with the corresponding macrophage. In some embodiments, the CAR specifically binds to ROR1.

[0008] An aspect of the present disclosure is a therapeutic macrophage overexpressing an IL12p70 dimer and comprising a disruption in a genomic B2M gene. In some embodiments, the therapeutic macrophage is differentiated from an induced pluripotent stem cell (iPSC). In some embodiments, the iPSC is reprogrammed from a somatic cell. In some embodiments, the therapeutic macrophage comprises a disruption in both alleles of the B2M gene.

[0009] An aspect of the present disclosure is a nucleic acid molecule comprising a sequence encoding a fusion protein that comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, and an HLA- G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the nucleic acid molecule has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

[0010] An aspect of the present disclosure is a method of engineering a cell, the method comprising inserting into the genome of the cell a sequence encoding a fusion protein, thereby producing a gene-edited cell, wherein the fusion protein comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA- E, an HLA-F, and an HLA-G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, and wherein the coding sequence for the linker in the sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

[0011] An aspect of the present disclosure is a method of generating a therapeutic myeloid cell, the method comprising contacting a stem cell with a nucleic acid molecule to result in persistent overexpression of a cytokine by the stem cell, thereby producing an engineered stem cell overexpressing the cytokine; and differentiating the engineered stem cell into the therapeutic myeloid cell overexpressing the cytokine. In some embodiments, the cytokine comprises IL-12. In some embodiments, the IL-12 comprises an IL-12p70 dimer. In some embodiments, the IL- 12p70 dimer is encoded by a sequence comprising at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 59 or SEQ ID NO: 61. In some embodiments, the IL-12p70 dimer comprises a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the flexible linker is a bovine elastin motif (BEM) linker. In some embodiments, the nucleic acid molecule comprises an engineered chromatin opening sequence (ECOS). In some embodiments, the therapeutic myeloid cell comprises a disruption in a B2M -3-WSGR Docket No.61057-724.601 gene in its genome. In some embodiments, the disruption comprises insertion of a B2M-HLA-E construct. In some embodiments, the B2M-HLA-E construct comprises a linker. In some embodiments, the linker comprises a (G4S)4 linker sequence. In some embodiments, the coding sequence for the (G4S)4 linker sequence has a GC content of at most 70%, 65%, 64%, 63%, or 62%. In some embodiments, the nucleic acid molecule encodes a poly-adenylation signal. In some embodiments, the poly-adenylation signal comprises a human growth hormone poly-adenylation (hGHpA) signal. In some embodiments, the method further comprises contacting the stem cell with a nucleic acid molecule encoding a transgene. In some embodiments, the transgene comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR specifically binds to CD19. In some embodiments, the contacting comprises transfecting the stem cell with one or more synthetic RNA molecules encoding one or more gene-editing proteins, thereby resulting in the stem cell expressing the one or more gene-editing proteins. In some embodiments, the nucleic acid molecule comprises an EF1a promoter. In some embodiments, the nucleic acid molecule comprises an ECOS-EF1a-IL12-hGHpA repair template. In some embodiments, the ECOS-EF1a- IL12-hGHpA repair template comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 58 or SEQ ID NO: 60. In some embodiments, the therapeutic myeloid cell comprises a therapeutic macrophage. In some embodiments, the therapeutic macrophage phagocytoses 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% more SK-OV-3 cancer cells, when co-cultured with the PBMC-derived macrophage, as compared to a PBMC-derived macrophage. In some embodiments, the therapeutic macrophage infiltrates a spheroid solid tumor model comprising SK-OV-3 cells, when contacted with the spheroid solid tumor model, by 100 µm, 150 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900 µm, 950 µm, or 1000 µm. In some embodiments, the therapeutic macrophage induces 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% less CD69 upregulation in CD4+ or CD8+ T cells, when co-incubated with the CD4+ or CD8+ T cells and the wildtype macrophage, as compared to a wildtype macrophage.

[0012] An aspect of the present disclosure is a method for intratumoral therapeutic delivery, the method comprising: contacting a tumor tissue with a nucleic acid molecule that encodes a cytokine. In some embodiments, the cytokine comprises IL-12. In some embodiments, the IL-12 comprises an IL-12p70 dimer. In some embodiments, the IL-12p70 dimer is encoded by a sequence comprising at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 59 or SEQ ID NO: 61. In some embodiments, the IL-12p70 dimer comprises a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the flexible linker is a bovine elastin motif (BEM) linker. In some embodiments, the nucleic acid molecule comprises a sequence encoding an ECOS-EF1a-IL12-hGHpA construct. In some embodiments, -4-WSGR Docket No.61057-724.601 the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 58 or SEQ ID NO: 60.

[0013] An aspect of the present disclosure is a therapeutic myeloid cell persistently overexpressing a cytokine, wherein the therapeutic myeloid cell comprises a genomic insertion. In some embodiments, the cytokine comprises IL-12. In some embodiments, the IL-12 comprises an IL-12p70 dimer. In some embodiments, the IL-12p70 dimer is encoded by a sequence comprising at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 59 or SEQ ID NO: 61. In some embodiments, the IL-12p70 dimer comprises a peptide linker. In some embodiments, the peptide linker is a flexible linker. In some embodiments, the flexible linker is a bovine elastin motif (BEM) linker. In some embodiments, the genomic insertion comprises a sequence encoded by a repair template. In some embodiments, the repair template comprises an engineered chromatin opening sequence (ECOS). In some embodiments, the repair template comprises a sequence encoding an ECOS-EF1a-IL12-hGHpA construct. In some embodiments, the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 58 or SEQ ID NO: 60. In some embodiments, the therapeutic myeloid cell is a therapeutic macrophage. In some embodiments, the therapeutic myeloid cell is differentiated from an engineered stem cell. In some embodiments, the engineered stem cell is a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an induced pluripotent stem cell. In some embodiments, the therapeutic myeloid cell is differentiated from the engineered stem cell in vitro.

[0014] An aspect of the present disclosure is a pharmaceutical composition comprising a nucleic acid molecule encoding a cytokine, wherein the cytokine comprises a bovine elastin motif (BEM) linker.

[0015] An aspect of the present disclosure is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of the present disclosure.

[0016] An aspect of the present disclosure is a method of generating an engineered immune cell comprising contacting an immune cell with a nucleic acid molecule, wherein the nucleic acid molecule comprises a sequence encoding a chimeric antigen receptor (CAR), thereby generating the engineered immune cell. In some embodiments, the CAR specifically binds to CD19. In some embodiments, the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NO: 55-57. In some embodiments, the method further comprises contacting the immune cell with one or more synthetic RNA molecules encoding one or more gene editing proteins. In some embodiments, the one or more gene editing proteins target the TRAC locus and the CD52 locus. In some embodiments, the engineered -5-WSGR Docket No.61057-724.601 immune cell overexpresses EGFR. In some embodiments, a single administration of the nucleic acid molecule and the one or more synthetic RNA molecules simultaneously results in a disruption in the genome of the engineered immune cell at the TRAC locus and the CD52 locus. In some embodiments, the administration comprises electroporation. In some embodiments, the engineered immune cell lyses at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more RAJI cancer cells as compared to an immune cell which does not comprise the nucleic acid molecule when co-cultured with the RAJI cancer cells.

[0017] Any aspect or embodiment herein may be combined with any other aspect or embodiment as disclosed herein. INCORPORATION BY REFERENCE

[0018] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 shows a schematic overview of the differentiation of B2M-HLA-E expressing iPSCs.

[0020] FIG.2 shows EB002 ssDNA run on a gel.

[0021] FIG.3 shows a gel comprising EB002 PCR product.

[0022] FIG.4 shows EB002 single colonies analyzed for insertion.

[0023] FIG. 5 shows flow cytometry data of HLA-E expression in F13 cells after IFNγ stimulation.

[0024] FIG. 6 shows flow cytometry data of HLA-E expression in WT cells after IFNγ stimulation.

[0025] FIG.7 shows diagrams of IL-12 fusion and Il-12 P2A constructs.

[0026] FIG.8 shows quantification of production of IL-12p70 in THP-1 cells.

[0027] FIG.9 shows quantification of the production of IL-12p70 in iPSC-derived macrophage and PBMC-T cell co-culture.

[0028] FIG.10 shows quantification of the production of IFNγ in iPSC-derived macrophage and PBMC-T cell co-culture.

[0029] FIG.11 shows real-time fluorescence assay data.

[0030] FIG. 12 shows a schematic of the co-culture of IL-12-transfected B2M- / -iPSC-derived macrophages with PBMC T cells.

[0031] FIG.13 shows quantification of a comparison of PBMC T-cell activation marker CD69.

[0032] FIG.14 shows HLA-DR expression. -6-WSGR Docket No.61057-724.601

[0033] FIG. 15 shows a schematic of experiments done to test the cytotoxicity of IL-12- transfected B2M- / -iPSC-derived Macrophage and ROR1-CAR PBMC T cells against MDA-MB- 231 cancer cells.

[0034] FIG.16 shows quantification of lysis of MDA-MB-231 cells.

[0035] FIG.17 shows a schematic of a V2-ROR1-CAR (top) and its construct (bottom).

[0036] FIG.18 shows quantification of lysis of MDA-MB-231 cells.

[0037] FIG. 19 shows flow cytometry data of B2Me2, CIITAe1, CD52e1, or TRACe1 gene targeting in T cells.

[0038] FIG.20 shows flow cytometry data for simultaneous targeting of CD52e1 and TRACe1 in T cells.

[0039] FIG. 21 shows a schematic of homologous DNA templates for use with gene editing of the TRAC locus.

[0040] FIG. 22 shows gel electrophoresis of genomic DNA amplifying the TRAC locus in primary T cells following electroporation with mRNA encoding a gene editing protein and a homologous DNA template encoding GFP (top) or a CD19 CAR (bottom) and shows efficient on- target genomic integration 3 and 7 days after electroporation across n=3 PBMC donors.

[0041] FIG. 23 shows flow cytometry data of T cells after electroporation with a gene editing protein targeting the TRACe1 gene co-delivered with a homologous DNA repair template encoding epidermal growth factor receptor (EGFR) as well as different combinations of promoters and polyadenylation signals.

[0042] FIGs.24A-B show flow cytometry data of CD52e1 and TRACe1 edited T cells expressing EGFR.

[0043] FIG.25 shows flow cytometry data of T cells after electroporation with TRAC targeting mRNA with or without a CD19 CAR dsDNA repair template.

[0044] FIG. 26 shows efficacy of RAJI B cell lysis of T cells transfected with TRAC targeting mRNA alone or with a CD19 CAR dsDNA repair template.

[0045] FIG.27 shows ELISA results of clonally isolated iPSCs showing elevated levels of IL12 secretion in the supernatant, while unmodified iPSCs showed no detectable IL12 protein secretion (data not shown).

[0046] FIG. 28 shows PCR and gel electrophoresis of clonally isolated iPSCs following electroporation and single-cell deposition showing two colonies with large genomic inserts in the AAVS1 locus.

[0047] FIG.29 shows a cartoon schematic of iPSC to iMacrophage bioreactor differentiation.

[0048] FIG. 30 shows graphs showing that iPSC-derived macrophages display consistent expression of key identify markers CD14, CD45, and CD64 after differentiation, with no -7-WSGR Docket No.61057-724.601 detectable CD66b, TRA-1-60, or TRA-1-81 expression (representative results of at least 4 differentiation runs).

[0049] FIG. 31 shows graphs showing that CD14+ cells isolated from PBMCS (monocytes) display consistent expression of key identify markers CD14, CD45, and CD64, with minimally detectable CD66b, TRA-1-60, or TRA-1-81 expression (representative results of at least 2 PBMC donors).

[0050] FIG. 32 shows representative brightfield images of iPSC-derived macrophages (left) or PBMC CD14+ monocyte-derived macrophages (right).

[0051] FIG. 33 shows a graph showing differentiation yields of macrophages from small-scale vertical-flywheel differentiation across 4 independent iPS cell lines.

[0052] FIG. 34 shows a graph of an ELISA of IL12 modified iPSCs and differentiated macrophages showing sustained IL12 expression through the completion of differentiation.

[0053] FIG.35 shows flow cytometry data quantifying phagocytosis of tumor cells by engineered macrophages.

[0054] FIG.36 shows a representative image of an iPSC-derived macrophage (GFP+) engulfing two opsonized SKOV3 cells after a 2 hour co-culture.

[0055] FIG.37 shows aggregated flow cytometry results from 2 separate PBMC CD14+ donors and 3 separate iPSC-derived macrophage differentiations. Phagocytosis % is the proportion of CD14+ cells also positive for eFluor670 (eFLuo670+CD14+ / CD14+) p=0.016

[0056] FIG.38 shows mouse tumor tissue stained via IHC for GFP-specific antibody following intravenous injection of engineered iMacrophages shows accumulation of cell therapy at tumor site 72 hours after treatment.

[0057] FIG. 39 shows a graph showing B2M- / - and B2M-HLA-E iMacrophages demonstrated lower upregulation of the early activation marker CD69 across both CD4+ and CD8+ T cells when co-cultured for 72 hours (results from 3 independent PBMC donors).

[0058] FIG.40 shows representative maximally projected Z-stack images of GFP fields from a SKOV3 spheroid cytotoxicity time course showing the IL12 Macrophages + T cells lysing the spheroids within the 96 hour co-culture, faster than any of the other groupings.

[0059] FIG. 41 shows a graph showing that macrophages enhance lysis of GFP+ SKOV3 spheroids when compared to T cells alone, with the IL12 macrophages showing the greatest effect.

[0060] FIG.42 shows a graph showing that IL12 expressing Macrophages enhance lysis of GFP+ SKOV3 spheroids when compared to T cells alone, or supplemented with increasing titrations of recombinant IL12p70 protein.

[0061] FIG.43 shows a graph showing that IL12 expressing Macrophages enhance lysis of GFP+ SKOV3 spheroids when compared to T cells alone, including when supplemented with the -8-WSGR Docket No.61057-724.601 immunosuppressive recombinant protein IL-4 (2 ng) known to be present in ovarian cancer microenvironments at high levels.

[0062] FIG.44 shows a graph showing that IL12 Macrophages enhance lysis of GFP+ SKOV3 spheroids when compared to T cells alone, with results across 3 independent PBMC donors shown. DETAILED DESCRIPTION

[0063] In one aspect of the present disclosure, there is provided an isolated stem cell or immune cell comprising a genetically engineered disruption in a beta-2-microglobulin (B2M) gene, e.g., a loss of function, optionally in both alleles, of the B2M gene, wherein the immune cell is selected from a lymphoid cell or a myeloid cell. In some cases the lymphoid cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage. In embodiments the immune cell is an NK cell. In some cases, the stem cell is a pluripotent stem cell or a mesenchymal stem cell. In some cases, the pluripotent stem cell is an induced pluripotent stem cell (iPSC).

[0064] Methods of the present disclosure may comprise disruption of a B2M gene. Disruption of the B2M gene can be achieved through gene editing. Disruption of the B2M gene may be monoallelic or bi-allelic. Disruption of the B2M gene may comprise deletion of a portion of exon 2.

[0065] In another aspect of the present disclosure, there is provided a method of making an engineered immune cell, comprising (a) reprogramming a somatic cell to an iPS cell, the reprogramming comprising contacting the iPS cell with a ribonucleic acid (RNA) encoding one or more reprogramming factors; (b) disrupting a B2M gene in the iPS cell, the disrupting comprising gene-editing the cell by contacting the cell with RNA encoding one or more gene- editing proteins; and (c) differentiating the iPS cell into an immune cell, wherein the immune cell is selected from a lymphoid cell or a myeloid cell. In some cases the lymphoid cell is a T cell, e.g., a cytotoxic T cell or gamma-delta T cell; an NK cell; or an NK-T cell. In some cases, the myeloid cell is a macrophage, e.g., an M1 macrophage or an M2 macrophage.

[0066] In some embodiments, a stem cell of the present disclosure is an induced pluripotent stem cell (iPSC). The iPSC may be derived from a human. The iPSC may be derived from a subject who is not intended to receive a therapy comprising administration of the iPSC. The iPSC may be allogeneic to a patient intended to receive a therapy comprising administration of the iPSC. The iPSC of the present disclosure may be suitable for an “off the shelf” cell therapy for the treatment of a disease without substantial host immune rejection of the iPSC. The iPSC may be from a -9-WSGR Docket No.61057-724.601 master cell bank. A therapy of the present disclosure may comprise administration of an iPSC or any variant or combination thereof. The iPSC, iMSC, EiMSC, or any cell from which the iPSC is derived or is differentiated into may be allogeneic to a patient intended to receive a therapy comprising the iPSC or any variant or combination thereof.

[0067] An RNA molecule of the present disclosure may be a synthetic RNA molecule. The synthetic RNA molecule may be an mRNA molecule comprising one or more non-canonical nucleotides that substantially avoid cellular toxicity. The synthetic RNA molecule may be an mRNA molecule comprising one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5- aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5- methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5- hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5- carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5- amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5- hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio- 5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5- azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5- aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5- methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino- 5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5- azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2- thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5- hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5- hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2- thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4- methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl- 5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4- methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5- aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4- aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5- hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4- amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5- -10-WSGR Docket No.61057-724.601 aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4- hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5- azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4- hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4- methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5- hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5- azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5- methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5- hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5- hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5- azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5- methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5- hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4- hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4- hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4- hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4- hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6- hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl- 8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7- deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6- hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7- deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7- deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8- azaguanosine. In some cases, the synthetic RNA molecule is in vitro transcribed.

[0068] In some embodiments, methods of the present disclosure may comprise reprogramming a somatic cell into a stem cell. In some embodiments, the stem cell was reprogrammed from a somatic cell. The reprogramming may be non-viral. Reprogramming may comprise the use of one or more reprogramming factors. The one or more reprogramming factors may be encoded by synthetic RNA or mRNA. The one or more reprogramming factors may comprise one or more of Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, and Lin28. In some embodiments, the reprogramming is performed by transfecting cells with one or more nucleic acids encoding one or more reprogramming factors, including, but not limited to Oct4 protein, Sox2 protein, Klf4 -11-WSGR Docket No.61057-724.601 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro- RNA and biologically active fragments, analogues, variants and family-members thereof. In some embodiments, reprogramming the cell is performed in vivo. In one embodiment, the cell in vivo is reprogrammed by transfecting the cell with one or more nucleic acids encoding one or more reprogramming factors. In one embodiment, the one or more nucleic acids includes an RNA molecule that encodes Oct4 protein. In another embodiment, the one or more nucleic acids also includes one or more RNA molecules that encodes Sox2 protein, Klf4 protein, and c-Myc protein. In yet another embodiment, the one or more nucleic acids also includes an RNA molecule that encodes Lin28 protein. In one embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a pluripotent stem cell. In another embodiment, the cell is a human skin cell, and the human skin cell is reprogrammed to a glucose-responsive insulin-producing cell. Examples of other cells that can be reprogrammed and other cells to which a cell can be reprogrammed include, but are not limited to skin cells, pluripotent stem cells, MSCs, β-cells, retinal pigmented epithelial cells, hematopoietic cells, cardiac cells, airway epithelial cells, neural stem cells, neurons, glial cells, bone cells, blood cells, and dental pulp stem cells. In one embodiment, the cell is contacted with a medium that supports the reprogrammed cell. In one embodiment, the medium also supports the cell.

[0069] In some embodiments, methods of the present disclosure comprise formulating a cell of the present disclosure in a composition for therapeutic use. The composition may be suitable for use in the treatment of amyotrophic lateral sclerosis (ALS), spinal cord injury, degenerative disc disease, coronary artery disease, acute myocardial infarction, alcoholic liver cirrhosis, hepatitis C virus (HCV)-induced cirrhosis, multiple sclerosis (MS), osteoarthritis (OA), osteoarthritis of the knee, kidney allograft, critical limb ischemia, ischemic cardiomyopathy, Crohn’s disease, idiopathic pulmonary fibrosis, anal fistula, spinal cord injury, systemic lupus erythematosus (SLE), acute respiratory distress syndrome (ARDS), acute graft-versus-host disease (aGvHD), preterm bronchopulmonary dysplasia (BPD), autism nonischemic heart failure, and / or Type 2 diabetes mellitus. The composition may be suitable for use in the treatment of an infectious disease, optionally selected from an infection with a pathogen, optionally a bacterium, virus, fungus, or parasite. In some cases, the pathogen is a virus. The virus may be selected from but is not limited to an influenza virus, optionally selected from Type A, Type B, Type C, and Type D influenza viruses, or a member of the Coronaviridae family, optionally selected from a betacoronavirus, optionally selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome—Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43, or an alphacoronavirus, optionally selected from HCoV-NL63 -12-WSGR Docket No.61057-724.601 and HCoV-229E. B2M-HLA fusion construct and applications thereof

[0070] An aspect of the present disclosure is a nucleic acid molecule comprising a sequence encoding a fusion protein that comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides; and a linker of sequence (GGGGS)4 or (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the nucleic acid molecule has a GC content of at most 70%, 65%, 64%, 63%, or 62%. The GC content of the (G4S)4 linker may be 83.3%, 71.7%, or 61.7%. GC content may be understood to refer to the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). GC content may indicate the proportion of G and C bases out an implied total of 4 bases in a DNA or RNA molecule. GC content may refer to a molecule or a fragment of a molecule. GC content may be associated with stability of a molecule. In some embodiments, the coding sequence for the linker comprises the sequence of any one of SEQ ID NOs: 13-15. The linker may be a flexible linker. The flexible linker may comprise a bovine elastin motif (BEM) linker. The flexible linker may comprise one or more glycine residues, serine residues, and / or threonine residues.

[0071] In some embodiments, the B2M polypeptide comprises the sequence of SEQ ID NO: 1. The B2M polypeptide may comprise a fragment of a wildtype B2M protein. In some cases, the B2M polypeptide comprises a fragment of a wildtype human B2M protein. The coding sequence for the B2M polypeptide in the nucleic acid molecule may optionally not include the sequence of exon 1 or exon 2 of the B2M gene. The HLA polypeptide may comprise a sequence of a wildtype HLA protein selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G proteins. The HLA polypeptide may comprise HLA-E or HLA-G. The HLA-E polypeptide comprise the sequence of SEQ ID NO: 5. The HLA polypeptide may comprise a sequence of a wildtype human HLA protein. The nucleic acid molecule may comprise one or more regions of homology that comprise sequences homologous to sequences of a B2M gene for targeted insertion. The nucleic acid molecule may comprise one or more regions of homology that comprise sequences homologous to sequences within exon 3 of a B2M gene for targeted insertion into exon 3. In some embodiments, the nucleic acid molecule may comprise one or more regions of homology that comprise sequences homologous to sequences outside of exon 3 of a B2M gene for targeted insertion into exon 3. The sequence encoding the fusion protein may be inserted immediately prior to the B2M stop codon. The nucleic acid molecule may comprise the sequence of any one of SEQ ID NO: 10-12. In some embodiments, the nucleic acid molecule is a DNA plasmid or a viral vector. The nucleic acid molecule may be single-stranded.

[0072] An aspect of the present disclosure is a method of engineering a cell, the method -13-WSGR Docket No.61057-724.601 comprising inserting into genome of the cell a sequence encoding a fusion protein, thereby producing a gene-edited cell, wherein the fusion protein comprises: a B2M polypeptide; a HLA polypeptide selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, and wherein coding sequence for the linker in the sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%. The method may further comprise creating a double-strand break or a single-strand break in a target site of the genome of the cell. The method may comprise creating the double-strand break or the single-break by introducing to the cell one or more gene editing proteins that comprise a DNA binding domain and a cleavage domain of a nuclease.

[0073] The one or more gene editing proteins may be introduced to the cell by transfecting the cell with one or more RNA molecules that encode the one or more gene editing proteins. The target site may be within a B2M gene in the genome. In some cases, the target site is within exon 3 of the B2M gene. The target site may be immediately prior to the B2M stop codon.

[0074] The coding sequence for the linker of the present disclosure may comprise the sequence of any one of SEQ ID NOs: 13-15. The linker may comprise a sequence (GGGGS)4 or (G4S)4. The coding sequence for the linker may have a GC content of at most 70%, 65%, 64%, 63%, or 62%. The GC content of the (G4S)4 linker may be 83.3%, 71.7%, or 61.7%.

[0075] The B2M polypeptide of the present disclosure may comprise the sequence of SEQ ID NO: 1. The B2M polypeptide may comprise a fragment of a wildtype B2M protein. The B2M polypeptide may comprise a fragment of a wildtype human B2M protein. The coding sequence for the B2M polypeptide in the nucleic acid molecule may not comprise the sequence of exon 1 or exon 2 of the B2M gene.

[0076] The HLA polypeptide of the present disclosure may comprise a sequence of a wildtype HLA protein selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G proteins. In preferred embodiments, the HLA polypeptide may comprise HLA-E or HLA-G. The HLA-E polypeptide may comprise the sequence of SEQ ID NO: 5. The HLA polypeptide may comprise the sequence of a wildtype human HLA protein.

[0077] A method of the present disclosure may comprise introducing a nucleic acid molecule comprising a sequence encoding a fusion protein into a cell. The introducing may be viral or non- viral. The inserting may comprise transfecting the cell with the nucleic acid molecule. The nucleic acid molecule may be a DNA plasmid or a viral vector. The nucleic acid molecule may comprise one or more regions of homology that comprise sequences homologous to sequences of a B2M gene for targeted insertion. The one or more regions of homology may be referred to as homology arms. The nucleic acid molecule may comprise one or more regions of homology that comprise -14-WSGR Docket No.61057-724.601 sequences homologous to sequences within exon 3 of a B2M gene for targeted insertion into exon 3. The nucleic acid molecule may comprise the sequence of any one of SEQ ID NO: 10-12. The nucleic acid molecule may be a DNA plasmid or a viral vector. The nucleic acid molecule may be single-stranded.

[0078] The cell of the present disclosure may be a stem cell. The stem cell may be a pluripotent stem cell. The cell may be an induced pluripotent stem cell (iPSC). The iPSC may be derived from a human subject. The iPSC may be derived from a subject who is not intended to receive a therapy comprising administration of the iPSC. The iPSC may be allogeneic to a patient intended to receive a therapy comprising administration of the iPSC. The iPSC of the present disclosure may be suitable for an “off the shelf” cell therapy for the treatment of a disease without substantial host immune rejection of the iPSC. The iPSC may be from a master cell bank. A therapy of the present disclosure may comprise administration of an iPSCor any variant or combination thereof. The iPSC, iMSC, EiMSC, or any cell from which the iPSC is derived or is differentiated into may be allogeneic to a patient intended to receive a therapy comprising the iPSC, or any variant or combination thereof.

[0079] The method of any one of claims

[0072] to

[0078] , wherein the gene-edited cell expresses the HLA polypeptide upon interferon-γ (IFNγ) stimulation. The cell may be an iPSC. Methods of the present disclosure may comprise producing a population of gene-edited iPSCs. Methods of the present disclosure may further comprise differentiating the population of gene-edited iPSCs into a population of engineered induced mesenchymal stem cells (EiMSCs). In some cases, at least 50%, 60%, 70%, 80%, or 90% of the population of iPSCs or population of EiMSCs expresses the fusion protein upon IFNγ stimulation. The percentage of the iPSCs or EiMSCs in the population that expresses the fusion protein upon IFNγ stimulation may be at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold higher than the percentage of cells in a corresponding population of iPSCs or EiMSCs that are otherwise the same but comprise a coding sequence for fusion protein comprising a linker with higher than 70%, 80%, or 90% GC content.

[0080] An aspect of the present disclosure is a population of cells comprising the iPSCs or EiMSCs produced by any one of the methods of the present disclosure. The population of cells may be a clonal line or be derived from a clonal line. The clonal line may be generated without the use of a reporter system. A cell obtained from a population of cells of the present disclosure or a cell used to generate a population of cells of the present disclosure may be a stem cell, optionally a pluripotent stem cell. The cell may be an induced pluripotent stem cell (iPSC). The iPSC may be derived from a human subject or human patient. The iPSC may be derived from a subject who is not intended to receive a therapy comprising administration of the iPSC. The iPSC may be allogeneic to a patient intended to receive a therapy comprising administration of the iPSC. -15-WSGR Docket No.61057-724.601 The iPSC may be from a master cell bank. The cell of the present disclosure may be a macrophage or a lymphocyte. The cell of the present disclosure may be differentiated into a macrophage or lymphocyte. The cell may be a T cell, a B cell, or an NK cell.

[0081] Another aspect of the present disclosure is a cell comprising an exogenous sequence encoding a fusion protein that comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G polypeptides; and a linker of sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the exogenous sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%. The GC content of the (G4S)4 linker may be 83.3%, 71.7%, or 61.7%. GC content may be understood to refer to the percentage of nitrogenous bases in a DNA or RNA molecule that are either guanine (G) or cytosine (C). GC content may indicate the proportion of G and C bases out an implied total of 4 bases in a DNA or RNA molecule. GC content may refer to a molecule or a fragment of a molecule. GC content may be associated with stability of a molecule. In several cases, the linker comprises four to twelve glycines. In many cases, the linker comprises one or more serines. In some cases, the linker comprises one or more GGGGS (G4S) repeats. In various cases, the linker comprises two to six G4S repeats.

[0082] A further aspect of the present disclosure is a pharmaceutical composition comprising a population of cells of the present disclosure and a pharmaceutically acceptable excipient. Excipients of the present disclosure may include liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.

[0083] An aspect of the present disclosure is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of the present disclosure. The disease or condition may be a proliferative disease, autoimmune disease, inflammatory disease, or an infectious disease. The disease or condition may be amyotrophic lateral sclerosis (ALS), spinal cord injury, degenerative disc disease, coronary artery disease, acute myocardial infarction, alcoholic liver cirrhosis, hepatitis C virus (HCV)- induced cirrhosis, multiple sclerosis (MS), osteoarthritis (OA), osteoarthritis of the knee, kidney allograft, critical limb ischemia, ischemic cardiomyopathy, Crohn’s disease, idiopathic pulmonary fibrosis, anal fistula, spinal cord injury, systemic lupus erythematosus (SLE), acute respiratory distress syndrome (ARDS), acute graft-versus-host disease (aGvHD), preterm bronchopulmonary dysplasia (BPD), autism nonischemic heart failure, or Type 2 diabetes mellitus. The disease or condition may be an infectious disease, optionally selected from an infection with a pathogen, -16-WSGR Docket No.61057-724.601 optionally a bacterium, virus, fungus, or parasite. The disease or condition may be a cancer. Cancer may include but is not limited to basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, and Meigs’ syndrome. IL-12 overexpressing constructs and applications thereof

[0084] An aspect of the present disclosure is a method of manufacturing a therapeutic macrophage, the method comprising contacting the macrophage with a nucleic acid molecule to result in overexpression of an IL-12p70 dimer by the macrophage, thereby producing the therapeutic macrophage overexpressing the IL12p70 dimer. The macrophage may be differentiated from an induced pluripotent stem cell (iPSC). The macrophage may be differentiated from the iPSC in vitro.

[0085] The method of the present disclosure may further comprise differentiating a stem cell into the macrophage in vitro. The stem cell may be a mesenchymal stem cell (MSC). The stem cell may be an iPSC. The method of the present disclosure may further comprise reprogramming a somatic cell into the iPSC. The reprogramming may comprise introducing into the somatic cell one or more synthetic RNA molecules encoding one or more reprogramming factors, resulting in the somatic cell expressing the one or more reprogramming factors. -17-WSGR Docket No.61057-724.601

[0086] An iPSC-derived macrophage of the present disclosure engineered to overexpress IL-12 may be useful for delivery to a tumor microenvironment. The iPSC-derived macrophage of the present disclosure may comprise a disruption in a B2M gene. The disruption may be bi-allelic. The disruption may affect both alleles. The disruption of both alleles of the B2M gene may be designed to reduce alloreactivity of a transplanted iPSC-derived macrophage.

[0087] In some embodiments, the macrophage comprises a disruption in a B2M gene in its genome. The macrophage may comprise disruption of both alleles of the B2M gene. The method of the present disclosure may comprise providing a gene-edited iPSC that comprises a disruption in a B2M gene in its genome and differentiating the iPSC into the macrophage. The method of the present disclosure may further comprise introducing one or more gene editing proteins into an iPSC to result in disruption in a B2M gene in the genome of the iPSC, thereby generating the gene-edited iPSC. The introducing may comprise transfecting the iPSC with one or more synthetic RNA molecules encoding the one or more gene editing proteins, thereby resulting in the iPSC expressing the one or more gene editing proteins.

[0088] A nucleic acid molecule of the present disclosure may comprise a sequence encoding IL- 12α, IL-12β, or both. The nucleic acid molecule may comprise a sequence encoding IL-12α and IL-12β. In some cases, the nucleic acid molecule comprises a sequence encoding a fusion protein that comprises IL-12α and IL-12β, and wherein the IL-12α and IL-12β are fused via a peptide linker.

[0089] The nucleic acid molecule of the present disclosure may further comprise a coding sequence for a stagger peptide. The coding sequence for the stagger peptide may be located between coding sequence for the IL-12α and coding sequence for the IL-12β. The stagger peptide may comprise a 2A sequence or a 2A-like sequence. The stagger peptide may comprise a sequence with a C-terminal sequence that is GP. The stagger peptide may comprise a sequence with a C- terminal consensus sequence that is D(V / I)ExNPG P, where x = any amino acid. In some cases, the stagger peptide comprises at least one of GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP. The nucleic acid molecule of the present disclosure may be a synthetic RNA molecule. The nucleic acid molecule may be delivered to the macrophage via a viral vector. “Stagger peptide” and “peptide linker” may be used interchangeably throughout the present disclosure.

[0090] Constructs of the present disclosure may comprise IL-12 subunits joined by a stagger peptide. The stagger peptide may comprise a self-cleaving peptide. The self-cleaving peptide may comprise P2A from porcine teschovirus-1. The self-cleaving peptide may comprise E2A self- -18-WSGR Docket No.61057-724.601 cleaving peptide derived from equine rhinitis A virus, F2A self-cleaving peptide derived from foot-and-mouth disease virus 18, or T2A self-cleaving peptide derived from thosea asigna virus 2A self-cleaving peptide. The self-cleaving peptide may be located between two coding sequences (CDSs) of polypeptides. The self-cleaving peptide may act through ribosomal skipping to allow for the encoding of two polypeptides which can dissociate into individual proteins upon translation.

[0091] Constructs of the present disclosure may comprise IL-12 subunits joined by a flexible linker. The flexible linker may comprise a peptide. The flexible linker may comprise a motif. The flexible linker may comprise a motif comprising glycine, serine, and / or threonine, or a combination thereof. The flexible linker may comprise a bovine elastin motif (BEM) linker. In some embodiments, the BEM linker may allow for higher levels of IL-12 expression as compared to a self-cleaving peptide. In some embodiments, a BEM linker may allow for higher IL-12 bioactivity as compared to a self-cleaving peptide. In some embodiments, a BEM linker may allow for higher IL-12 bioavailability as compared to a self-cleaving peptide.

[0092] In some embodiments, the therapeutic macrophage secretes interferon γ (IFNγ) when co- cultured with T cells. The therapeutic macrophage may secrete IFNγ when co-cultured with peripheral blood mononuclear cells (PBMCs). In some cases, the therapeutic macrophage does not substantially secrete IFNγ when not co-cultured with T cells. In various cases, T cells, when co-cultured with the therapeutic macrophage, induce greater cell lysis of cancer cells that are in contact with the T cells as compared to when co-cultured with a corresponding macrophage that lacks overexpression of the IL-12p70 dimer and optionally is otherwise the same. In many cases, PBMC-T cells, when co-cultured with the therapeutic macrophage, induce at least 1.2-fold, 1.5- fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold greater cell lysis of cancer cells that are in contact with the PBMC-T cells as compared to when co-cultured with the corresponding macrophage that is otherwise the same but lacks overexpression of the IL-12p70 dimer. T cells that express a chimeric antigen receptor (CAR), when co-cultured with the therapeutic macrophage, may induce at least 1.2-fold, 1.5-fold, 1.8-fold, 1.9-fold, or 2-fold greater cell lysis of cancer cells that are in contact with the T cells as compared to when the T cells are contacted to the cancer cells alone without co-culture with the macrophage. The CAR may specifically bind to ROR1.T cells that express a chimeric antigen receptor (CAR), when co- cultured with the therapeutic macrophage, may induce at least 5%, 8%, 10%, 12%, 15%, 16%, 17%, 18%, or 20% greater cell lysis of cancer cells that are in contact with the T cells as compared to when co-cultured with the corresponding macrophage that is otherwise the same but lacks overexpression of the IL-12p70 dimer.

[0093] An aspect of the present disclosure is a therapeutic macrophage overexpressing IL12p70 -19-WSGR Docket No.61057-724.601 dimer and comprising a disruption in a genomic B2M gene. The therapeutic macrophage may be differentiated from an induced pluripotent stem cell (iPSC). The therapeutic the macrophage may be differentiated from the iPSC in vitro. The iPSC may be reprogrammed from a somatic cell. The iPSC may be reprogrammed from a somatic cell by introducing into the somatic cell one or more synthetic RNA molecules encoding one or more reprogramming factors, resulting in the somatic cell expressing the one or more reprogramming factors. The therapeutic macrophage may comprise disruption of both alleles of the B2M gene. In some cases, the therapeutic macrophage is produced by providing a gene-edited iPSC that comprises a disruption in a B2M gene in its genome and differentiating the iPSC into the macrophage. The gene-edited iPSC may be generated by introducing one or more gene editing proteins into an iPSC to result in disruption in a B2M gene in the genome of the iPSC, thereby generating the gene-edited iPSC. The introducing may comprise transfecting the iPSC with one or more synthetic RNA molecules encoding the one or more gene editing proteins, thereby resulting in the iPSC expressing the one or more gene editing proteins.

[0094] A therapeutic macrophage of the present disclosure may comprise an exogenous nucleic acid sequence encoding IL-12α, IL-12β, or both. The therapeutic macrophage may comprise an exogenous nucleic acid sequence encoding IL-12αand IL-12β. The exogenous nucleic acid sequence may encode a fusion protein that comprises IL-12αand IL-12β and wherein the IL-12αand IL-12β are fused via a peptide linker. The exogenous nucleic acid sequence may further comprise a coding sequence for a stagger peptide, and wherein the coding sequence for the stagger peptide is located between coding sequence for the IL-12αand coding sequence for the IL-12β. The stagger peptide may comprise a 2A sequence or a 2A-like sequence. The stagger peptide may comprise a sequence with a C-terminal sequence that is GP. The stagger peptide may comprise a sequence with a C-terminal consensus sequence that is D(V / I)ExNPG P, where x = any amino acid. In some cases, the stagger peptide comprises at least one of GDVESNPGP, GDIEENPGP, VEPNPGP, IETNPGP, GDIESNPGP, GDVELNPGP, GDIETNPGP, GDVENPGP, GDVEENPGP, GDVEQNPGP, IESNPGP, GDIELNPGP, HDIETNPGP, HDVETNPGP, HDVEMNPGP, GDMESNPGP, GDVETNPGP, GDIEQNPGP, and DSEFNPGP. The nucleic acid molecule of the present disclosure may be a synthetic RNA molecule. The nucleic acid molecule may be delivered to the macrophage via a viral vector. The exogenous nucleic acid sequence may be a synthetic RNA molecule. The exogenous nucleic acid sequence may be inserted into genome of the therapeutic macrophage.

[0095] In some embodiments, the therapeutic macrophage secretes interferon γ (IFNγ) when co- cultured with T cells. The therapeutic macrophage may secrete IFNγ when co-cultured with peripheral blood mononuclear cells (PBMCs).The therapeutic macrophage may not substantially -20-WSGR Docket No.61057-724.601 secrete IFNγ in absence of T cell stimulation. In some cases, T cells, when co-cultured with the therapeutic macrophage, induce greater cell lysis of cancer cells that are in contact with the T cells as compared to when co-cultured with a corresponding macrophage that lacks overexpression of the IL-12p70 dimer and optionally is otherwise the same. PBMC-T cells, when co-cultured with the therapeutic macrophage, may induce at least 1.2-fold, 1.5-fold, 1.8-fold, 1.9-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold greater cell lysis of cancer cells that are in contact with the PBMC-T cells as compared to when co-cultured with the corresponding macrophage that is otherwise the same but lacks overexpression of the IL-12p70 dimer. T cells that express a chimeric antigen receptor (CAR), when co-cultured with the therapeutic macrophage, may induce at least 1.2-fold, 1.5-fold, 1.8-fold, 1.9-fold, or 2-fold greater cell lysis of cancer cells that are in contact with the T cells as compared to when the T cells are contacted to the cancer cells alone without co-culture with the macrophage. The CAR may specifically bind to ROR1. T cells that express a chimeric antigen receptor (CAR) of the present disclosure, when co-cultured with the therapeutic macrophage, may induce at least 5%, 8%, 10%, 12%, 15%, 16%, 17%, 18%, or 20% greater cell lysis of cancer cells that are in contact with the T cells as compared to when co- cultured with the corresponding macrophage that is otherwise the same but lacks overexpression of the IL-12p70 dimer.

[0096] An aspect of the present disclosure is a population of cells comprising the therapeutic macrophages of the present disclosure. A further aspect of the present disclosure is a pharmaceutical composition comprising the population of cells of the present disclosure and a pharmaceutically acceptable excipient. Excipients of the present disclosure may include liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.

[0097] An aspect of the present disclosure is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of the present disclosure. The disease or condition may be a proliferative disease, autoimmune disease, inflammatory disease, or an infectious disease. The disease or condition may be a cancer. Cancer may include but is not limited to one or more of basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, -21-WSGR Docket No.61057-724.601 adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, and Meigs’ syndrome. Engineered pluripotent stem cells

[0098] The pluripotent stem cell of the present disclosure may be an induced pluripotent stem (iPS) cell. The iPS cell may be from a master cell bank. In many embodiments, the iPS cell is self- renewing. The iPS cell may be derived from a differentiated cell by transfecting the differentiated cell with one or more RNA molecules encoding one or more reprogramming factors. In many embodiments, the pluripotent stem cell is an embryonic stem cell. The pluripotent stem cell may be a human cell. The pluripotent stem cell may be a bone marrow derived mesenchymal stem cell.

[0099] iPS cells of the present disclosure may express pluripotency markers such as TRA-1-60 and / or TRA-1-81. iPSC cells may be differentiated into various cell types using static culture. iPS cells may be differentiated into lymphocytes, macrophages, immune cells, mesenchymal stem cells, stem cells, blood cells, neuronal cells, or any combination thereof.

[0100] Disclosed herein are methods of making and using MSCs or iMSCs that are derived from induced pluripotent stem cells (iPSCs), e.g., via mRNA-based reprogramming. Such methods, which include the step of assaying the MSCs or iMSCs for a protein expression and / or secretion signature, yield superior cell populations and / or therapeutic effect. Further, such methods yield MSCs that demonstrate consistency among samples / batches, thus providing therapeutic reliability.

[0101] The cell in a more differentiated state of the present disclosure may be selected from but not limited to the following: a mesenchymal stem cell, a hematopoietic cell, a macrophage, a lymphocyte, a hepatocyte, a fibroblast, a keratinocyte, a melanocyte, an oligodendrocyte, a glial cell, a hematopoietic cell, a hematopoietic stem cell, a bone marrow cell, a bone marrow stem cell, -22-WSGR Docket No.61057-724.601 an adipose cell, a tissue-specific resident cell, a neuron, a muscle cell, a bone cell, a pancreatic cell, an immune cell, a blood cell, an epithelial cell, or an endothelial cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage.

[0102] In various embodiments, the method of the present disclosure comprises introducing into the pluripotent stem cell a nucleic acid molecule encoding a gene editing protein, thereby resulting in expression of the gene editing protein in the pluripotent stem cell. The nucleic acid molecule of the present disclosure encoding the gene editing protein may be a DNA molecule. In some cases, the nucleic acid molecule encoding the gene editing protein is an RNA molecule. In many cases, the RNA molecule is a synthetic RNA molecule. The RNA molecule may comprise one or more non-canonical nucleotides that avoid substantial cellular toxicity. The one or more non-canonical nucleotides may comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5- hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5- methoxyuridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine, optionally at an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-canonical nucleotides.

[0103] Engineered cells of the present disclosure may exhibit sustained transgene expression. Transgene expression may be sustained for an extended period of time. Without wishing to be bound by any certain theory, sustained transgene expression may be facilitated by an engineered chromatin opening sequence (ECOS) according to some embodiments of the present disclosure. An ECOS may comprise a CBX3-ECOS, an A2ECOS, a SRF-ECOS, an ECOS-455, an ECOS- 63, or a biologically active fragment thereof. The SRF-ECOS may comprise one or more promoters of a SURF1 and / or SURF2 gene. The SRF-ECOS may comprise the intergenic region between the SURF1 and SURF2 promoters. The SRF-ECOS may comprise part of or all of the regulatory region between the transcription start sites of SURF1 and SURF2 genes. The SRF- ECOS may comprise the first exon and the first intron of SURF1 and SURF2 genes. The ECOS may comprise a sequence having at least 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of any one of SEQ ID NOs: 28-33. The ECOS may comprise the sequence of any one of SEQ ID NOs: 28-33.

[0104] In various embodiments, the ECOS inserted in the target DNA sequence is located between a coding sequence and a promoter sequence that drives the expression of the coding sequence. The ECOS may maintain expression of the coding sequence in the engineered cell for at least 8, 9, 10, 20, 30, 40, 50, 60, or 90 days, or at least 4, 5, 6, 7, 8, 9, or 12 months. The inserted ECOS -23-WSGR Docket No.61057-724.601 may maintain expression of the coding sequence in the engineered cell as long as the engineered cell remains alive. The inserted ECOS may maintain transgene or coding sequence expression during and after differentiation or transdifferentiation. For example, the ECOS may maintain expression of a transgene or coding sequence in an iPSC that is differentiated into a lymphocyte or a macrophage.

[0105] Promoter sequences of the present disclosure may comprise an elongation factor-1 alpha (Ef1α) promoter, a synthesis friendly constitutive (SFC) promoter, a phosphoglycerate kinase (PGK) promoter, a cytomegalovirus (CMV) promoter, a spleen focus forming virus (SFFV) promoter, a JeT promoter, an miR223 promoter, an H1 promoter, a SP146 promoter, or a biologically active fragment thereof. The inserted sequence may comprise from upstream to downstream: the promoter sequence, the ECOS, and the coding sequence. The promoter sequence may comprise a CAG promoter. The CAG promoter may comprise the following sequences: a cytomegalovirus early enhancer element; a promoter, first exon, and first intron of chicken beta- actin gene; and a splice acceptor of a rabbit-globin gene.

[0106] In some embodiments, the method disclosed herein comprises producing a population of the engineered cells. In some embodiments, expression of the coding sequence in at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population persists through at least 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, 10 passages, 11 passages, or 12 passages when the population is cultured in vitro. In several embodiments, expression of the coding sequence in at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population persists after one freeze-thaw cycle, two, three, four, or more freeze-thaw cycles. The percentage of cells that expresses the coding sequence in the population of engineered cells may be at least 5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35- fold, 40-fold, or 50-fold higher than percentage of cells that expresses the coding sequence in a corresponding population of engineered cells that are otherwise the same but do not have the ECOS inserted in the target DNA sequence. The coding sequence in at most 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of cells in the population of engineered cells may undergo gene silencing during differentiation of the engineered cells. In some cases, expression of the coding sequence in the population of engineered cells persists in vivo when the population is transplanted into a human or animal subject.

[0107] The coding sequence of the present disclosure may encode any transgene. The transgene may include but is not limited to a fluorescent indicator molecule, a reporter molecule, a fluorescent molecule, a green fluorescent protein (GFP), cytokine, a homing factor, a transcription factor, a immunomodulatory protein, an antibody or antigen-binding fragment thereof, a chimeric antigen receptor (CAR), a T cell receptor, a B cell receptor, a hormone, an epigenetic modulator, -24-WSGR Docket No.61057-724.601 a ligand, a receptor, an enzyme, an antigen, a secreted protein, a non-coding RNA, or any combination thereof. In some embodiments, the coding sequence encodes IL7, IL15, IL4, IL10, IL12, IL2, IL21, or any combination thereof. In some embodiments the coding sequence further encodes a homing factor. In some embodiments, the homing factor comprises C-C chemokine receptor type 2 (CCR2), C-X-C chemokine receptor type 4 (CXCR4), P-selectin, E-selectin, L- selectin, or any combination thereof. In various embodiments, the coding sequence encodes Indoleamine 2,3-dioxygenase 1 (IDO1).

[0108] In some embodiments, expression levels of a transgene or coding sequence described herein are determined by the combination of an ECOS and a promoter sequence inserted into a construct comprising the transgene or coding sequence. Expression may be modulated by selection of a particular combination of an ECOS and a promoter as described herein.

[0109] The ECOS may comprise a CBX3-ECOS, an A2ECOS, a SRF-ECOS, an ECOS-455, an ECOS-63, or a biologically active fragment thereof. The ECOS may comprise a sequence having at least 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence of any one of SEQ ID NOs: 28- 33. The ECOS may comprise the sequence of any one of SEQ ID NOs: 28-33. The ECOS may maintain expression of the coding sequence for at least 8, 9, 10, 20, 30, 40, 50, 60, or 90 days, or at least 4, 5, 6, 7, 8, 9, or 12 months. Expression of the coding sequence may be greater when the coding sequence is expressed with an ECOS than when the coding sequence is expressed without an ECOS.

[0110] The SRF-ECOS of the present disclosure may comprise one or more promoters of a SURF1 and / or SURF2 gene. The SRF-ECOS may comprise the intergenic region between the SURF1 and SURF2 promoters. The SRF-ECOS may comprise part of or all of the regulatory region between the transcription start sites of SURF1 and SURF2 genes. The SRF-ECOS may comprise the first exon and the first intron of SURF1 and SURF2 genes.

[0111] Methods of the present disclosure may comprise producing a population of the engineered pluripotent stem cell and differentiating the population of engineered pluripotent stem cells in vitro into a population of cells in a more differentiated state. In some embodiments, expression of the coding sequence in at least 50%, 60%, 70%, 80%, 85% 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population persists during the differentiation. In many embodiments, expression of the coding sequence in at least 50%, 60%, 70%, 80%, 85% 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population persists through at least 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, 10 passages, 11 passages, or 12 passages. Expression of the coding sequence in at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population may persist after one freeze-thaw cycle, two, three, four, or more freeze-thaw cycles. Expression of the -25-WSGR Docket No.61057-724.601 coding sequence in at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population of cells in a more differentiated state may persist through at least 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, 10 passages, 11 passages, or 12 passages. Expression of the coding sequence in at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% of cells in the population of cells in a more differentiated state may persist after one freeze-thaw cycle, two, three, four, or more freeze-thaw cycles. The percentage of cells that expresses the coding sequence in the population of cells in a more differentiated state may be at least 5-fold, 10- fold, 11-fold, 12-fold, 13-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, or 50-fold higher than the percentage of cells that expresses the coding sequence in a corresponding population of cells that are otherwise the same but do not have the ECOS inserted in the target DNA sequence. In some cases, the coding sequence in at most 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of cells in the population of cells in a more differentiated state undergoes gene silencing during the differentiation. In some embodiments, expression of the coding sequence in the population of cells in a more differentiated state persists in vivo when the population is transplanted into a human or animal subject. In some embodiments, expression of the coding sequence is first detected after differentiating the engineered pluripotent stem cell into the cell in a more differentiated state. In some embodiments expression of the coding sequence is not detected prior to differentiating the engineered pluripotent stem cell into the cell in a more differentiated state. In some embodiments, expression of the coding sequence is at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% higher in the cell in a more differentiated state compared to the engineered pluripotent stem cell. In some embodiments, expression of the coding sequence is first detected prior to a first passage, prior to a second passage, prior to a third passage, prior to a fifth passage, prior to a sixth passage, prior to a seventh passage, prior to a eighth passage, prior to a ninth or more passage. In many embodiments, the percentage of cells that expresses the coding sequence in the population of engineered cells is at least 5-fold, 10-fold, 11-fold, 12-fold, 13-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, or 50-fold higher than percentage of cells that expresses the coding sequence in the engineered pluripotent cell prior to differentiating.

[0112] In many embodiments, a cell of the present disclosure is a stem cell. The stem cell may be a pluripotent stem cell. The stem cell may be a bone marrow-derived mesenchymal stem cell. In some cases, the pluripotent stem cell is an induced pluripotent stem (iPS) cell. In several cases, the iPS cell is from a master cell bank. In many cases, the iPS cell is self-renewing. The iPS cell may be derived from a differentiated cell by transfecting the differentiated cell with one or more RNA molecules encoding one or more reprogramming factors. In various embodiments, the pluripotent stem cell is an embryonic stem cell. The cell may be a human cell. The cell may be an -26-WSGR Docket No.61057-724.601 animal cell. The animal may be a mouse, a rat, a pig, a monkey, a cow, or a mammal. The cell may be a mesenchymal stem cell. In many embodiments, the cell is a hematopoietic cell. The cell may be a macrophage or a lymphocyte. In various embodiments, the cell is a hepatocyte, a fibroblast, a keratinocyte, a melanocyte, an oligodendrocyte, a glial cell, a hematopoietic cell, a bone marrow cell, a bone marrow stem cell, an adipose cell, a tissue-specific resident cell, a neuron, a muscle cell, a bone cell, a pancreatic cell, an immune cell, a blood cell, an epithelial cell, or an endothelial cell. In some cases, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. The cell may be differentiated from an iPS cell.

[0113] A further aspect of the present disclosure is a composition comprising an in vitro population of the cells of the present disclosure. The in vitro population of cells may comprise a single cell type. The in vitro population of cells may be a clonal line or be derived from a clonal line. The clonal line may be generated without the use of a reporter system. The in vitro population of cells may comprise iPSCs, iMSCs, engineered iMSCs (EiMSCs), macrophages, lymphocytes, stem cells, mesenchymal stem cells, fibroblasts, adipocytes, immune cells, or any combination thereof.

[0114] In many embodiments, expression of the coding sequence in the population persists in vivo when the population is transplanted into a human or animal subject. Any one of the populations of cells described herein may be injected or infused into a human patient or animal. The cells of the population may engraft into bone marrow. The cells of the population may continue to sustain transgene expression for an extended period of time.

[0115] An aspect of the present disclosure is a pharmaceutical composition comprising a pharmaceutically acceptable excipient, and the composition of the present disclosure or the population of cells of the present disclosure. Excipients of the present disclosure may include liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.

[0116] Another aspect of the present disclosure is a method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the composition of the present disclosure, or the population of cells of the present disclosure, or the pharmaceutical composition of the present disclosure. The disease or condition may comprise a metabolic disease, a proliferative disease, an autoimmune disease, a neurological disease, or any combination thereof.

[0117] In some embodiments, the iPS cell is derived from a differentiated cell by transfecting the differentiated cell with one or more RNA molecules encoding one or more reprogramming factors. The pluripotent stem cell may be an embryonic stem cell. The cell may be a human cell. The cell -27-WSGR Docket No.61057-724.601 may be a mesenchymal stem cell. In certain embodiments, the cell is a hematopoietic cell. In various embodiments, the cell is a macrophage or a lymphocyte. The cell may be a hepatocyte, a fibroblast, a keratinocyte, a melanocyte, an oligodendrocyte, a glial cell, a hematopoietic cell, a bone marrow cell, a bone marrow stem cell, an adipose cell, a tissue-specific resident cell, a neuron, a muscle cell, a bone cell, a pancreatic cell, an immune cell, a blood cell, an epithelial cell, or an endothelial cell. In many embodiments, the immune cell is a T cell, a B cell, a monocyte, a natural killer cell, a dendritic cell, or a macrophage. The cell may be differentiated from an iPS cell. Reprogramming Methods

[0118] In embodiments, the present disclosure relates to RNA-based modifications, e.g., reprogramming and / or gene-editing. In some embodiments, an RNA molecule encodes a gene-editing protein. In some embodiments, an RNA molecule encodes a reprogramming factor.

[0119] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides.

[0120] In various embodiments, the present invention relates to the reprogramming of induced pluripotent stem cells (iPSCs) to iPSC-derived mesenchymal stromal cells (iMSCs), using non-viral, RNA-based means. iPSCs, namely pluripotent or less differentiated cells, can be reprogrammed from non-pluripotent or differentiated cells, including fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipose cells, and tissue-resident progenitor cells.

[0121] In some embodiments, the method for reprogramming a non-pluripotent cell comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; and (c) transfecting the non- pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro- RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed; and wherein step (c) occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0122] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more -28-WSGR Docket No.61057-724.601 reprogramming factors selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed to a less differentiated state; and wherein step (c) occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0123] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors; and wherein step (c) may be performed at least twice and the amount of one or more synthetic RNA molecules transfected in one or more later transfections may be greater than the amount transfected in one or more earlier transfections to result in the cell being reprogrammed to a less differentiated state and occurs in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0124] In some embodiments, the method for reprogramming a non-pluripotent cell, comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; and (c) transfecting the non- pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed; and wherein step (c) may be performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell.

[0125] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; and (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors; wherein the transfecting results in the cell expressing the one or more reprogramming factors to result in the cell being reprogrammed to a less differentiated state; and wherein step (c) may be performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell to a less differentiated state. -29-WSGR Docket No.61057-724.601

[0126] In some embodiments, the method for reprogramming a non-pluripotent cell, comprises: (a) providing a non-pluripotent cell; (b) culturing the non-pluripotent cell; (c) transfecting the non- pluripotent cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (d) repeating step (c) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections may be greater than the amount transfected in one or more earlier transfections, to result in the non-pluripotent cell being reprogrammed, wherein steps (c) and (d) occur in the presence of a medium containing ingredients that support reprogramming of the non-pluripotent cell.

[0127] In some embodiments, the method for reprogramming a differentiated cell to a less differentiated state, comprises: (a) providing a differentiated cell; (b) culturing the differentiated cell; (c) transfecting the differentiated cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (d) repeating step (c) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections may be greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state, wherein steps (c) and (d) occur in the presence of a medium containing ingredients that support reprogramming of the differentiated cell to a less differentiated state.

[0128] In some embodiments, the method for reprogramming a non-pluripotent cell comprises: (a) providing a non-pluripotent cell, the non-pluripotent cell being derived from a biopsy of a human subject; (b) culturing the non-pluripotent cell; and (c) transfecting the non-pluripotent cell with a synthetic RNA molecule, wherein: the synthetic RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the non-pluripotent cell expressing the one or more reprogramming factor(s) which reprograms the non-pluripotent cell; and step (c) may be performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the non-pluripotent cell.

[0129] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) providing a non-pluripotent cell; (b) culturing the cell; and (c) transfecting the cell with -30-WSGR Docket No.61057-724.601 a synthetic RNA molecule, wherein: the RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state, and step (c) may be performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a medium containing ingredients that support reprogramming of the cell to a less differentiated state.

[0130] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) providing a non-pluripotent cell; (b) culturing the cell in a medium containing ingredients that support reprogramming of the cell to a less differentiated state; and (c) transfecting the cell with a synthetic RNA molecule, wherein: the RNA molecule encodes one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state, and step (c) may be performed without using irradiated human neonatal fibroblast feeder cells and occurs in the presence of a feeder cell conditioned medium.

[0131] In some embodiments, the method for reprogramming a cell to a less differentiated state comprises: (a) providing a non-pluripotent cell; (b) culturing the cell in a medium containing albumin and ingredients that support reprogramming of the cell to a less differentiated state, wherein the albumin may be treated with an ion-exchange resin or charcoal; (c) transfecting the cell with a synthetic RNA molecule, wherein the RNA molecule encoding one or more reprogramming factor(s) selected from the group consisting of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof, wherein the transfecting results in the cell expressing the one or more reprogramming factor(s) which reprograms the cell to a less differentiated state.

[0132] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: (a) culturing a differentiated cell with a reprogramming medium; (b) transfecting the cell -31-WSGR Docket No.61057-724.601 with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules include at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and (c) repeating step (b) at least twice during 5 consecutive days, wherein the amount of one or more synthetic RNA molecules transfected in one or more later transfections may be greater than the amount transfected in one or more earlier transfections, to result in the cell being reprogrammed to a less differentiated state, wherein steps (a)-(c) are performed without using feeder cells and occur in the presence of a feeder cell conditioned medium.

[0133] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: a. culturing a differentiated cell with a reprogramming medium containing albumin, wherein the albumin may be treated with an ion-exchange resin or charcoal; b. transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding one or more reprogramming factors and wherein the transfecting results in the cell expressing the one or more reprogramming factors; and c. repeating step (b) at least twice during 5 consecutive days to result in the cell being reprogrammed to a less differentiated state.

[0134] In some embodiments, the method for reprogramming a cell to a less differentiated state, comprises: a. culturing a differentiated cell with a reprogramming medium containing albumin, wherein the albumin may be treated with sodium octanoate; brought to a temperature of at least about 40°C; and treated with an ion-exchange resin or charcoal; b. transfecting the cell with one or more synthetic RNA molecules, wherein the one or more synthetic RNA molecules includes at least one RNA molecule encoding one or more reprogramming transcription factors and wherein the transfecting results in the cell expressing the one or more synthetic RNA molecules; and c. repeating step (b) at least twice during about 5 consecutive days to result in the cell being reprogrammed to a less differentiated state.

[0135] In embodiments, the reprogramming may be non-viral. In embodiments, the reprogramming factor may be one or more of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro- RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof.

[0136] In some embodiments, iPSCs are obtained and iMSCs are generated via cell reprogramming with non-immunogenic messenger RNA (mRNA) encoding one or more reprogramming factors in a defined, animal component-free process. In some embodiments the process may be immunosuppressant-free. In some embodiments, the process may be animal component-free. In some -32-WSGR Docket No.61057-724.601 embodiments, the process may be defined.

[0137] Cells can be reprogrammed by exposing them to specific extracellular cues and / or by ectopic expression of specific proteins, microRNAs, etc. While several reprogramming methods have been previously described, most that rely on ectopic expression require the introduction of exogenous DNA, which can carry mutation risks. DNA-free reprogramming methods based on direct delivery of reprogramming proteins have been reported. However, these methods are too inefficient and unreliable for commercial use. In addition, RNA-based reprogramming methods have been described (see, e.g., Angel. MIT Thesis.2008.1-56; Angel et al. PloS ONE.2010.5,107; Warren et al. Cell Stem Cell.2010.7,618-630; Angel. MIT Thesis.2011.1-89; and Lee et al., Cell.2012.151,547-558; the contents of all of which are hereby incorporated by reference). However, existing RNA-based reprogramming methods are slow, unreliable, and inefficient when performed on adult cells, require many transfections (resulting in significant expense and opportunity for error), can reprogram only a limited number of cell types, can reprogram cells to only a limited number of cell types, require the use of immunosuppressants, and require the use of multiple human-derived components, including blood-derived HSA and human fibroblast feeders. The many drawbacks of previously disclosed RNA- based reprogramming methods make them undesirable for research, therapeutic or cosmetic use.

[0138] In some embodiments, reprogramming may be performed by transfecting cells with one or more nucleic acids encoding one or more reprogramming factors, including, but not limited to Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In one embodiment, the cell may be a human skin cell, and the human skin cell may be reprogrammed to a pluripotent stem cell.

[0139] Importantly, infecting skin cells with viruses encoding Oct4, Sox2, Klf4, and c-Myc, combined with culturing the cells in a medium that supports the growth of cardiomyocytes, has been reported to cause reprogramming of the skin cells to cardiomyocytes, without first reprogramming the skin cells to pluripotent stem cells (See Efs et al Nat Cell Biol.2011;13:215-22, the contents of which are hereby incorporated by reference). In certain situations, direct reprogramming (reprogramming one somatic cell to another somatic cell without first reprogramming the somatic cell to a pluripotent stem cell, also known as “transdifferentiation”) may be desirable, in part because culturing pluripotent stem cells can be time-consuming and expensive, the additional handling involved in establishing and characterizing a stable pluripotent stem cell line can carry an increased risk of contamination, and the additional time in culture associated with first producing pluripotent stem cells can carry an increased risk of genomic instability and the acquisition of mutations, including point mutations, copy-number -33-WSGR Docket No.61057-724.601 variations, and karyotypic abnormalities.

[0140] In embodiments, fewer total transfections may be required to reprogram a cell according to the methods of the present invention than according to other methods. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein from about 1 to about 12 transfections are performed during about 20 consecutive days, or from about 4 to about 10 transfections are performed during about 15 consecutive days, or from about 4 to about 8 transfections are performed during about 10 consecutive days. It is recognized that when a cell is contacted with a medium containing nucleic acid molecules, the cell may likely come into contact with and / or internalize more than one nucleic acid molecule either simultaneously or at different times. A cell can therefore be contacted with a nucleic acid more than once, e.g., repeatedly, even when a cell is contacted only once with a medium containing nucleic acids.

[0141] Of note, nucleic acids can contain one or more non-canonical or “modified” residues as described herein. For instance, any of the non-canonical nucleotides described herein can be used in the present reprogramming methods. In one embodiment, pseudouridine-5′-triphosphate can be substituted for uridine-5′-triphosphate in an in vitro-transcription reaction to yield synthetic RNA, wherein up to 100% of the uridine residues of the synthetic RNA may be replaced with pseudouridine residues. In vitro-transcription can yield RNA with residual immunogenicity, even when pseudouridine and 5-methylcytidine are completely substituted for uridine and cytidine, respectively (see, e.g., Angel. Reprogramming Human Somatic Cells to Pluripotency Using RNA [Doctoral Thesis]. Cambridge, MA: MIT; 2011, the contents of which are hereby incorporated by reference). For this reason, it is common to add an immunosuppressant to the transfection medium when transfecting cells with RNA. In certain situations, adding an immunosuppressant to the transfection medium may not be desirable, in part because the recombinant immunosuppressant most commonly used for this purpose, B18R, can be expensive and difficult to manufacture. In one embodiment, the immunosuppressant is B18R or a biologically active fragment, analogue, variant or family-member thereof or dexamethasone or a derivative thereof. In one embodiment, the transfection medium does not contain an immunosuppressant, and the nucleic-acid dose is chosen to prevent excessive toxicity. In another embodiment, the nucleic-acid dose is less than about 1mg / cm2of tissue or less than about 1mg / 100,000 cells or less than about 10mg / kg.

[0142] In various cases, transfection of a cell with synthetic nucleic acids for reprogramming the cell may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells. The system relates to use of a compound of Formula (A): -34-WSGR Docket No.61057-724.601

[0143] where R21, R23, and R24are independently selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C5-10 aryl, 5- to 10-membered heteroaryl, or C3-6 cycloalkyl; n is 1 to 20; m is 6, 7, 8, 9, or 10; p is 1, 2, 3, or 4; q is 1, 2, 3, 4, or 5; and r is 1, 2, 3, 4, 5, or 6.

[0144] The system relates to use of a compound of Formula (IV):where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0145] Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or more of US20210009505A1, US 10,501,404, and WO2021003462. The entire contents of which are incorporated by reference in their entirety.

[0146] In any of the herein-disclosed aspects or embodiments, a synthetic RNA molecule may be in the form of a circular RNA (circRNA). The circRNA are manufactured by methods do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA, the circRNA are manufactured by methods that do not require ribozymes to yield a circRNA, and / or the circRNA are manufactured by methods that do not require HPLC-based purification, e.g., post-ligation. A nucleic acid that can be manufactured into a circRNA has the structure: 5’-X-Y-A-IRES-B-CDS-C-Y’-Z 3’. Here, Y and Y’ each independently comprise one or more nucleotides and Y and Y’ are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null. The CDS of a circRNA may encode one or more proteins of interest, the protein of interest being one or more reprogramming factors, optionally selected from Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, Lin28, Glis1, Utf1, -35-WSGR Docket No.61057-724.601 Aicda, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof. In some cases, the CDS encodes two, three, four, five, six, seven, eight, nine, ten, eleven, or more reprogramming factor(s). Additional details regarding circRNAs useful in the present disclosure are described in WO2022232291A1, the contents of which are incorporated herein by reference in its entirety.

[0147] Reprogrammed cells produced according to certain embodiments of the present invention are suitable for therapeutic and / or cosmetic applications as they do not contain undesirable exogenous DNA sequences, and they are not exposed to animal-derived or human-derived products, which may be undefined, and which may contain toxic and / or pathogenic contaminants. Furthermore, the high speed, efficiency, and reliability of certain embodiments of the present invention may reduce the risk of acquisition and accumulation of mutations and other chromosomal abnormalities. Certain embodiments of the present invention can thus be used to generate cells that have a safety profile adequate for use in therapeutic and / or cosmetic applications. For example, reprogramming cells using RNA and the medium of the present invention, wherein the medium does not contain animal or human-derived components, can yield cells that have not been exposed to allogeneic material. Certain embodiments are therefore directed to a reprogrammed cell that has a desirable safety profile. In one embodiment, the reprogrammed cell has a normal karyotype. In another embodiment, the reprogrammed cell has fewer than about 5 copy-number variations (CNVs) relative to the patient genome, such as fewer than about 3 copy-number variations relative to the patient genome, or no copy- number variations relative to the patient genome. In yet another embodiment, the reprogrammed cell has a normal karyotype and fewer than about 100 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 50 single nucleotide variants in coding regions relative to the patient genome, or fewer than about 10 single nucleotide variants in coding regions relative to the patient genome.

[0148] Endotoxins and nucleases can co-purify and / or become associated with other proteins, such as serum albumin. Recombinant proteins, in particular, can often have high levels of associated endotoxins and nucleases, due in part to the lysis of cells that can take place during their production. Endotoxins and nucleases can be reduced, removed, replaced or otherwise inactivated by many of the methods of the present invention, including, for example, by acetylation, by addition of a stabilizer such as sodium octanoate, followed by heat treatment, by the addition of nuclease inhibitors to the albumin solution and / or medium, by crystallization, by contacting with one or more ion-exchange resins, by contacting with charcoal, by preparative electrophoresis or by affinity chromatography. In embodiments, partially or completely reducing, removing, replacing, or otherwise inactivating endotoxins and / or nucleases from a medium and / or from one or more components of a medium is -36-WSGR Docket No.61057-724.601 provided and this can increase the efficiency with which cells can be transfected and reprogrammed. Certain embodiments are therefore directed to a method for transfecting a cell with one or more nucleic acids, wherein the transfection medium is treated to partially or completely reduce, remove, replace or otherwise inactivate one or more endotoxins and / or nucleases. Other embodiments are directed to a medium that causes minimal degradation of nucleic acids. In one embodiment, the medium contains less than about 1EU / mL, or less than about 0.1EU / mL, or less than about 0.01EU / Ml.

[0149] In certain situations, protein-based lipid carriers such as serum albumin can be replaced with non-protein-based lipid carriers such as methyl-beta-cyclodextrin. The medium of the present invention can also be used without a lipid carrier, for example, when transfection is performed using a method that may not require or may not benefit from the presence of a lipid carrier, for example, using one or more lipid-based transfection reagents, polymer-based transfection reagents or peptide- based transfection reagents or using electroporation. Many protein-associated molecules, such as metals, can be highly toxic to cells in vivo. This toxicity can cause decreased viability, as well as the acquisition of mutations. Certain embodiments thus have the additional benefit of producing cells that are free from toxic molecules.

[0150] The associated-molecule component of a protein can be measured by suspending the protein in solution and measuring the conductivity of the solution. Certain embodiments are therefore directed to a medium that contains a protein, wherein about a 10% solution of the protein in water has a conductivity of less than about 500 µmho / cm. In one embodiment, the solution has a conductivity of less than about 50 µmho / cm. In another embodiment, less than about 0.65% of the dry weight of the protein comprises lipids and / or less than about 0.35% of the dry weight of the protein comprises free fatty acids.

[0151] Certain embodiments are therefore directed to a method for transfecting a cell with a nucleic acid, wherein the cell is transfected more than once, and wherein the amount of nucleic acid delivered to the cell is different for two of the transfections. In one embodiment, the cell proliferates between two of the transfections, and the amount of nucleic acid delivered to the cell is greater for the second of the two transfections than for the first of the two transfections. In another embodiment, the cell is transfected more than twice, and the amount of nucleic acid delivered to the cell is greater for the second of three transfections than for the first of the same three transfections, and the amount of nucleic acid delivered to the cells is greater for the third of the same three transfections than for the second of the same three transfections. In yet another embodiment, the cell is transfected more than once, and the maximum amount of nucleic acid delivered to the cell during each transfection is sufficiently low to yield at least about 80% viability for at least two consecutive transfections.

[0152] In embodiments, there are provided methods in which modulating the amount of nucleic acid delivered to a population of proliferating cells in a series of transfections can result in both an increased -37-WSGR Docket No.61057-724.601 effect of the nucleic acid and increased viability of the cells. In embodiments, when cells are contacted with one or more nucleic acids encoding one or more reprogramming factors in a series of transfections, the efficiency of reprogramming can be increased when the amount of nucleic acid delivered in later transfections is greater than the amount of nucleic acid delivered in earlier transfections, for at least part of the series of transfections. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein one or more nucleic acids is repeatedly delivered to the cell in a series of transfections, and the amount of the nucleic acid delivered to the cell is greater for at least one later transfection than for at least one earlier transfection. In one embodiment, the cell is transfected from about 2 to about 10 times, or from about 3 to about 8 times, or from about 4 to about 6 times. In another embodiment, the one or more nucleic acids includes at least one RNA molecule, the cell is transfected from about 2 to about 10 times, and the amount of nucleic acid delivered to the cell in each transfection is the same as or greater than the amount of nucleic acid delivered to the cell in the most recent previous transfection. In yet another embodiment, the amount of nucleic acid delivered to the cell in the first transfection is from about 20ng / cm2to about 250ng / cm2, or from 100ng / cm2to 600ng / cm2. In yet another embodiment, the cell is transfected about 5 times at intervals of from about 12 to about 48 hours, and the amount of nucleic acid delivered to the cell is about 25ng / cm2for the first transfection, about 50ng / cm2for the second transfection, about 100ng / cm2for the third transfection, about 200ng / cm2for the fourth transfection, and about 400ng / cm2for the fifth transfection. In yet another embodiment, the cell is further transfected at least once after the fifth transfection, and the amount of nucleic acid delivered to the cell is about 400ng / cm2.

[0153] Several molecules can be added to media by conditioning. Certain embodiments are therefore directed to a medium that is supplemented with one or more molecules that are present in a conditioned medium. In one embodiment, the medium is supplemented with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In another embodiment, the medium is supplemented with TGF-β or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, a cell is reprogrammed according to the method of the present invention, wherein the medium is not supplemented with TGF- β for from about 1 to about 5 days and is then supplemented with TGF-β for at least about 2 days. In yet another embodiment, the medium is supplemented with IL-6, IL-6R or a biologically active fragment, analogue, variant, agonist, or family-member thereof. In yet another embodiment, the medium is supplemented with a sphingolipid or a fatty acid. In still another embodiment, the sphingolipid is lysophosphatidic acid, lysosphingomyelin, sphingosine-1-phosphate or a biologically active analogue, variant or derivative thereof.

[0154] In addition to mitotically inactivating cells, under certain conditions, irradiation can change the gene expression of cells, causing cells to produce less of certain proteins and more of certain other -38-WSGR Docket No.61057-724.601 proteins than non-irradiated cells, for example, members of the Wnt family of proteins. In addition, certain members of the Wnt family of proteins can promote the growth and transformation of cells. In embodiments, the efficiency of reprogramming can be greatly increased by contacting a cell with a medium that is conditioned using irradiated feeders instead of mitomycin-c-treated feeders. In embodiments, the increase in reprogramming efficiency observed when using irradiated feeders is caused in part by Wnt proteins that are secreted by the feeders. Certain embodiments are therefore directed to a method for reprogramming a cell, wherein the cell is contacted with Wnt1, Wnt2, Wnt3, Wnt3a or a biologically active fragment, analogue, variant, family-member or agonist thereof, including agonists of downstream targets of Wnt proteins, and / or agents that mimic one or more of the biological effects of Wnt proteins, for example, 2-amino-4-[3,4-(methylenedioxy)benzylamino]- 6-(3-methoxyphenyl)pyrimidine.

[0155] Because of the low efficiency of many DNA-based reprogramming methods, these methods may be difficult or impossible to use with cells derived from patient samples, which may contain only a small number of cells. In contrast, the high efficiency of certain embodiments of the present invention can allow reliable reprogramming of a small number of cells, including single cells. Certain embodiments are directed to a method for reprogramming a small number of cells. Other embodiments are directed to a method for reprogramming a single cell. In one embodiment, the cell is contacted with one or more enzymes. In another embodiment, the enzyme is collagenase. In yet another embodiment, the collagenase is animal-component free. In one embodiment, the collagenase is present at a concentration of from about 0.1mg / mL to about 10mg / mL, or from about 0.5mg / mL to about 5mg / mL. In another embodiment, the cell is a blood cell. In yet another embodiment, the cell is contacted with a medium containing one or more proteins that is derived from the patient’s blood. In still another embodiment, the cell is contacted with a medium comprising: DMEM / F12 + 2mM L- alanyl-L-glutamine + from about 5% to about 25% patient-derived serum, or from about 10% to about 20% patient-derived serum, or about 20% patient-derived serum.

[0156] In embodiments, transfecting cells with a mixture of RNA encoding Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro- RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof using the medium of the present invention can cause the rate of proliferation of the cells to increase. When the amount of RNA delivered to the cells is too low to ensure that all of the cells are transfected, only a fraction of the cells may show an increased proliferation rate. In certain situations, such as when generating a personalized therapeutic, increasing the proliferation rate of cells may be desirable, in part because doing so can reduce the time necessary to generate the therapeutic, and therefore can reduce the cost -39-WSGR Docket No.61057-724.601 of the therapeutic. Certain embodiments are therefore directed to a method for transfecting a cell with a mixture of RNA encoding Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro- RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In one embodiment, the cell exhibits an increased proliferation rate. In another embodiment, the cell is reprogrammed.

[0157] While detailed examples are provided herein for the production of specific types of cells and for the production of therapeutics comprising specific types of cells, it is recognized that the methods of the present invention can be used to produce many other types of cells, and to produce therapeutics comprising one or more of many other types of cells, for example, by reprogramming a cell according to the methods of the present invention, and culturing the cell under conditions that mimic one or more aspects of development by providing conditions that resemble the conditions present in the cellular microenvironment during development.

[0158] Other embodiments are directed to a method for reprogramming a cell. In one embodiment, the cell is reprogrammed by contacting the cell with one or more nucleic acids. In one embodiment, the cell is contacted with a plurality of nucleic acids encoding at least one of Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro- RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family-members thereof. In another embodiment, the cell is contacted with a plurality of nucleic acids encoding a plurality of proteins including: Oct4 protein, Sox2 protein, Klf4 protein, c-Myc protein, l-Myc protein, TERT protein, Nanog protein, Lin28 protein, Glis1 protein, Utf1 protein, Aicda protein, miR200 micro-RNA, miR291 micro-RNA, miR294 micro-RNA and miR295 micro-RNA, miR302 micro-RNA, miR367 micro-RNA, miR369 micro-RNA and biologically active fragments, analogues, variants and family- members thereof.

[0159] Illustrative subjects or patients refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0160] In some embodiments, a synthetic RNA molecule is used to reprogram iPSCs into iPSC- derived iMSCs (EiMSCs). In embodiments, the synthetic RNA molecule is mRNA. In embodiments, -40-WSGR Docket No.61057-724.601 the synthetic RNA molecule is in vitro transcribed. In embodiments, the synthetic RNA is a circRNA.

[0161] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides. In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0162] In some embodiments, the synthetic RNA comprises a 5’ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5’-UTR which comprises a sequence that increases RNA stability in vivo, and the 5’-UTR optionally comprises an alpha-globin or beta-globin 5’-UTR. In some embodiments, the synthetic RNA comprises a 3’-UTR which comprises a sequence that increases RNA stability in vivo, and the 3’-UTR optionally comprises an alpha-globin or beta-globin 3’-UTR. In some embodiments, the synthetic RNA comprises a 5’-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3’-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3’ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3’ poly(A) tail which comprises from about 20 nucleotides to about 250 nucleotides.

[0163] Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′- UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between -41-WSGR Docket No.61057-724.601 about 20nt and about 250nt or between about 120nt and about 150nt long. In a further embodiment, the 3′-poly(A) tail is about 20nt, or about 30nt, or about 40nt, or about 50nt, or about 60nt, or about 70nt, or about 80nt, or about 90nt, or about 100nt, or about 110nt, or about 120nt, or about 130nt, or about 140nt, or about 150nt, or about 160nt, or about 170nt, or about 180nt, or about 190nt, or about 200nt, or about 210nt, or about 220nt, or about 230nt, or about 240nt, or about 250nt long.

[0164] In some embodiments, the RNA comprises a tail composed of a plurality of adenines with one or more guanines.

[0165] In embodiments, the RNA comprises (a) a sequence encoding a protein, and (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides.

[0166] In embodiments, the one or more other nucleotides comprises deoxyguanosine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxyguanosine residues. In embodiments, the tail region comprises more than 50% deoxyguanosine residues.

[0167] In embodiments, the one or more other nucleotides comprises deoxycytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxycytidine residues. In embodiments, the tail region comprises more than 50% deoxycytidine residues.

[0168] In embodiments, the one or more other nucleotides comprises deoxythymidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxythymidine residues. In embodiments, the tail region comprises more than 50% deoxythymidine residues.

[0169] In embodiments, the one or more other nucleotides comprise deoxyguanosine residues and deoxycytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% deoxyadenosine residues. In embodiments, the tail region comprises fewer than 50% deoxyadenosine residues.

[0170] In embodiments, the one or more other nucleotides comprises guanosine residues.

[0171] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues. In embodiments, the tail region comprises more than 50% guanosine residues.

[0172] In embodiments, the one or more other nucleotides comprises cytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytidine residues. In embodiments, the tail region comprises more than 50% cytidine residues.

[0173] In embodiments, the one or more other nucleotides comprises uridine residues. In -42-WSGR Docket No.61057-724.601 embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues. In embodiments, the tail region comprises more than 50% uridine residues.

[0174] In embodiments, the one or more other nucleotides comprise guanosine residues and cytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0175] In embodiments, the tail region comprises fewer than 50% adenosine residues.

[0176] In embodiments, the tail is (A)150. In embodiments, the tail is (A39G)3(A)30. In embodiments, the tail is (A19G)7(A)10. In embodiments, the tail is (A9G)15.

[0177] In embodiments, the length of the tail region is between about 80 nucleotides and about 120 nucleotides, about 120 nucleotides and about 160 nucleotides, about 160 nucleotides and about 200 nucleotides, about 200 nucleotides and about 240 nucleotides, about 240 nucleotides and about 280 nucleotides, or about 280 nucleotides and about 320 nucleotides.

[0178] In embodiments, the length of the tail region is greater than 320 nucleotides.

[0179] In embodiments, the RNA comprises a 5’ cap structure. In embodiments, the RNA 5’-UTR comprises a Kozak consensus sequence. In embodiments, the RNA 5’-UTR comprises a sequence that increases RNA stability in vivo, and the 5’-UTR may comprise an alpha-globin or beta-globin 5’- UTR.

[0180] In embodiments, the RNA 3’-UTR comprises a sequence that increases RNA stability in vivo, and the 3’-UTR may comprise an alpha-globin or beta-globin 3’-UTR. In embodiments, the RNA comprises a 3’ poly(A) tail. In embodiments, the RNA 3’ poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

[0181] In embodiments, the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0182] In embodiments, the RNA is prepared by in vitro transcription. In embodiments, the RNA is synthetic.

[0183] In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides. In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0184] Further description of reprogramming is found in one or more of WO2013 / 086008, WO2014 / 071219, WO2015 / 117021, WO2016 / 131052, WO2018 / 035377, WO2019 / 191341, WO2021 / 003462, WO2021 / 231549, WO2021 / 222389, WO2022187704, or PCT / US2023 / 066464. The entire contents of which are incorporated by reference in their entirety. Gene-Editing

[0185] In any herein disclosed aspect or embodiment, a cell is gene-edited. -43-WSGR Docket No.61057-724.601

[0186] In embodiments, gene-editing a cell comprises contacting the cell with a synthetic nucleic acid encoding one or more gene-editing proteins, optionally selected from a nuclease, a transcription activator-like effector nuclease (TALEN), a zinc-finger nuclease, a meganuclease, a nickase, a clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein, CRISPR / Cas9, Cas9, xCas9, Cas12a (Cpf1), Cas13a, Cas14, CasX, CasY, a Class 1 Cas protein, a Class 2 Cas protein, MAD7, and a gene-editing protein comprising a repeat sequence comprising LTPvQVVAIAwxyz, or a natural or engineered variant, family member, orthologue, fragment or fusion construct thereof.

[0187] In embodiments, the gene-editing protein comprises: (i) a DNA-binding domain comprising a plurality of repeat sequences and (ii) the nuclease domain comprising a catalytic domain of a nuclease. In embodiments, the at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzα and is optionally between 36 and 39 amino acids long, where: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G, or null, z is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, GKQALETVQRLLPVLCQAHG, GGKQALETVQRLLPVLCQD or GGKQALETVQRLLPVLCQA, and α is four consecutive amino acids.

[0188] In embodiments, α comprises at least one glycine (G) residue. In embodiments, α comprises at least one histidine (H) residue. In embodiments, α comprises at least one histidine (H) residue at any one of positions 33, 34, or 35. In embodiments, α comprises at least one aspartic acid (D) residue. In embodiments, α comprises at least one, or two, or three of a glycine (G) residue, a histidine (H) residue, and an aspartic acid (D) residue.

[0189] In embodiments, α comprises one or more hydrophilic residues, optionally selected from: a polar and positively charged hydrophilic amino acid, optionally selected from arginine © and lysine (K); a polar and neutral of charge hydrophilic amino acid, optionally selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), and cystei©(C); a polar and negatively charged hydrophilic amino acid, optionally selected from aspartate (D) and glut©te (E), and an aromatic, polar and positively charged hydrophilic amino acid, optionally selected from histidine (H).

[0190] In some embodiments, α comprises one or more polar and positively charged hydrophilic amino acids selected from ©inine (R) and lysine (K). In some embodiments, α comprises one or more polar and neutral of charge hydrophilic amino acids selected from asparagine (N), glutamine (Q), serine (S), threonine (T), proline (P), a©cysteine (C). In some embodiments, α comprises one or more -44-WSGR Docket No.61057-724.601 polar and negatively charged hydrophilic amino acids selected from aspartate (D)©d glutamate (E). In some embodiments, α comprises one or more aromatic, polar and positively charged hydrophilic amino acids selected from histidine (H).

[0191] In embodiments, α comprises one or more hydrophobic residues, optionally selected from: a hydrophobic, aliphatic amino acid, optionally selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V), and a hydrophobic, aromatic amino acid, optionally selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In some embodiments, α comprises one or more hydrophobic, aliphatic amino acids selected from glycine (G), alanine (A), leucine (L), isoleucine (I), methionine (M), and valine (V). In some embodiments, α comprises one or more aromatic amino acids selected from phenylalanine (F), tryptophan (W), and tyrosine (Y). In embodiments, the DNA-binding domain comprises about 15, or about, 16, or about 17, or about 18, or about 18.5 repeat sequences.

[0192] In embodiments, α is selected from GHGG, HGSG, HGGG, from GGHD, GAHD, AHDG, PHDG, GPHD, GHGP, PHGG, PHGP, AHGA, LHGA, VHGA, IVHG, IHGM, RHGD, RDHG, RHGE, HRGE, RHGD, HRGD, GPYE, NHGG, THGG, GTHG, GSGS, GSGG, GGGG, GRGG, and GKGG.

[0193] In embodiments, the gene-editing protein has a DNA binding domain having at least one repeat of LTPEQVVAIAS*RVD*GGKQALETVQRLLPVLCQAGHGG (the “*RVD*” corresponds to the dinucleotide “xy” of the sequences of the present disclosure).

[0194] In embodiments, the repeat sequence is 33 or 34 amino acids long. In embodiments, the repeat sequence is 36-39 amino acids long. In some embodiments, the repeat sequence is 36 amino acids long. In some embodiments, the repeat sequence is 37 amino acids long. In some embodiments, the repeat sequence is 38 amino acids long. In some embodiments, the repeat sequence is 39 amino acids long.

[0195] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzGHGG or LTPvQVVAIAwxyzGTHG and is from 36 to 39 amino acids long, wherein: “v” is Q, D or E, “w” is S or N, “x” is H, N, or I, “y” is D, A, I, N, G, H, K, S, or null, and “z” is GGKQALETVQRLLPVLCQD or GGKQALETVQRLLPVLCQA and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In some embodiments, a gene-editing protein comprises a C-terminal GTHG produces more efficient editing at the target locus than TALENs at 33°C. GTHG. In various embodiments, a gene-editing protein comprises a C-terminal GTHG produces more efficient editing at the target locus than TALENs at 37°C.

[0196] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid -45-WSGR Docket No.61057-724.601 sequence: LTPvQVVAIAwxyzα and is from 36 to 39 amino acids long, wherein: v is Q, D or E, w is S or N, x is I, H, N, or I, y is D, A, I, N, H, K, S, G or null, z is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, GKQALETVQRLLPVLCQAHG, GGKQALETVQRLLPVLCQD or GGKQALETVQRLLPVLCQA, α is four consecutive amino acids; and (ii) a nuclease domain comprising a catalytic domain of a nuclease. In embodiments, α is selected from GHGG, HGSG, HGGG, from GGHD, GAHD, AHDG, PHDG, GPHD, GHGP, PHGG, PHGP, AHGA, LHGA, VHGA, IVHG, IHGM, RHGD, RDHG, RHGE, HRGE, RHGD, HRGD, GPYE, NHGG, THGG, GTHG, GSGS, GSGG, GGGG, GRGG, and GKGG. In some embodiments, a gene-editing protein comprises a C-terminal GTHG produces more efficient editing at the target locus than TALENs at 33°C. GTHG. In various embodiments, a gene-editing protein comprises a C-terminal GTHG produces more efficient editing at the target locus than TALENs at 37°C.

[0197] Certain embodiments are directed to a nucleic acid molecule encoding a non-naturally occurring fusion protein comprising a first region that recognizes a predetermined nucleotide sequence and a second region with endonuclease activity, wherein the first region contains an artificial TAL effector repeat domain comprising one or more repeat units about 36 amino acids in length which differ from each other by no more than seven amino acids, and wherein the repeat domain is engineered for recognition of the predetermined nucleotide sequence. In one embodiment, the first region contains the amino acid sequence: LTPXQVVAIAS where X can be either E or Q. In another embodiment, the amino acid sequence LTPXQVVAIAS of the encoded non-naturally occurring fusion protein is immediately followed by an amino acid sequence selected from: HD, NG, NS, NI, NN, and N. In a further embodiment, the fusion protein comprises restriction endonuclease activity.

[0198] In embodiments, the gene-editing protein comprises (i) a DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises the amino acid sequence: LTPvQVVAIAwxyzHG wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is any amino acid or no amino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, or GKQALETVQRLLPVLCQAHG. In another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG wherein “v” is D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, or GKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG wherein “v” is D or E, “w” is -46-WSGR Docket No.61057-724.601 S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, or GKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAIwyzHG, wherein “v” is D or E, “w” is S or N, “y” is any amino acid other than G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, or GKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzHG, wherein “v” is D or E, “w” is S or N, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, or GKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG, wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, or GKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzHG, wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQDHG, GGKQALETVQRLLPVLCQAHG, GKQALETVQRLLPVLCQDHG, or GKQALETVQRLLPVLCQAHG. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx, wherein “v” is D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy, wherein “v” is D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is any amino a©id or no a©ino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E, “w” is S or N, “x” is N, H or I, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E, “w” is S or N, “x” is any amino acid other than N, H and I, “y” is any amino acid or no amino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwyzGHGG, wherein “v” is Q, D or E, “w” is S or N, “y” -47-WSGR Docket No.61057-724.601 is any amino acid other than G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwIAzGHGG, wherein “v” is Q, D or E, “w” is S or N, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, “y” is any amino acid or no amino acid, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxyzGHGG, wherein “v” is Q, D or E, V is S or N, “x” is S, T or Q, “y” is selected from: D, A, I, N, H, K, S, and G, and “z” is GGRPALE, GGKQALE, GGKQALETVQRLLPVLCQD, GGKQALETVQRLLPVLCQA, GKQALETVQRLLPVLCQD or GKQALETVQRLLPVLCQA. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwx, wherein “v” is Q, D or E, “w” is S or N, and “x” is S, T or Q. In yet another embodiment, the repeat sequence comprises: LTPvQVVAIAwxy, wherein “v” is Q, D or E, “w” is S or N, “x” is S, T or Q, and “y” is selected from: D, A, I, N, H, K, S, and G.

[0199] The above-mentioned gene-editing proteins comprise a repeat variable di-residue (RVD) at residue 12 or 13, e.g., at “x” and “y” in the various above-mentioned repeat sequences, e.g., LTPvQVVAIAwxyzα, which targets the DNA-binding domain to a target DNA molecule. In embodiments, the RVD recognizes one base pair in the nucleic acid molecule. In embodiments, the RVD recognizes a C residue in the nucleic acid molecule and is selected from HD, N(null), HA, ND, and HI. In embodiments, the RVD recognizes a G residue in the nuclei© acid molecule and is selected from NN, NH, NK, HN, and NA. In embodiments, the RVD recognizes an A residue in the nucleic acid molecule and is selected from Nl and NS. In embodiments, the RVD recognizes a T residue in the nucleic acid molecule and is selected from NG, HG, H(null), and IG.

[0200] In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HD. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is N(null). In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HA. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is ND. In some embodiments, the RVD recognizing a C residue in the nucleic acid molecule is HI. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NN. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NH. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NK. In some embodiments, the RVD recognizing a G residue in the nucleic acid molecule is HN. In some -48-WSGR Docket No.61057-724.601 embodiments, the RVD recognizing a G residue in the nucleic acid molecule is NA. In some embodiments, the RVD recognizing an A residue in the nucleic acid molecule is Nl. In some embodiments, the RVD recognizing an A residue in the nucleic acid molecule is NS. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is NG. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is HG. In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is H(null). In some embodiments, the RVD recognizing a T residue in the nucleic acid molecule is IG.

[0201] In some embodiments, alternative DNA binding domains are employed.

[0202] For example, the alternative DNA binding domains described herein are, in embodiments, paired with the novel engineered nuclease domains described herein.

[0203] For example, the alternative DNA binding domains described herein are, in embodiments, used in the conditional activity: temperature dependence methods described herein.

[0204] For example, the alternative DNA binding domains described herein are, in embodiments, used in the conditional activity: methylation status methods described herein.

[0205] In embodiments, the engineered gene-editing proteins do not require a thymine (T) in the zero position of the target site (“T0”).

[0206] In embodiments, the engineered gene-editing proteins that comprise DNA-binding domains comprise alterations in the in the N-terminal region to remove the T0 requirement.

[0207] In embodiments, there is provided a method of gene-editing a cell with one or more of the present gene-editing proteins, optionally with also using a linear DNA repair template, optionally also using conditional activity methods described herein, where the target site lacks a thymine (T) in the zero position.

[0208] Wild type N-terminal region is characterized by –he sequence: Asp–25 - IVGVGKQWSGARAL - Glu240 (DIVGVGKQWSGARALE). In embodiments, there is provided the engineered N-termi–al region of Asp–25 - IVGVGKQKRGARAL - Glu240 (underlining showing the change WS->KR) (DIVGVGKQKRGARALE).

[0209] In embodiments, there is provided an engineered N-terminal region in which KQWS is replaced with one or more amino acids, e.g., about 2-10 amino acids, or about 4-10 amino acids, or about 6-10 amino acids, or about 8-10 amino acids, or about 4 amino acids, or about 6 amino acids, or about 8 amino acids, or about 10 amino acids.

[0210] In embodiments, there is provided the engineered N-termi–al region of Asp225 – IVGVGGSKRGAGSGARAL - Glu244 (underlining showing the change KQWS -> GSKRGAGS) (DIVGVGGSKRGAGSGARALE).

[0211] In some cases, a cell is contacted with a demethylating agent during the process of gene- editing. In embodiments, the demethylating agent is selected from 5-az’citidine and 5-aza-2'- -49-WSGR Docket No.61057-724.601 deoxycitidine (decitabine).

[0212] In some embodiments, the gene-editing protein comprises: (a) the DNA-binding domain comprising a plurality of repeat sequences and at least one of the repeat sequences comprises a repeat variable di-residue (RVD) at residue 12 or 13; and (b) the nuclease domain comprising a catalytic domain, the catalytic domain comprising a hybrid of the catalytic domains of Fokl and Stsl, comprising the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Fokl with at least one of the domains of Fokl being substituted in whole or in part with the α1, α2, α3, α4, α5, α6, β1, β2, β3, β4, β5, and β6 domains of Stsl and optionally comprising at least one mutation, e.g., in the catalytic site of the nuclease thereby preventing the mutant nuclease domain from creating a break in a DNA site. In embodiments, the nuclease domain is capable of binding to the DNA and forming a dimer with another nuclease domain. In embodiments, the nuclease domain comprising a mutation is incapable of incapable of creating a single-stranded break in a DNA site.

[0213] In some embodiments, certain fragments of an endonuclease cleavage domain are used, including fragments that are truncated at the N-terminus, fragments that are truncated at the C- terminus, fragments that have internal deletions, and fragments that combine N-terminus, C-terminus, and / or internal deletions, which maintain part or all of the catalytic activity of the full endonuclease cleavage domain. Determining whether a fragment can maintain part, or all of the catalytic activity of the full domain can be accomplished by, for example, synthesizing a gene-editing protein that contains the fragment according to the methods of the present invention, inducing cells to express the gene- editing protein according to the methods of the present invention, and measuring the efficiency of gene editing. In some embodiments, a measurement of gene-editing efficiency is used to ascertain whether any specific fragment maintains part or all of the catalytic activity of the full endonuclease cleavage domain. Certain embodiments are therefore directed to a biologically active fragment of an endonuclease cleavage domain. In one embodiment, the endonuclease cleavage domain is selected from: FokI, StsI, StsI-HA, StsI-HA2, StsI-UHA, StsI-UHA2, StsI-HF, and StsI-UHF or a natural or engineered variant or biologically active fragment thereof, or a hybrid or chimera thereof.

[0214] In embodiments, the gene-editing protein comprises a linker. In another embodiment, the linker connects a DNA-binding domain to a nuclease domain. In a further embodiment, the linker is between about 1 and about 10 amino acids long. In some embodiments, the linker is about 1, about 2, or about 3, or about 4, or about 5, or about 6, or about 7, or about 8, or about 9, or about 10 amino acids long. In one embodiment, the gene-editing protein is capable of generating a nick or a double- strand break in a target DNA molecule.

[0215] In embodiments, the gene-editing protein is any of those described in International Patent Publication No. WO2014 / 071219, WO2021 / 231549, or PCT / US2023 / 066464, hereby incorporated by reference in their entireties. -50-WSGR Docket No.61057-724.601

[0216] In various embodiments, the cell is transfected (e.g., contacted) with a synthetic nucleic acid encoding the gene-editing protein at about 30°C to about 35°C, e.g., without limitation about 33°C. In embodiments, the contacting occurs at about 30°C. In some embodiments, the contacting occurs at about 31 °C. In some embodiments, the contacting occurs at about 32°C. In some embodiments, the contacting occurs at about 33°C. In some embodiments, the contacting occurs at about 34°C. In some embodiments, the contacting occurs at about 35°C. In embodiments, the gene-editing protein is functionally temperature-switchable. In embodiments, the method further c©rises the step of (c) culturing the contacted cell at about 30°C to about 35°C. In embodiments, the method furthe©omprises the step of (c) culturing the contacted cell at about 30°C. In embodiments, the method fur©r comprises the step of (c) culturing the contacted cell at about 31 °C. In embodiments, the method ©ther comprises the step of (c) culturing the contacted cell at about 32°C. In embodiments, the meth©further comprises the step of (c) culturing the contacted cell at about 33°C. In embodiments, the m©od further comprises the step of (c) culturing the contacted cell at about 34°C. In embodiments, th©ethod further comprises the step of (c) culturing the contacted cell at about 35°C.

[0217] Further description of temperature-sensitive gene-editing is found in WO2021 / 231549. The entire contents of which are incorporated by reference in their entirety.

[0218] In embodiments, the synthetic nucleic acid encoding the gene-editing protein is transfected along with a repair template. In some cases, the repair template is a double stranded synthetic oligodeoxynucleotide (dsODNs). In embodiments, the dsODNs comprises a repair template and comprises the TTAGGG motif. In some cases, the dsODN comprises a repair template and lacks the TTAGGG motif and a separate dsODNs comprising the TTAGGG motif is transfected into the cell.

[0219] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides. In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0220] In some embodiments, the synthetic RNA comprises a 5’ cap structure. In some embodiments, -51-WSGR Docket No.61057-724.601 the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5’-UTR which comprises a sequence that increases RNA stability in vivo, and the 5’-UTR optionally comprises an alpha-globin or beta-globin 5’-UTR. In some embodiments, the synthetic RNA comprises a 3’-UTR which comprises a sequence that increases RNA stability in vivo, and the 3’-UTR optionally comprises an alpha-globin or beta-globin 3’-UTR. In some embodiments, the synthetic RNA comprises a 5’-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3’-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3’ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3’ poly(A) tail which comprises from about“20 nucleotides to about 250 nucleotides. "Poly(A) tail” and “poly-adenylation signal” may be used interchangeably throughout the present disclosure.

[0221] In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides. In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0222] In various cases, transfection of a cell with synthetic nucleic acids for gene-editing the cell may be facilitated by use of the ToRNAdo™ Nucleic-Acid Delivery System. This system relates to new lipids that find use, inter alia, in improved delivery of biological payloads, e.g., nucleic acids, to cells.

[0223] The system relates to use of a compound of Formula (A):

[0224] where R21, R23, and R24are independently selected from hydrogen, C1-6alkyl, C1-6haloalkyl, C1-6hydroxyalkyl, C1-6alkoxyalkyl, C5-10aryl, 5- to 10-membered heteroaryl, or C3-6cycloalkyl; n is 1 to 20; m is 6, 7, 8, 9, or 10; p is 1, 2, 3, or 4; q is 1, 2, 3, 4, or 5; and r is 1, 2, 3, 4, 5, or 6.

[0225] The system relates to use of a compound of Formula (IV) -52-WSGR Docket No.61057-724.601

[0226] where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0227] Further description of ToRNAdo™ Nucleic-Acid Delivery System is found in one or more of US20210009505A1, US 10,501,404, and WO2021003462. The entire contents of which are incorporated by reference in their entirety.

[0228] In any of the herein-disclosed aspects or embodiments, a synthetic RNA molecule encoding the gene-editing protein may be in the form of a circular RNA (circRNA). The circRNA are manufactured by methods do not require a linear oligonucleotide (splint) to pre-orient the two reacting ends of a linear RNA to assist in ligation to yield a circRNA, the circRNA are manufactured by methods that do not require ribozymes to yield a circRNA, and / or the circRNA are manufactured by methods that do not require HPLC-based purification, e.g., post-ligation. A nucleic acid that can be manufactured into a circRNA has the structure: 5'-X-Y-A-IRES-B-CDS-C-Y'-Z 3'. Here, Y and Y' each independently comprise one or more nucleotides and Y and Y' are substantially complementary; X and Z each independently comprise one or more nucleotides and X and Z are not substantially complementary; IRES comprises an internal ribosome entry site; CDS comprises a coding sequence; and A, B, and C are each independently a spacer comprising one or more nucleotides or null. The CDS of a circRNA encodes the gene-editing protein(s). Additional details regarding circRNAs useful in the present disclosure are described in WO2022232291A1, the contents of which are incorporated herein by reference in its entirety.

[0229] In some embodiments, the synthetic RNA comprises a 5’ cap structure. In some embodiments, the synthetic RNA comprises a Kozak consensus sequence. In some embodiments, the synthetic RNA comprises a 5’-UTR which comprises a sequence that increases RNA stability in vivo, and the 5’-UTR optionally comprises an alpha-globin or beta-globin 5’-UTR. In some embodiments, the synthetic RNA comprises a 3’-UTR which comprises a sequence that increases RNA stability in vivo, and the 3’-UTR optionally comprises an alpha-globin or beta-globin 3’-UTR. In some embodiments, the synthetic RNA comprises a 5’-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3’-UTR which comprises a microRNA binding site that modulates RNA stability in a cell type-specific manner. In some embodiments, the synthetic RNA comprises a 3’ poly(A) tail. In some embodiments, the synthetic RNA comprises a 3’ poly(A) tail which comprises from about 20 nucleotides to about -53-WSGR Docket No.61057-724.601 250 nucleotides.

[0230] Certain embodiments are directed to a nucleic acid comprising a 5′-cap structure selected from Cap 0, Cap 1, Cap 2, and Cap 3 or a derivative thereof. In one embodiment, the nucleic acid comprises one or more UTRs. In another embodiment, the one or more UTRs increase the stability of the nucleic acid. In a further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 5′- UTR. In a still further embodiment, the one or more UTRs comprise an alpha-globin or beta-globin 3′-UTR. In a still further embodiment, the RNA molecule comprises an alpha-globin or beta-globin 5′-UTR and an alpha-globin or beta-globin 3′-UTR. In one embodiment, the 5′-UTR comprises a Kozak sequence that is substantially similar to the Kozak consensus sequence. In another embodiment, the nucleic acid comprises a 3′-poly(A) tail. In a further embodiment, the 3′-poly(A) tail is between about 20nt and about 250nt or between about 120nt and about 150nt long. In a further embodiment, the 3′-poly(A) tail is about 20nt, or about 30nt, or about 40nt, or about 50nt, or about 60nt, or about 70nt, or about 80nt, or about 90nt, or about 100nt, or about 110nt, or about 120nt, or about 130nt, or about 140nt, or about 150nt, or about 160nt, or about 170nt, or about 180nt, or about 190nt, or about 200nt, or about 210nt, or about 220nt, or about 230nt, or about 240nt, or about 250nt long.

[0231] In some embodiments, the RNA comprises a tail composed of a plurality of adenines with one or more guanines.

[0232] In embodiments, the RNA comprises (a) a sequence encoding a protein, and (b) a tail region comprising deoxyadenosine nucleotides and one or more other nucleotides.

[0233] In embodiments, the one or more other nucleotides comprises deoxyguanosine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxyguanosine residues. In embodiments, the tail region comprises more than 50% deoxyguanosine residues.

[0234] In embodiments, the one or more other nucleotides comprises deoxycytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxycytidine residues. In embodiments, the tail region comprises more than 50% deoxycytidine residues.

[0235] In embodiments, the one or more other nucleotides comprises deoxythymidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% deoxythymidine residues. In embodiments, the tail region comprises more than 50% deoxythymidine residues.

[0236] In embodiments, the one or more other nucleotides comprise deoxyguanosine residues and deoxycytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% deoxyadenosine residues. In embodiments, the tail region comprises fewer than 50% -54-WSGR Docket No.61057-724.601 deoxyadenosine residues.

[0237] In embodiments, the one or more other nucleotides comprises guanosine residues.

[0238] In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% guanosine residues. In embodiments, the tail region comprises more than 50% guanosine residues.

[0239] In embodiments, the one or more other nucleotides comprises cytidine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% cytidine residues. In embodiments, the tail region comprises more than 50% cytidine residues.

[0240] In embodiments, the one or more other nucleotides comprises uridine residues. In embodiments, the tail region comprises about 1%, about 2%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% uridine residues. In embodiments, the tail region comprises more than 50% uridine residues.

[0241] In embodiments, the one or more other nucleotides comprise guanosine residues and cytidine residues. In embodiments, the tail region comprises about 99%, about 98%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, or about 50% adenosine residues.

[0242] In embodiments, the tail region comprises fewer than 50% adenosine residues.

[0243] In embodiments, the tail is (A)150. In embodiments, the tail is (A39G)3(A)30. In embodiments, the tail is (A19G)7(A)10. In embodiments, the tail is (A9G)15.

[0244] In embodiments, the length of the tail region is between about 80 nucleotides and about 120 nucleotides, about 120 nucleotides and about 160 nucleotides, about 160 nucleotides and about 200 nucleotides, about 200 nucleotides and about 240 nucleotides, about 240 nucleotides and about 280 nucleotides, or about 280 nucleotides and about 320 nucleotides.

[0245] In embodiments, the length of the tail region is greater than 320 nucleotides.

[0246] In embodiments, the RNA comprises a 5’ cap structure. In embodiments, the RNA 5’-UTR comprises a Kozak consensus sequence. In embodiments, the RNA 5’-UTR comprises a sequence that increases RNA stability in vivo, and the 5’-UTR may comprise an alpha-globin or beta-globin 5’- UTR.

[0247] In embodiments, the RNA 3’-UTR comprises a sequence that increases RNA stability in vivo, and the 3’-UTR may comprise an alpha-globin or beta-globin 3’-UTR. In embodiments, the RNA comprises a 3’ poly(A) tail. In embodiments, the RNA 3’ poly(A) tail is from about 20 nucleotides to about 250 nucleotides in length.

[0248] In embodiments, the RNA is from about 200 nucleotides to about 5000 nucleotides in length.

[0249] In embodiments, the RNA is prepared by in vitro transcription. In embodiments, the RNA is -55-WSGR Docket No.61057-724.601 synthetic.

[0250] In some embodiments, the synthetic RNA comprises about 200 nucleotides to about 5000 nucleotides. In some embodiments, the synthetic RNA comprises from about 500 to about 2000 nucleotides, or about 500 to about 1500 nucleotides, or about 500 to about 1000 nucleotides.

[0251] The gene-editing proteins disclosed herein are useful for inserting a nucleic acid relevant to the present

[0252] Further description of gene-editing is found in one or more of WO2013 / 086008, WO2014 / 071219, WO2015 / 117021, WO2016 / 131052, WO2018 / 035377, WO2019 / 191341, WO2021 / 003462, WO2021 / 231549, WO2021 / 222389, WO2022187704, or PCT / US2023 / 066464. The entire contents of which are incorporated by reference in their entirety. RNA Modifications

[0253] In embodiments, the present disclosure relates to RNA-based modifications, e.g., reprogramming and / or gene-editing. In some embodiments, an RNA molecule encodes a gene-editing protein. In some embodiments, a RNA molecule encodes a reprogramming factor.

[0254] In embodiments, the RNA is mRNA. In embodiments, the RNA is modified mRNA. In embodiments, the modified mRNA comprises one or more non-canonical nucleotides.

[0255] In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides.

[0256] In some embodiments, the synthetic RNA molecule contains one or more non-canonical nucleotides that include one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine can be less toxic than synthetic RNA molecules containing only canonical nucleotides, due in part to the ability of substitutions at these positions to interfere with recognition of synthetic RNA molecules by proteins that detect exogenous nucleic acids, and furthermore, that substitutions at these positions can have minimal impact on the efficiency with which the synthetic RNA molecules can be translated into protein, due in part to the lack of interference of substitutions at these positions with base-pairing and base-stacking interactions.

[0257] In embodiments, the synthetic RNA molecule is mRNA comprising one or more non- canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5- methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5- hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5- ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5-carboxyuridine, 5- carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine, 5-amino-5- azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5-aminopseudouridine, 5- -56-WSGR Docket No.61057-724.601 hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio-5-methyluridine, 4-thio-5- aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4-thio-5-aminopseudouridine, 4-thio- 5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4- aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5- methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5- methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5- azacytidine, N4-methylpseudoisocytidine, 2-thio-5-azacytidine, 2-thiopseudoisocytidine, 2-thio-N4- methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2- thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5- azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5- aminopseudoisocytidine, 2-thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2- thio-N4-methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4- methyl-5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5- aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4- aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5- hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4-amino-5- hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4- hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-hydroxy-5- azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4- hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5- aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2- thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4- methyl-5-methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl- 5-hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2- thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5- hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4- amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxypseudoisocytidine, 2-thio-N4- hydroxy-5-azacytidine, 2-thio-N4-hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl- -57-WSGR Docket No.61057-724.601 5-azacytidine, 2-thio-N4-hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5- azacytidine, 2-thio-N4-hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5- aminopseudoisocytidine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6- aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7- deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8- azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8- azaadenosine, N6-hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7-deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7- deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8- azaguanosine.

[0258] In some embodiments, the one or more non-canonical nucleotides are selected from 5- hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5- hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5- methoxypseudouridine. In some embodiments, at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of the non-canonical nucleotides are one or more of 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5- methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5- hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5- methoxypseudouridine.

[0259] In some embodiments, at least about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of cytidine residues are non-canonical nucleotides selected from 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine.

[0260] In some embodiments, at least about 20%, or about 30%, or about 40%, or about 50%, or at least about 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 100% of uridine residues are non-canonical nucleotides selected from 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5- methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5- formylpseudouridine, and 5-methoxypseudouridine.

[0261] In some embodiments, at least about 10% (e.g., 10%, or about 20%, or about 30%, or about -58-WSGR Docket No.61057-724.601 40%, or about 50%) of guanosine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine. In some embodiments, the RNA contains no more than about 50% 7-deazaguanosine in place of guanosine residues.

[0262] In some embodiments, the synthetic RNA molecule does not contain non-canonical nucleotides in place of adenosine residues.

[0263] Other non-canonical nucleotides that can be used in place of or in combination with 5-methyluridine include but are not limited to: pseudouridine and 5-methylpseudouridine (a.k.a. “1- methylpseudouridine”, a.k.a. “N1-methylpseudouridine”) or one or more derivatives thereof. Other non-canonical nucleotides that can be used in place of or in combination with 5-methylcytidine and / or 5-hydroxymethylcytidine include, but are not limited to: pseudoisocytidine, 5-methylpseudoisocytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, N4- methylcytidine, N4-acetylcytidine or one or more derivatives thereof. In certain embodiments, for example, when performing only a single transfection or when the cells being transfected are not particularly sensitive to transfection-associated toxicity or innate-immune signaling, the fractions of non-canonical nucleotides can be reduced. Reducing the fraction of non-canonical nucleotides can be beneficial, in part, because reducing the fraction of non-canonical nucleotides can reduce the cost of the nucleic acid. In certain situations, for example, when minimal immunogenicity of the nucleic acid is desired, the fractions of non-canonical nucleotides can be increased.

[0264] Note that alternative naming schemes exist for certain non-canonical nucleotides. For example, in certain situations, 5-methylpseudouridine can be referred to as “3-methylpseudouridine” or “N3-methylpseudouridine” or “1-methylpseudouridine” or “N1-methylpseudouridine”. Nucleotides that contain the prefix “amino” can refer to any nucleotide that contains a nitrogen atom bound to the atom at the stated position of the nucleotide, for example, 5-aminocytidine can refer to 5-aminocytidine, 5-methylaminocytidine, and 5-nitrocytidine. Similarly, nucleotides that contain the prefix “methyl” can refer to any nucleotide that contains a carbon atom bound to the atom at the stated position of the nucleotide, for example, 5-methylcytidine can refer to 5-methylcytidine, 5- ethylcytidine, and 5-hydroxymethylcytidine, nucleotides that contain the prefix “thio” can refer to any nucleotide that contains a sulfur atom bound to the atom at the given position of the nucleotide, and nucleotides that contain the prefix “hydroxy” can refer to any nucleotide that contains an oxygen atom bound to the atom at the given position of the nucleotide, for example, 5-hydroxyuridine can refer to 5-hydroxyuridine and uridine with a methyl group bound to an oxygen atom, wherein the oxygen atom is bound to the atom at the 5C position of the uridine.

[0265] In some embodiments, non-canonical nucleotides are incorporated into RNA to increase the efficiency with which the RNA can be translated into protein and can decrease the toxicity of the RNA. In embodiments, the RNA molecule comprises one or more non-canonical nucleotides. In some -59-WSGR Docket No.61057-724.601 embodiments, the nucleic acid comprises one or more non-canonical nucleotide members of the 5 methylcytidine de-methylation pathway. In some embodiments, the nucleic acid comprises at least one of: 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, and 5-carboxycytidine or a derivative thereof. In some embodiments, the nucleic acid comprises at least one of: pseudouridine, 5- methylpseudouridine, 5-methyluridine, 5-methylcytidine, 5-hydroxymethylcytidine, N4- methylcytidine, N4-acetylcytidine, and 7-deazaguanosine or a derivative thereof.

[0266] Certain non-canonical nucleotides can be incorporated more efficiently than other non- canonical nucleotides into RNA molecules by RNA polymerases that are commonly used for in vitro transcription, due in part to the tendency of these certain non-canonical nucleotides to participate in standard base-pairing interactions and base-stacking interactions, and to interact with the RNA polymerase in a manner similar to that in which the corresponding canonical nucleotide interacts with the RNA polymerase. As a result, certain nucleotide mixtures containing one or more non-canonical nucleotides can be beneficial in part because in vitro-transcription reactions containing these nucleotide mixtures can yield a large quantity of RNA. Certain embodiments are therefore directed to a nucleotide mixture containing one or more nucleotides that includes one or more substitutions at the 2C and / or 4C and / or 5C positions in the case of a pyrimidine or the 6C and / or 7N and / or 8C positions in the case of a purine. Nucleotide mixtures include, but are not limited to (numbers preceding each nucleotide indicate an exemplary fraction of the non-canonical nucleotide triphosphate in an in vitro- transcription reaction, for example, 0.2 pseudoisocytidine refers to a reaction containing adenosine-5′- triphosphate, guanosine-5′-triphosphate, uridine-5′-triphosphate, cytidine-5′-triphosphate, and pseudoisocytidine-5′-triphosphate, wherein pseudoisocytidine-5′-triphosphate is present in the reaction at an amount approximately equal to 0.2 times the total amount of pseudoisocytidine-5′- triphosphate + cytidine-5′-triphosphate that is present in the reaction, with amounts measured either on a molar or mass basis, and wherein more than one number preceding a nucleoside indicates a range of exemplary fractions): 1.0 pseudouridine, 0.1 – 0.82-thiouridine, 0.1 – 0.85-methyluridine, 0.2 – 1.05-hydroxyuridine, 0.2 – 1.05-methoxyuridine, 0.1 – 1.05-aminouridine, 0.1 – 1.04-thiouridine, 0.1 – 1.02-thiopseudouridine, 0.1 – 1.04-thiopseudouridine, 0.1 – 1.05-hydroxypseudouridine, 0.2 – 15-methylpseudouridine, 0.2 – 1.05-methoxypseudouridine, 0.1 – 1.05-aminopseudouridine, 0.2 – 1.0 2-thiocytidine, 0.1 – 0.8 pseudoisocytidine, 0.2 – 1.0 5-methylcytidine, 0.2 – 1.0 5- hydroxycytidine, 0.2 – 1.0 5-hydroxymethylcytidine, 0.2 – 1.0 5-methoxycytidine, 0.1 – 1.0 5- aminocytidine, 0.2 – 1.0 N4-methylcytidine, 0.2 – 1.0 5-methylpseudoisocytidine, 0.2 – 1.0 5- hydroxypseudoisocytidine, 0.2 – 1.0 5-aminopseudoisocytidine, 0.2 – 1.0 N4- methylpseudoisocytidine, 0.2 – 1.02-thiopseudoisocytidine, 0.2 – 1.07-deazaguanosine, 0.2 – 1.06- thioguanosine, 0.2 – 1.06-thio-7-deazaguanosine, 0.2 – 1.08-azaguanosine, 0.2 – 1.07-deaza-8- azaguanosine, 0.2 – 1.0 6-thio-8-azaguanosine, 0.1 – 0.5 7-deazaadenosine, and 0.1 – 0.5 N6- -60-WSGR Docket No.61057-724.601 methyladenosine.

[0267] In some embodiments, the RNA comprising one or more non-canonical nucleotides composition or synthetic polynucleotide composition (e.g., which may be prepared by in vitro transcription) contains substantially or entirely the canonical nucleotide at positions having adenine or “A” in the genetic code. The term “substantially” in this context refers to at least 90%. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) 7-deazaguanosine at positions with “G” in the genetic code as well as the corresponding canonical nucleotide “G”, and the canonical and non-canonical nucleotide at positions with G may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three or four) of 5-hydroxycytidine, 5-methylcytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine at positions with “C” in the genetic code as well as the canonical nucleotide “C”, and the canonical and non-canonical nucleotide at positions with C may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non-canonical nucleotide at positions of “C” are as described in the preceding paragraph. In these embodiments, the RNA composition or synthetic polynucleotide composition may further contain (e.g., consist of) one or more (e.g., two, three, or four) of 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, pseudouridine, 5-hydroxypseudouridine, 5-methylpseudouridine, 5- hydroxymethylpseudouridine, 5-carboxypseudouridine, 5-formylpseudouridine, and 5- methoxypseudouridine at positions with “U” in the genetic code as well as the canonical nucleotide “U”, and the canonical and non-canonical nucleotide at positions with “U” may be in the range of 5:1 to 1:5, or in some embodiments in the range of 2:1 to 1:2. In some embodiments, the level of non- canonical nucleotide at positions of “U” are as described in the preceding paragraph.

[0268] In embodiments, combining certain non-canonical nucleotides can be beneficial in part because the contribution of non-canonical nucleotides to lowering the toxicity of RNA molecules can be additive. Certain embodiments are therefore directed to a nucleotide mixture, wherein the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, for example, the nucleotide mixture contains both pseudoisocytidine and 7-deazaguanosine or the nucleotide mixture contains both N4-methylcytidine and 7-deazaguanosine, etc. In one embodiment, the nucleotide mixture contains more than one of the non-canonical nucleotides listed above, and each of the non-canonical nucleotides is present in the mixture at the fraction listed above, for example, the nucleotide mixture contains 0.1 – 0.8 pseudoisocytidine and 0.2 – 1.0 7-deazaguanosine or the nucleotide mixture contains 0.2 – 1.0 N4-methylcytidine and 0.2 – 1.07-deazaguanosine, etc.

[0269] In certain situations, for example, when it may not be necessary or desirable to maximize the -61-WSGR Docket No.61057-724.601 yield of an in vitro-transcription reaction, nucleotide fractions other than those given above may be used. The exemplary fractions and ranges of fractions listed above relate to nucleotide-triphosphate solutions of typical purity (greater than 90% purity). Larger fractions of these and other nucleotides can be used by using nucleotide-triphosphate solutions of greater purity, for example, greater than about 95% purity or greater than about 98% purity or greater than about 99% purity or greater than about 99.5% purity, which can be achieved, for example, by purifying the nucleotide triphosphate solution using existing chemical-purification technologies such as high-pressure liquid chromatography (HPLC) or by other means. In one embodiment, nucleotides with multiple isomers are purified to enrich the desired isomer.

[0270] In some embodiments, the one or more non-canonical nucleotides avoids substantial cellular toxicity.

[0271] In some embodiments, the non-canonical nucleotides comprise one or more of 5- hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5- methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5- formylpseudouridine, and 5-methoxypseudouridine, optionally at an amount of at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the non-canonical nucleotides.

[0272] In some embodiments, at least about 50% of cytidine residues are non-canonical nucleotides, and which are selected from 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5- carboxycytidine, 5-formylcytidine, and 5-methoxycytidine.

[0273] In some embodiments, at least about 75% or at least about 90% of cytidine residues are non- canonical nucleotides, and the non-canonical nucleotides are selected from 5-hydroxycytidine, 5- methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, and 5- methoxycytidine.

[0274] In some embodiments, at least about 20% of uridine, or at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5- hydroxypseudouridine, 5-methylpseudouridine, 5-hydroxymethylpseudouridine, 5- carboxypseudouridine, 5-formylpseudouridine, and 5-methoxypseudouridine.

[0275] In some embodiments, at least about 40%, or at least about 50%, or at least about 75%, or at about least 90% of uridine residues are non-canonical nucleotides, and the non-canonical nucleotides are selected from pseudouridine, 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-formyluridine, 5-methoxyuridine, 5-hydroxypseudouridine, 5- -62-WSGR Docket No.61057-724.601 methylpseudouridine, 5-hydroxymethylpseudouridine, 5-carboxypseudouridine, 5- formylpseudouridine, and 5-methoxypseudouridine.

[0276] In some embodiments, at least about 10% of guanine residues are non-canonical nucleotides, and the non-canonical nucleotide is optionally 7-deazaguanosine. In some embodiments, the synthetic RNA comprises no more than about 50% 7-deazaguanosine in place of guanosine residues. In some embodiments, the synthetic RNA does not comprise non-canonical nucleotides in place of adenosine residues.

[0277] Any RNA disclosed herein may comprise non-canonical nucleotides. Myeloid Cells with Persistent Cytokine Expression

[0278] In embodiments, provided herein are therapeutic myeloid cells with persistent cytokine expression.

[0279] In some embodiments, the therapeutic myeloid cells may comprise macrophages, monocytes, dendritic cells, and / or granulocytes, or combinations thereof. The therapeutic myeloid cells may be differentiated from stem cells. The stem cells may be pluripotent stem cells. The stem cells may be induced pluripotent stem cells. The stem cells may originate from a human subject. The stem cells may originate from a human subject who is not intended to receive the therapeutic myeloid cells as therapy. The stem cells may originate from a stem cell bank. The stem cells may be genetically edited prior to differentiation. The stem cells may be modified with a transgene construct prior to differentiation. The therapeutic myeloid cells may be primary myeloid cells. Any of the modifications described herein may be applied to a stem cell or an immune cell differentiated therefrom, or both.

[0280] The therapeutic myeloid cells may be engineered to comprise a transgene. The therapeutic myeloid cells may be contacted with a nucleic acid molecule. The nucleic acid molecule may be single-stranded DNA or double-stranded DNA. The nucleic acid molecule may be a DNA repair template. The DNA repair template may encode a transgene. The transgene may comprise a cytokine. The cytokine may comprise an interleukin (IL). The interleukin may comprise but it not limited to IL-12 or IL-4. The cytokine may comprise an IL-12 dimer. The IL-12 dimer may comprise a linker peptide. The nucleic acid molecule may comprise one or more transgenes. The nucleic acid molecule may comprise one, two, three, four, or five transgenes. The transgene may comprise a chimeric antigen receptor (CAR). The CAR may specifically recognize for example CD19, CD20, CD22, BCMA, HER2, EGFR, GD2, ROR1, or mesothelin. The transgene may be encoded by a construct inserted into the genome of the therapeutic myeloid cell. The construct may be inserted into the genome into a particular locus. The locus may comprise for example the TRAC locus, the B2M locus, the CD52 locus, or the AAVS1 locus. The construct may encode a CD19 specific CAR. The construct encoding the CD19 specific CAR may be inserted into the -63-WSGR Docket No.61057-724.601 B2M locus. The insertion of the CD19 CAR into the B2M locus may result in a disruption of the B2M locus. The disruption may be bi-allelic.

[0281] The therapeutic myeloid cell may be engineered to comprise a construct. The construct may be inserted into the genome. The construct may comprise a DNA sequence. The construct may comprise a B2M-HLA-E construct of the present disclosure. The construct may be inserted into a particular locus. The construct may be inserted for example into the TRAC locus, the CD52 locus, or the AAVS1 locus. The B2M-HLA-E construct may be inserted into the B2M locus. The insertion of the B2M-HLA-E construct into the B2M locus may result in a disruption of the B2M locus. The disruption may comprise a knockout.

[0282] In some embodiments, the therapeutic myeloid cell may comprise a cytokine construct having persistent expression. In some embodiments, the cytokine may comprise an interleukin. In some embodiments, the interleukin may comprise an interleukin-12 (IL-12). In some embodiments, the IL-12 may comprise a dimer. In some embodiments, the dimer may comprise an IL-12 alpha and / or an IL-12 beta. In some embodiments, the IL-12 dimer may comprise an IL- 12p70 dimer. In some embodiments, the IL-12 dimer may be encoded by a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 59 or SEQ ID NO: 61. In some embodiments, the IL-12 dimer may comprise a peptide linker. In some embodiments, the peptide linker may comprise a flexible linker. In some embodiments, the flexible linker may comprise a bovine elastin motif (BEM) linker. In some embodiments, the IL- 12 dimer may be encoded by a sequence comprising an engineered chromatin opening sequence (ECOS). In some embodiments, the ECOS may comprise ECOS-63. In some embodiments, the ECOS-63 may comprise a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the therapeutic myeloid cell may comprise a therapeutic macrophage. In some embodiments, the therapeutic macrophage may be differentiated from a stem cell. In some embodiments, the stem cell is contacted with a nucleic acid sequence comprising a repair template. In some embodiments, the repair template may comprise an ECOS- EF1a-IL12-hGHpA repair template. In some embodiments, the ECOS-EF1a-IL12-hGHpA repair template may comprise a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 58 or SEQ ID NO: 60. In some embodiments, the stem cell is contacted with a gene editing protein of certain embodiments of the present disclosure. In some embodiments, the stem cell may comprise an induced pluripotent stem cell (iPSC). In some embodiments, the therapeutic myeloid cell may comprise a disruption in a B2M locus in its genome. In some embodiments, the disruption may comprise a knockout. In some embodiments, the disruption may comprise insertion of a B2M-HLA-E construct. In some embodiments, the B2M-HLA-E construct may comprise a linker sequence. In some embodiments, the linker -64-WSGR Docket No.61057-724.601 sequence may comprise a (G4S)4 linker sequence. In some embodiments, the coding sequence for the (G4S)4linker sequence may have a GC content of at most 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, or 60%. In some embodiments, the cytokine construct may comprise a poly-adenylation signal. In some embodiments, the B2M-HLA-E construct may comprise a poly- adenylation signal. In some embodiments, any construct described herein may comprise a poly- adenylation signal. In some embodiments, the poly-adenylation signal may comprise a human growth hormone poly-adenylation (hGHpA) signal.

[0283] The constructs of the present disclosure, including but not limited to constructs encoding one or more transgenes, may comprise a linker sequence. The linker sequence may comprise a peptide linker. The linker sequence may comprise a motif. The motif may comprise glycine residues and serine residues. The motif may comprise a (G4S)4 motif. The linker sequence may comprise a self-cleaving linker. The self-cleaving linker may comprise a P2A linker. The self- cleaving linker may comprise an E2A linker. The linker sequence may comprise a flexible linker. The flexible linker may comprise a bovine elastin motif (BEM) linker.

[0284] The constructs of the present disclosure, including but not limited to constructs encoding one or more transgenes, may encode a poly-adenylation signal. The poly-adenylation signal may comprise but is not limited to polyadenylation signal 1 (PAS1), PAS2, PAS3, SV40 late polyadenylation signal, bovine growth hormone (BGH) hormone polyadenylation signal, human growth hormone polyadenylation (hGHpA) signal, beta-globin polyadenylation signal, TK polyadenylation signal, CMV polyadenylation signal, or an alternative PAS.

[0285] The therapeutic myeloid cells may be engineered to comprise a disruption in the genome. The disruption in the genome may comprise a knockout. The disruption in the genome may comprise the insertion of a construct. The disruption in the genome may be located at for example the B2M locus, the CD52 locus, the TRAC locus, the TRBC locus, the CCR5 locus, the AAVS1 locus, the ROSA26 locus, the PDCD1 (PD-1) locus, or the IL2RA locus. The disruption in the genome may be located at more than one locus. The disruption in the genome may be located at the TRAC locus and the CD52 locus. The disruption in the genome may be generated by a gene- editing protein of certain embodiments of the present disclosure.

[0286] The therapeutic myeloid cells may be engineered for persistent expression of a transgene. The persistent expression may be driven by an engineered chromatin opening sequence (ECOS) of the present disclosure. The ECOS may comprise ECOS-63. The ECOS may be comprise a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 28. The ECOS may comprise a sequence having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, -65-WSGR Docket No.61057-724.601 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 28-32. The therapeutic myeloid cells may comprise an ECOS in the genome which drives persistent expression of a transgene. The transgene may comprise a cytokine. The cytokine may comprise an interleukin. The interleukin may comprise IL-12.

[0287] The therapeutic myeloid cells of the present disclosure may be engineered to comprise a construct. The construct may encode a sequence that is inserted into the genome. The construct may comprise a repair template. The construct may comprise an ECOS-EF1a-IL12-hGHpA construct.

[0288] In some embodiments, when co-cultured with peripheral blood mononuclear cell (PBMC)- derived macrophages, the therapeutic myeloid cells of the present disclosure may phagocytose 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% more SK-OV-3 cancer cells as compared to a PBMC-derived macrophages. In some embodiments, the therapeutic myeloid cell may infiltrate a spheroid solid tumor model comprising SK-OV-3 cells when contacted with the spheroid tumor model by 100 µm, 150 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900 µm, 950 µm, or 1000 µm. In some embodiments, the therapeutic myeloid cell may induce 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% less CD69 upregulation in CD4+ or CD8+ T cells, when co-incubated with the CD4+ or CD8+ T cells and a wildtype macrophage, as compared to the wildtype macrophage.

[0289] Provided herein are methods and compositions for contacting a tumor tissue with an engineered cell of the present disclosure. In some embodiments, the contacting results in cell killing. In some embodiments, the cell killing comprises killing of cancer cells. In some embodiments, the cancer cells comprise SK-OV-3 cells. In some embodiments, the cancer cells may comprise but are not limited to cancer cells from any one of the following cell lines: HeLa, MCF-7, A549, PC3, U87, HCT116, SKOV3, K562, Jurkat, BT-474, MDA-MB-231, T47D, HL- 60, SW480, Caco-2, DU145, H1299, SH-SY5Y, NCI-H460, RPMI-8226, LNCaP, THP-1, HepG2, HEP3B, BxPC-3, PANC-1, Calu-3, A2780, NB4, and Hs578T. Provided herein are methods and compositions for treating a disease that may comprise but is not limited to cancer. In some embodiments, the cancer may comprise one or more of the following: basal cell carcinoma; biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; -66-WSGR Docket No.61057-724.601 larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and sarcomas; and post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with phakomatoses, edema, and Meigs’ syndrome. Chimeric Antigen Receptor (CAR)-Bearing Immune Cells

[0290] In embodiments, the present immune cells (e.g., cells that are gene-edited and reprogrammed into an immune cell) are engineered with chimeric antigen receptors (CARs), e.g., the present immune cells are CAR-NK cells or CAR-T.

[0291] In embodiments, the immune cell, optionally NK cell or T cell, is genetically modified to express a recombinant chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain comprising an antigen binding region. In embodiments, the intracellular signaling domain comprises at least one immunoreceptor tyrosine based activation motif (ITAM)-containing domain.

[0292] In embodiments, the intracellular signaling domain is from one of CD3-zeta, CD28, CD27, CD134 (OX40), and CD137 (4‐1BB).

[0293] In embodiments, the transmembrane domain is from one of CD28 or a CD8.

[0294] In embodiments, the antigen binding region binds one antigen. In embodiments, the binding region binds two antigens.

[0295] In embodiments, the extracellular domain comprising an antigen binding region comprises: (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv). In embodiments, the extracellular domain comprising an antigen binding region comprises two of (a) a natural ligand or receptor, or fragment thereof, or (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv). In embodiments, the extracellular domain comprising an antigen binding region comprises -67-WSGR Docket No.61057-724.601 one of each of: (a) a natural ligand or receptor, or fragment thereof, and (b) an immunoglobulin domain, optionally a single-chain variable fragment (scFv).

[0296] In embodiments, the antigen binding region binds a tumor antigen.

[0297] In embodiments, the antigen binding region comprises one or more of: (i) CD94 / NKG2a, which optionally binds HLA-E on a tumor cell; (ii) CD96, which optionally binds CD155 on a tumor cell; (iii) TIGIT, which optionally binds CD155 or CD112 on a tumor cell; (iv) DNAM-1, which optionally binds CD155 or CD112 on a tumor cell; (v) KIR, which optionally binds HLA class I on a tumor cell; (vi) NKG2D, which optionally binds NKG2D-L on a tumor cell; (vii) CD16 (e.g., CD16a or CD16b), which optionally binds an antibody / antigen complex on a tumor cell and / or wherein the CD16a is optionally a high affinity variant, optionally homozygous or heterozygous for F158V; (viii) NKp30, which optionally binds B7-H6 on a tumor cell; (ix) NKp44; and (x) NKp46.

[0298] In embodiments, the antigen binding region comprises an immunoglobulin domain, optionally an scFv directed against HLA-E, CD155, CD112 HLA class I, NKG2D-L, or B7-H6, as well as any variant thereof.

[0299] In embodiments, the antigen binding region binds an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, as well as any variant thereof. In embodiments, an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, as well as any variant thereof can be used as a single-target CAR, dual-target CAR, mAb, or any combination of any of those

[0300] In embodiments, the antigen binding region binds two antigen, the antigens being: (a) an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, -68-WSGR Docket No.61057-724.601 Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2, as well as any variant thereof and (b) an antigen selected from AFP, APRIL, BCMA, CD123 / IL3Ra, CD133, CD135 / FLT3, CD138, CD147, CD19, CD20, CD22, CD239 (BCAM), CD276 (B7-H3), CD30, CD314 / NKG2D, CD319 / CS1 / SLAMF7, CD326 / EPCAM / TROP1, CD37, CD38, CD44v6, CD5, CD7, CD70, CLDN18.2, CLDN6, cMET, EGFRvIII, EPHA2, FAP, FR alpha, GD2, GPC3, IL13Ralpha2, Integrin B7, Lewis Y (LeY), MESO, MG7 antigen, MUC1, NECTIN4, NKG2DL, PSCA, PSMA / FOL1, ROBO1, ROR1, ROR2, TNFRSF13B / TACI, TRBC1, TRBC2, and TROP 2, as well as any variant thereof.

[0301] In embodiments, the antigen binding region binds two antigen, the antigens being: (a) an antigen selected from CD16, CD64, CD78, CD96,CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvlll), CD19, CD20, CD30, CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma- associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-l, IGF-I I, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, RORl, Nkp30, N KG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (FAP); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7- 1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphoblastic leukemia antigen, such as one selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5, a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, as well as any variant thereof and (b) an antigen selected from CD16, CD64, CD78, CD96,CLL1, CD116, CD117, CD71, CD45, CD71, CD123 and CD138, a tumor-associated surface antigen, such as ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant I II (EGFRvl ll), CD19, CD20, CD30, -69-WSGR Docket No.61057-724.601 CD40, disialoganglioside GD2, ductal-epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin- reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, hsp70-2, M-CSF, prostase, prostase specific antigen (PSA), PAP, NY-ESO-1, LAGA-la, p53, prostein, PSMA, surviving and telomerase, prostate- carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGFl)-l, IGF-I I, IGFI receptor, mesothelin, a major histocompatibility complex (MHC) molecule presenting a tumor-specific peptide epitope, 5T4, RORl, Nkp30, N KG2D, tumor stromal antigens, the extra domain A (EDA) and extra domain B (EDB) of fibronectin and the Al domain of tenascin-C (TnC Al) and fibroblast associated protein (FAP); a lineage-specific or tissue specific antigen such as CD3, CD4, CD8, CD24, CD25, CD33, CD34, CD133, CD138, CTLA-4, B7- 1 (CD80), B7-2 (CD86), GM-CSF, cytokine receptors, endoglin, a major histocompatibility complex (MHC) molecule, BCMA (CD269, TNFRSF 17), multiple myeloma or lymphoblastic leukemia antigen, such as one selected from TNFRSF17, SLAMF7, GPRC5D, FKBP11, KAMP3, ITGA8, and FCRL5, a virus-specific surface antigen such as an HIV-specific antigen (such as HIV gpl20); an EBV-specific antigen, a CMV-specific antigen, a HPV-specific antigen, a Lasse Virus-specific antigen, an Influenza Virus-specific antigen, as well as any variant thereof.

[0302] In embodiments, the extracellular domain of the recombinant CAR comprises the extracellular domain of an NK cell activating receptor or a scFv.

[0303] In embodiments, the NK cell comprises a gene-edit in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1.

[0304] In embodiments, the gene-edit in one or more of IL-7, CCL17, CCR4, IL-6, IL-6R, IL-12, IL-15, NKG2A, NKG2D, KIR, TRAIL, TRAC, PD1, and HPK1 is caused by contacting the cell with RNA encoding one or more gene-editing proteins. In embodiments, the gene-edit of causes a reduction or elimination of expression and / or activity of IL-6, NKG2A, NKG2D, KIR, TRAC, PD1, and / or HPK1. In embodiments, the gene-edit causes an increase of expression and / or activity of IL-7, CCL17, CCR4, IL-6R, IL-12, IL-15, and / or TRAIL.

[0305] In embodiments, the immune cell, e.g., a T cell, NK cell, or macrophage, further comprises one or more recombinant genes capable of encoding a suicide gene product. In embodiments, the suicide gene product comprises a protein selected from the group consisting of thymidine kinase and an apoptotic signaling protein.

[0306] Any immune cell disclosed herein (e.g., comprising a gene edit (e.g., in B2M), and / or expressing a fusion protein comprising B2M polypeptide and an HLA polypeptide) can be further genetically engineered to express a CAR. -70-WSGR Docket No.61057-724.601 Methods of In Vivo Engineering of Myeloid Cells in Tumor Microenvironment

[0307] Provided herein are methods and compositions for in vivo engineering of myeloid cells in a tumor microenvironment. Myeloid cells within a tumor microenvironment may be engineered via contact with a therapeutic payload. The therapeutic payload may comprise a nucleic acid molecule. The therapeutic payload may be comprised within or encoded by a nucleic acid molecule. The nucleic acid molecule may comprise an RNA or a DNA molecule. The RNA molecule may comprise a synthetic RNA molecule. The RNA molecule may comprise a messenger RNA (mRNA). The DNA molecule may comprise a ssDNA or a dsDNA molecule. The therapeutic payload may be delivered before, after, or simultaneously with a synthetic RNA molecule. The synthetic RNA molecule may encode a gene-editing protein of the present disclosure. The myeloid cell of the present disclosure may comprise a macrophage, a monocyte, a dendritic cell, or a granulocyte. Also provided herein are methods and compositions for generating an engineered immune cell, wherein the engineered immune cell may comprise a myeloid cell.

[0308] In some embodiments, the myeloid cell may comprise a disruption in its genome. In some embodiments, the disruption may occur at a TRAC locus, a CD52 locus, an AAVS1 locus, and / or a ROSA26 locus. In some embodiments, the disruption may occur at more than one locus. In some embodiments, the disruption may occur at a TRAC locus and a CD52 locus. In some embodiments, the disruption may occur at the TRAC locus and the CD52 locus simultaneously.

[0309] The nucleic acid molecule may encode a cytokine. The cytokine may comprise an interleukin such as for example IL-12 and IL-4 or an interferon such as for example interferon alpha. The nucleic acid molecule may comprise an engineered chromatin opening sequence (ECOS). The ECOS may comprise a CBX3-ECOS, an A2ECOS, a SRF-ECOS, an ECOS-455, an ECOS-63, or a biologically active fragment thereof. The nucleic acid molecule may comprise a promoter such as elongation factor-1 alpha (Ef1α) promoter, a synthesis friendly constitutive (SFC) promoter, a phosphoglycerate kinase (PGK) promoter, a cytomegalovirus (CMV) promoter, a spleen focus forming virus (SFFV) promoter, a JeT promoter, an miR223 promoter, an H1 promoter, a SP146 promoter, or a biologically active fragment thereof. The nucleic acid molecule may encode a poly-adenylation signal such as polyadenylation signal 1 (PAS1), PAS2, PAS3, SV40 late polyadenylation signal, bovine growth hormone (BGH) hormone polyadenylation signal, human growth hormone polyadenylation (hGHpA) signal, beta-globin polyadenylation signal, TK polyadenylation signal, CMV polyadenylation signal, or an alternative PAS.

[0310] In some embodiments, the nucleic acid molecule may comprise a sequence encoding a CAR. In some embodiments, the CAR may specifically recognize CD19. In some embodiments, -71-WSGR Docket No.61057-724.601 the nucleic acid molecule may comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NO: 55-57.

[0311] In some embodiments, the nucleic acid molecule may comprise a repair template. In some embodiments, the repair template may comprise a sequence encoding epidermal growth factor receptor (EGFR). In some embodiments, the repair template may comprise a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NO: 52- 54.

[0312] The nucleic acid molecule may be delivered via a viral vector. The nucleic acid molecule may be delivered via a plasmid vector. The delivery may comprise an injection. The delivery may further comprise delivery of a synthetic RNA molecule. The injection may comprise a direct intratumoral injection. The nucleic acid molecule and synthetic RNA molecule may contact a myeloid cell in vivo. The contacting may result in the myeloid cell internalizing the nucleic acid molecule and synthetic RNA molecule. The contacting may result in the myeloid cell overexpressing the therapeutic payload. The overexpression of the therapeutic payload may cause the myeloid cell to exhibit enhanced cancer cell killing. The overexpression of the therapeutic payload may cause the myeloid cell to more efficiently infiltrate a solid tumor. In some embodiments, the myeloid cell may lyse at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more RAJI cancer cells as compared to an immune cell which does not comprise the nucleic acid molecule when co-cultured with the RAJI cancer cells. Methods of Treatment

[0313] An aspect of the present disclosure is a pharmaceutical composition comprising a therapeutically-effective amount of any of the cells described herein of any herein-disclosed aspect or embodiment and a pharmaceutically-acceptable excipient. In many embodiments, the cells may be clonally related.

[0314] In various embodiments, the present methods and compositions find use in methods of treating, preventing, or ameliorating a disease, disorder, and / or condition. For instance, in some embodiments, the described methods of in vivo delivery, including administration strategies, and formulations are used in a method of treatment. In some methods, the described methods reduce symptoms associated with a disease. In some embodiments, the methods eliminate the underlying cause of the disease.

[0315] Pharmaceutical compositions of the present disclosure may be used to treat a disease, condition, or disorder. The disease, condition, or disorder may comprise a metabolic disease, a proliferative disease, an autoimmune disease, a neurological disease, or any combination thereof.

[0316] In various cases, the disease or disorder is any autoimmune disease or inflammatory disease. In numerous cases, the disease or disorder is Duchenne muscular dystrophy (DMD). In -72-WSGR Docket No.61057-724.601 various cases, the disease or disorder is acute graft-versus-host disease (aGvHD), acute myocardial infarction, acute respiratory distress syndrome (ARDS), alcoholic liver cirrhosis, amyotrophic lateral sclerosis (ALS), anal fistula, Alzheimer's disease, asthma, atherosclerosis, autism nonischemic heart failure, coronary artery disease, critical limb ischemia, Crohn’s disease, chronic traumatic encephalopathy, chronic obstructive pulmonary disease (COPD), degenerative disc disease, dementia, diabetes, encephalitis of viral, bacterial or autoimmune origin, endometriosis, epilepsy, heart arrhythmia such as atrial fibrillation (AF), heart disease, hepatitis C virus (HCV)-induced cirrhosis, Huntington's Disease, idiopathic pulmonary fibrosis, Inflammatory Bowel Disease (IBD), insulin resistance, ischemic cardiomyopathy, Keratoconus, kidney allograft, Lysosomal storage diseases, malaria with CNS degeneration, multiple sclerosis (MS), multiple system atrophy, neuro-AIDS, Niemann Pick disease, obesity, obesity-related complications, osteoarthritis (OA), osteoarthritis of the knee, osteoporosis, Parkinson's Disease, preterm bronchopulmonary dysplasia (BPD), progressive supranuclear palsy, prostatitis, renal failure, spinal cord injury, spinal cord injury, systemic lupus erythematosus (SLE), Tay-Sachs Disease, and / or Type 2 diabetes mellitus.

[0317] In some embodiments, the disease, condition, or disorder is cancer. In embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a solid tumor. In various embodiments, the cancer is selected from basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma (e.g., Kaposi’s sarcoma); skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; lymphoma including Hodgkin's and non- Hodgkin's lymphoma, as well as B-cell lymphoma (including low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; as well as other carcinomas and -73-WSGR Docket No.61057-724.601 sarcomas; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (e.g. that associated with brain tumors), and Meigs' syndrome.

[0318] In embodiments, the present methods relate to therapeutic use in autoimmune diseases or disorders. Examples of autoimmune diseases or disorders that may be treated or prevented by the present invention include, but are not limited to, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain- Barre syndrome, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), irritable bowel disease (IBD), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, Rheumatoid arthritis, sarcoidosis, scleroderma, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis, giant cell arteritis, ulcerative colitis, uveitis, vitiligo and Wegener's granulomatosis. Administration and Formulations

[0319] In some embodiments, the present disclosure relates to compositions described herein in the form of a pharmaceutical composition.

[0320] Therapeutic treatments comprise the use of one or more routes of administration and of one or more formulations that are designed to achieve a therapeutic effect at an effective dose, while minimizing toxicity to the subject to which treatment is administered. Illustrative formulations / compositions of the present disclosure include engineered cells along with a suitable delivery reagent, e.g., a liquid carrier.

[0321] Upon formulation, solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective, as described herein. The formulations may easily be administered in a variety of dosage forms such as injectable solutions and the like. For parenteral administration in an aqueous solution, for example, the solution generally is suitably -74-WSGR Docket No.61057-724.601 buffered, and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used, for example, for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Preferably, sterile aqueous media are employed as is known to those of skill in the art.

[0322] Pharmaceutical preparations may additionally comprise delivery reagents (a.k.a. “vehicles”, a.k.a. “delivery vehicles”) and / or excipients. Pharmaceutically acceptable delivery reagents, excipients, and methods of preparation and use thereof, including methods for preparing and administering pharmaceutical preparations to patients (a.k.a. “subjects”) are well known in the art, and are set forth in numerous publications, including, for example, in US Patent Appl. Pub. No. US 2008 / 0213377, the entirety of which is incorporated herein by reference.

[0323] Pharmaceutical compositions of the present disclosure can comprise excipients, including liquids such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0324] In embodiments, the composition is formulated for one or more of intrathecal, intra-lesional, intra-coronary, intravenous (IV), intra-articular, intramuscular, and intra-endobronchial administration and administration via intrapancreatic endovascular injection, intra-nucleus pulposus, lumbar puncture, intra-myocardium, transendocardium, intra-fistula tract, intermedullary space, intradural space and leg injection.

[0325] In embodiments, the composition is formulated for infusion. In some embodiments, the composition is formulated for infusion, wherein the composition is delivered to the bloodstream of a subject or patient through a needle in a vein of the subject or patient through a peripheral line, a central line, a tunneled line, an implantable port, and / or a catheter. In some embodiments, the subject or patient may also receive supportive medications or treatments, such as hydration, by infusion. In some embodiments, the composition is formulated for intravenous infusion. In some embodiments, the infusion is continuous infusion, secondary intravenous therapy (IV), and / or IV push. In some embodiments, the infusion of the composition may be administered through the use of equipment selected from one or more of an infusion pump, hypodermic needle, drip chamber, peripheral cannula, and pressure bag. -75-WSGR Docket No.61057-724.601

[0326] In embodiments, the method of treating a subject comprises administering a cell of the present disclosure to a subject in need thereof. In embodiments, the cell is formulated for therapeutic use. In embodiments, the cell is suitable for administration to a human subject. In embodiments, the method is conducted in vivo.

[0327] In numerous embodiments, the administering is intravenous, or intraarterial.

[0328] In some embodiments, the present invention relates to one or more administration techniques described in US Patent Nos.5,711,964; 5,891,468; 6,316,260; 6,413,544; 6,770,291; and 7,390,780, the entire contents of which are hereby incorporated by reference in their entireties. Lipids / Cell Contacting / Transfection

[0329] In embodiments, the present invention relates delivery mRNAs encoding a gene-editing protein and / or a reprogramming factor, as disclosed elsewhere herein, are delivered to a cell via a lipid.

[0330] In embodiments, the lipid is a compound of Formula (I)wherein: Q1, Q2, Q3, and Q4 are independently an atom or group capable of adopting a positive charge; A1 and A2 are independently null, H, or optionally substituted C1-C6 alkyl; L1, L2, and L3 are independently null, a bond, (C1-C20)alkanediyl, (halo)(C1-C20)alkanediyl, (hydroxy)(C1-C20)alkanediyl, (alkoxy)(C1-C20)alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, or any combination of the aforementioned optionally linked by one or more of an ether, an ester, an anhydride, an amide, a carbamate, a secondary amine, a tertiary amine, a quaternary ammonium, a thioether, a urea, a carbonyl, or an imine; R1, R2, R3, R4, R5, R6, R7, and R8 are independently null, H, (C1-C60)alkyl, (halo)(C1-C60)alkyl, (hydroxy)(C1-C60)alkyl, (alkoxy)(C1-C60)alkyl, (C2-C60)alkenyl, (halo)(C2-C60)alkenyl, (hydroxy)(C2- C60)alkenyl, (alkoxy)(C2-C60)alkenyl, (C2-C60)alkynyl, (halo)(C2-C60)alkynyl, (hydroxy)(C2- C60)alkynyl, (alkoxy)(C2-C60)alkynyl, wherein at least one of R1, R2, R3, R4, R5, R6, R7, and R8 comprises at least two unsaturated bonds; and x, y, and z are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0331] In embodiments, the lipid is a compound of Formula (A): -76-WSGR Docket No.61057-724.601where R21, R23, and R24are independently selected from hydrogen, C1-6 alkyl, C1-6 haloalkyl, C1-6 hydroxyalkyl, C1-6 alkoxyalkyl, C5-10 aryl, 5- to 10-membered heteroaryl, or C3-6 cycloalkyl; n is 1 to 20; m is 6, 7, 8, 9, or 10; p is 1, 2, 3, or 4; q is 1, 2, 3, 4, or 5; and r is 1, 2, 3, 4, 5, or 6.

[0332] In embodiments, the lipid is a compound of Formula (II):wherein: R9, R10, R11, R12, R13, R14, R15, R16, R17, R18, R19, R20, R21, R22, R23, R24, R25, R26, R27, and R28 are independently H, halo, OH, (C1-C6)alkyl, (halo)(C1-C6)alkyl, (hydroxy)(C1-C6)alkyl, (alkoxy)(C1-C6)alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclo; and i, j, k, m, s, and t are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0333] In embodiments, the lipid is a compound of Formula (III):wherein L4, L5, L6, and L7 are independently a bond, (C1-C20)alkanediyl, (halo)(C1- C20)alkanediyl, (hydroxy)(C1-C20)alkanediyl, (alkoxy)(C1-C20)alkanediyl, arylene, heteroarylene, cycloalkanediyl, heterocycle-diyl, -(CH2)v1-C(O)-, –((CH2)v1-O)v2-, or –((CH2)v1-C(O)-O)v2-; R29, R30, R31, R32, R33, R34, and R35 are independently H, (C1-C60)alkyl, (halo)(C1-C60)alkyl, -77-WSGR Docket No.61057-724.601 (hydroxy)(C1-C60)alkyl, (alkoxy)(C1-C60)alkyl, (C2-C60)alkenyl, (halo)(C2-C60)alkenyl, (hydroxy)(C2- C60)alkenyl, (alkoxy)(C2-C60)alkenyl, (C2-C60)alkynyl, (halo)(C2-C60)alkynyl, (hydroxy)(C2- C60)alkynyl, (alkoxy)(C2-C60)alkynyl, wherein at least one of R29, R30, R31, R32, R33, R34, and R35 comprises at least two unsaturated bonds; v, v1 and v2 are independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0334] In embodiments, the lipid is a compound of Formula (IV):wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0335] In embodiments, the lipid is a compound of Formula (V):

[0336] In embodiments, the lipid is a compound of Formula (VI):-78-WSGR Docket No.61057-724.601

[0337] In embodiments, the lipid is a compound of Formula (VII):

[0338] In embodiments, the lipid is a compound of Formula (VIII):

[0339] In embodiments, the lipid is a compound of Formula (IX):

[0340] In embodiments, the lipid is a compound of Formula (X):-79-WSGR Docket No.61057-724.601

[0341] In embodiments, the lipid is a compound of Formula (XI):

[0342] In embodiments, the lipid is a compound of Formula (XII):-80-WSGR Docket No.61057-724.601

[0343] In embodiments, the lipid is a compound of Formula (XIII):

[0344] In embodiments, the lipid is a compound of Formula (XIV):

[0345] In embodiments, the lipid is a compound of Formula (XV):wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. -81-WSGR Docket No.61057-724.601

[0346] In embodiments, the lipid is a compound of Formula (XVI):

[0347] In embodiments, the present compounds (e.g., of Formulae I-XVI) are components of a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier and / or a lipid nucleic-acid complex and / or a liposome and / or a lipid nanoparticle.

[0348] In embodiments, the present compounds (e.g., of Formulae I-XVI) are components of a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier and / or a lipid nucleic-acid complex and / or a liposome and / or a lipid nanoparticle which does not require an additional or helper lipid. In embodiments, the present compounds (e.g., of Formulae I-XVI) are components of a pharmaceutical composition and / or a lipid aggregate and / or a lipid carrier and / or a lipid nucleic-acid complex and / or a liposome and / or a lipid nanoparticle that further comprises a neutral lipid (e.g., dioleoylphosphatidylethanolamine (DOPE), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), or cholesterol) and / or a further cationic lipid (e.g., N-[1-(2,3-dioleoyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), 1,2-bis(oleoyloxy)-3-3-(trimethylammonium) propane (DOTAP), or 1,2-dioleoyl-3-dimethylammonium-propane (DODAP)).

[0349] In embodiments, the lipid is any of those described in International Patent Publication No. WO2021 / 003462, hereby incorporated by reference in its entirety.

[0350] In embodiments, the lipid is any of those of Table A. Table A. Illustrative Biocompatible Lipids and Polymers-82-WSGR Docket No.61057-724.601-83-WSGR Docket No.61057-724.601Protein Expression and / or Secretion Signature

[0351] Each type of cell expresses particular sets of proteins, within the cell, on the cell’s surface, and secreted into the extracellular space. The particular sets of proteins that each type of cell expresses depends on the general and immediate function of the cell. Protein expression is correlated with mRNA levels and thus can be assayed by methods that analyze the distribution, amount, and identity of particular mRNAs within a cell. There are several methods of quantitatively measuring mRNA, including northern blotting and reverse transcription-quantitative PCR (RT-qPCR). Hybridization microarrays may also be used to generate expression profiles or high-throughput analyses of a range of genes within a cell. Further, ‘tag based’ technologies, such as Serial analysis of gene expression (SAGE) and RNA-Seq can be used to determine the relative measure of the cellular concentration of different mRNAs.

[0352] In some embodiments, protein expression of specific cells is determined by determining concentration of different mRNAs by one or more of northern blotting, RT-qPCR, hybridization microarrays, and tag-based technologies, such as SAGE and RNA-Seq.

[0353] There are generally two strategies used for detection of proteins in the extracellular milieu: direct methods and indirect methods. The direct method comprises a one-step staining, and may involve a labeled antibody (e.g., FITC conjugated antiserum) reacting directly with the protein in the extracellular milieu. The indirect method comprises an unlabeled primary antibody that reacts with the protein in the extracellular milieu, and a labeled secondary antibody that reacts with the primary -84-WSGR Docket No.61057-724.601 antibody. Labels can include radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Methods of conducting these assays are well known in the art. See, e.g., Harlow et al. (Antibodies, Cold Spring Harbor Laboratory, NY, 1988), Harlow et al. (Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, NY, 1999), Virella (Medical Immunology, 6th edition, Informa HealthCare, New York, 2007), and Diamandis et al. (Immunoassays, Academic Press, Inc., New York, 1996). Kits for conducting these assays are commercially available from, for example, Clontech Laboratories, LLC. (Mountain View, CA). In some embodiments, proteins are detected in the extracellular milieu of monocytes or macrophages using detection methods comprising one or more antibodies. In some embodiments, the detection methods further comprise labels, including radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase.

[0354] In some embodiments, flow cytometry is used to determine whether cells express certain sets of proteins that are on the surface or that are secreted. In some embodiments, antibodies specific to particular proteins are used in combination with proteomic approaches to determine, e.g., the protein secretion signature of a particular cell. In some embodiments, the supernatant of a purified set of cell types is assayed using a Western blot to determine the concentrations of an array of secreted proteins, to which antibodies are available. In some embodiments, the protein secretion signatures of specific cell derived from different sources, such as iPSCs, skin cells, or bone marrow are determined and compared. DEFINITIONS

[0355] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting.

[0356] As used herein, “a,” “an,” or “the” can mean one or more than one.

[0357] Herein the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Further, the term “about” when used in connection with a referenced numeric -85-WSGR Docket No.61057-724.601 indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50” covers the range of 45 to 55. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0358] As used herein, the phrases “at least one”, “one or more”, and “and / or” are open-ended expressions that are both conjunctive and disjunctive in operation. For example, each of the expressions “at least one of A, B and C”, “at least one of A, B, or C”, “one or more of A, B, and C”, “one or more of A, B, or C” and “A, B, and / or C” mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.

[0359] As used herein, “or” may refer to “and”, “or,” or “and / or” and may be used both exclusively and inclusively. For example, the term “A or B” may refer to “A or B”, “A but not B”, “B but not A”, and “A and B”. In some cases, context may dictate a particular meaning.

[0360] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0361] As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0362] The terms “comprise”, “comprising”, “contain,” “containing,” “including”, “includes”, “having”, “has”, “with”, or variants thereof as used in either the present disclosure and / or in the claims, are intended to be inclusive in a manner similar to the term “comprising.” Although the open-ended term “comprising” is used herein to describe and claim the invention, the present invention, or embodiments thereof, may alternatively be described using alternative terms such as -86-WSGR Docket No.61057-724.601 “consisting of” or “consisting essentially of.”

[0363] The term “substantially” is meant to be a significant extent, for the most part; or essentially. In other words, the term substantially may mean nearly exact to the desired attribute or slightly different from the exact attribute. Substantially may be indistinguishable from the desired attribute. Substantially may be distinguishable from the desired attribute but the difference is unimportant or negligible.

[0364] The terms “increased”, “increasing”, or “increase” are used herein to generally mean an increase by a statically significant amount relative to a reference level. In some aspects, the terms “increased,” or “increase,” mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level. Other examples of “increase” include an increase of at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold or more as compared to a reference level.

[0365] The terms “decreased”, “decreasing”, or “decrease” are used herein generally to mean a decrease in a value relative to a reference level. In some aspects, “decreased” or “decrease” means a reduction by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level.

[0366] As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology.

[0367] The term “in vivo” refers to an event that takes place in a subject’s body.

[0368] The term “ex vivo” refers to an event which involves treating or performing a procedure on a cell, tissue and / or organ which has been removed from a subject’s body. Aptly, the cell, tissue and / or organ may be returned to the subject’s body in a method of treatment or surgery.

[0369] By preventing is meant, at least, avoiding the occurrence of a disease and / or reducing the likelihood of acquiring the disease.

[0370] By treating is meant, at least, ameliorating or avoiding the effects of a disease, including reducing a sign or symptom of the disease. -87-WSGR Docket No.61057-724.601

[0371] As used herein, the term “variant” encompasses but is not limited to nucleic acids or proteins which comprise a nucleic acid or amino acid sequence which differs from the nucleic acid or amino acid sequence of a reference by way of one or more substitutions, deletions and / or additions at certain positions. The variant may comprise one or more conservative substitutions. Conservative substitutions may involve, e.g., the substitution of similarly charged or uncharged amino acids.

[0372] “Carrier” or “vehicle” as used herein refer to carrier materials suitable for drug administration. Carriers and vehicles useful herein include any such materials known in the art, e.g., any liquid, gel, solvent, liquid diluent, solubilizer, surfactant, lipid or the like, which is nontoxic, and which does not interact with other components of the composition in a deleterious manner.

[0373] The phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0374] The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” are intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients. The use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the invention is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.

[0375] By “synthetic RNA molecule” is meant an RNA molecule that is produced outside of a cell or that is produced inside of a cell using bioengineering, by way of non-limiting example, an RNA molecule that is produced in an in vitro-transcription reaction, an RNA molecule that is produced by direct chemical synthesis or an RNA molecule that is produced in a genetically- engineered E. coli cell.

[0376] By “medium” is meant a solvent or a solution comprising a solvent and a solute, by way of non-limiting example, Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM + 10% fetal bovine serum (FBS), saline or water.

[0377] By “transfection medium” is meant a medium that can be used for transfection, by way of non-limiting example, Dulbecco’s Modified Eagle’s Medium (DMEM), DMEM / F12, saline or -88-WSGR Docket No.61057-724.601 water.

[0378] By “Oct4 protein” is meant a protein that is encoded by the POU5F1 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Oct4 protein, mouse Oct4 protein, Oct1 protein, a protein encoded by POU5F1 pseudogene 2, a DNA-binding domain of Oct4 protein or an Oct4-GFP fusion protein. In some embodiments the Oct4 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 20, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 20. In some embodiments, the Oct4 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 20. Or in other embodiments, the Oct4 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 20.

[0379] MAGHLASDFAFSPPPGGGGDGPGGPEPGWVDPRTWLSFQGPPGGPGIGPGVGPG SEVWGIPPCPPPYEFCGGMAYCGPQVGVGLVPQGGLETSQPEGEAGVGVESNSDGASP EPCTVTPGAVKLEKEKLEQNPEESQDIKALQKELEQFAKLLKQKRITLGYTQADVGLTL GVLFGKVFSQTTICRFEALQLSFKNMCKLRPLLQKWVEEADNNENLQEICKAETLVQA RKRKRTSIENRVRGNLENLFLQCPKPTLQQISHIAQQLGLEKDVVRVWFCNRRQKGKRS SSDYAQREDFEAAGSPFSGGPVSFPLAPGPHFGTPGYGSPHFTALYSSVPFPEGEAFPPVS VTTLGSPMHSN (SEQ ID NO: 20)

[0380] By “Sox2 protein” is meant a protein that is encoded by the SOX2 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Sox2 protein, mouse Sox2 protein, a DNA-binding domain of Sox2 protein or a Sox2-GFP fusion protein. In some embodiments the Sox2 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 21, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 21. In some embodiments, the Sox2 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 21. Or in other embodiments, the Sox2 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 21.

[0381] MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVW SRGQRRKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYK YRPRRKTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMN GWSNGSYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNG SPTYSMSYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYL PGAEVPEPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHM (SEQ ID NO: 21) -89-WSGR Docket No.61057-724.601

[0382] By “Klf4 protein” is meant a protein that is encoded by the KLF4 gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human Klf4 protein, mouse Klf4 protein, a DNA-binding domain of Klf4 protein or a Klf4-GFP fusion protein. In some embodiments the Klf4 protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 22, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 22. In some embodiments, the Klf4 protein comprises an amino acid sequence having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 22. Or in other embodiments, the Klf4 protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 22.

[0383] QPPGESDMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPP VLPGRPYDLAAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTH PPESVAATVSSSASASSSSSPSSSGPASAPSTCSFTYPIRAGNDPGVAPGGTGGGLLYGRE SAPPPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLS APGSEYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSN GHRPAAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQM QPQVPPLHYQELMPPGSCMPEEPKPKRGRRSWPRKRTATHTCDYAGCGKTYTKSSHLK AHLRTHTGEKPYHCDWDGCGWKFARSDELTRHYRKHTGHRPFQCQKCDRAFSRSDHL ALHMKRHF (SEQ ID NO: 22)

[0384] By “c-Myc protein” is meant a protein that is encoded by the MYC gene, or a natural or engineered variant, family-member, orthologue, fragment or fusion construct thereof, by way of non-limiting example, human c-Myc protein, mouse c-Myc protein, l-Myc protein, c-Myc (T58A) protein, a DNA-binding domain of c-Myc protein or a c-Myc-GFP fusion protein. In some embodiments the c-Myc protein comprises an amino acid sequence that has at least 70% identity with SEQ ID NO: 23, or in other embodiments, at least 75%, 80%, 85%, 90%, or 95% identity with SEQ ID NO: 23. In some embodiments, the c-Myc protein comprises an amino acid having from 1 to 20 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 23. Or in other embodiments, the c-Myc protein comprises an amino acid sequence having from 1 to 15 or from 1 to 10 amino acid insertions, deletions, or substitutions (collectively) with respect to SEQ ID NO: 23.

[0385] MDFFRVVENQQPPATMPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQS ELQPPAPSEDIWKKFELLPTPPLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQL EMVTELLGGDMVNQSFICDPDDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKD SGSPNPARGHSVCSTSSLYLQDLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPS SDSLLSSTESSPQGSPEPLVLHEETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGS -90-WSGR Docket No.61057-724.601 PSAGGHSKPPHSPLVLKRCHVSTHQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNN RKCTSPRSSDTEENVKRRTHNVLERQRRNELKRSFFALRDQIPELENNEKAPKVVILKK ATAYILSVQAEEQKLISEEDLLRKRREQLKHKLEQLRNSCA (SEQ ID NO: 23)

[0386] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.-91-WSGR Docket No.61057-724.601-92-WSGR Docket No.61057-724.601-93-WSGR Docket No.61057-724.601-94-WSGR Docket No.61057-724.601-95-WSGR Docket No.61057-724.601-96-WSGR Docket No.61057-724.601-97-WSGR Docket No.61057-724.601-98-WSGR Docket No.61057-724.601-99-WSGR Docket No.61057-724.601-100-WSGR Docket No.61057-724.601-101-WSGR Docket No.61057-724.601-102-WSGR Docket No.61057-724.601-103-WSGR Docket No.61057-724.601-104-WSGR Docket No.61057-724.601-105-WSGR Docket No.61057-724.601-106-WSGR Docket No.61057-724.601-107-WSGR Docket No.61057-724.601-108-WSGR Docket No.61057-724.601-109-WSGR Docket No.61057-724.601-110-WSGR Docket No.61057-724.601-111-WSGR Docket No.61057-724.601-112-WSGR Docket No.61057-724.601-113-WSGR Docket No.61057-724.601-114-WSGR Docket No.61057-724.601-115-WSGR Docket No.61057-724.601-116-WSGR Docket No.61057-724.601-117-WSGR Docket No.61057-724.601-118-WSGR Docket No.61057-724.601-119-WSGR Docket No.61057-724.601-120-WSGR Docket No.61057-724.601-121-WSGR Docket No.61057-724.601-122-WSGR Docket No.61057-724.601-123-WSGR Docket No.61057-724.601-124-WSGR Docket No.61057-724.601-125-WSGR Docket No.61057-724.601-126-WSGR Docket No.61057-724.601-127-WSGR Docket No.61057-724.601-128-WSGR Docket No.61057-724.601 EXAMPLES Example 1: B2M-HLA-E fusion expression using novel linker sequence

[0387] In this Example, an mRNA-engineered iPSC line was developed using a gene editing protein according to some embodiments of the present disclosure to express a B2M-HLA-E fusion transgene in lieu of the endogenous B2M gene product to mimic native B2M expression (e.g., upregulation when exposed to proinflammatory stimuli). Using a gene editing protein according to some embodiments of the present disclosure, conditions were optimized for insertion of a flexible linker-containing HLA-E transgene upstream of the stop codon of the native B2M gene in mRNA-reprogrammed iPSCs. Specifically, a single-stranded DNA template encoding a (GGGGS)4 flexible linker and HLA-E flanked by 125-nt sequences homologous to the B2M locus was co-delivered to iPSCs along with a gene editing protein according to some embodiments of the present disclosure mRNA targeting exon 3 of the B2M gene. It was found that the sequence of the flexible linker was susceptible to hairpin formation, and initial experiments resulted in insertion of a truncated sequence. Two new linkers were then designed and tested with GC content reduced from 83.3% to 71.7% and 61.7%, which increased the theoretical Gibb’s free energy from -6.0 to -3.92 and 0.64, respectively. It was found that the modifications of the nucleic acid sequence encoding the linker led to no significant difference in the rate of insertion but successful insertion of the full repair template was observed when using the two templates with reduced GC content. These edited populations were plated as single cells to obtain clonal mRNA- reprogrammed iPSC lines that express B2M-(GGGGS)4-HLA-E fusion protein.

[0388] FIG. 1 shows a schematic overview of the differentiation of B2M-HLA-E expressing iPSCs.

[0389] Primary human dermal fibroblasts were reprogrammed with mRNA as described herein. 100,000 iPSCs were electroporated with mRNA encoding a gene editing protein according to some embodiments of the present disclosure targeting exon 3 in the B2M gene (600ng each of B2M_e3_USL23_20 / TTTCTCCACTGTCTTTTTCA and B2M_e3_USR32_20 / TTACCTCCATGATGCTGCTT) and 700ng of ssDNA repair template (CR032 or EB001 or EB002) using a Neon Electroporation system with 21140V / 20 ms pulses. Genomic DNA was isolated 5 days after electroporation, amplified with primer pair CAGGGAGCAGCAGCAGCACT / GCCATACCTGGGGCCATACACC and analyzed with the T7E1 to confirm editing in the bulk population. Single cells were isolated 8 days after electroporation and expanded as colonies for 12 days. Genomic DNA was isolated, amplified with the same primer pair and Sanger sequenced to confirm bi-allelic insertion.

[0390] The B2M-HLA-E fusion protein was constructed by inserting (G4S)4-HLA-E (without the -129-WSGR Docket No.61057-724.601 HLA-E signal peptide) immediately prior to the B2M stop codon. This was done by editing exon 3 of B2M. The normal B2M stop codon and poly adenylation signal will thus apply. The GC content of the (G4S)4 linker was 83.3%, 71.7%, or 61.7% GC, and it was found that lowering the GC content increased the likelihood of repair template integration.

[0391] FIG. 2 shows an EB002 B2M-HLA-E ssDNA repair template run on a gel either as a plasmid (lane 2) or purified (lane 3) compared to a 100 bp ladder (lane 1).

[0392] The EB002 repair template was electroporated into iPSCs.600 ng of purified PCR product was run per well and 6 µg total was used. FIG.3 shows EB002 PCR product in lane 2 compared to 100 bp ladder in lane 1. The insertion band was extracted and purified. Single cells were deposited and colonies subsequently screened. FIG.4 shows EB002 single colonies analyzed for insertion. gDNA was extracted before PCR. F11 and F13 sequences were verified, and full bi- allelic insertion was confirmed. F11 and F13 were further expanded.

[0393] FIGs.5 and 6 show flow cytometry data of HLA-E expression in F13 (FIG.5) and WT (FIG.6) cells after IFNγ stimulation. F13 cells showed 80-90 % expression compared to 10-16% expression in WT cells when stained with HLA-E antibodies.

[0394] In some embodiments, HLA-G is be used in place of HLA-E. In some embodiments, both B2M-HLA-G and B2M-HLA-E are introduced into the cells. Example 2: iPSC-Derived Macrophages Engineered to Express IL-12 Enhance Cancer Cell Killing of a Multi-Cell Type Therapeutic Platform

[0395] In this Example, iPSC-derived macrophages were engineered to overexpress IL-12 for delivery to the tumor microenvironment. Bi-allelic knockout of the beta-2-microglobulin gene (B2M- / -) was designed to reduce alloreactivity of transplanted iPSC-derived macrophages. B2M- / - iPSC-macrophages transfected with mRNA encoding both IL-12 subunits joined by a self- cleaving peptide (IL-12 P2A) or a flexible linker (IL-12 fusion) resulted in the production of 20 and 40 ng / mL of the IL-12p70 heterodimer, respectively, as assessed via ELISA 24 hours after transfection. Both IL-12 mRNA constructs resulted in similar levels of IFNγ production when co- cultured with PBMC-derived T cells (17 ng / mL for IL-12 fusion and 15 ng / mL for IL-12 P2A) with no detectable IFNγ in the absence of IL-12 mRNA or T cell co-culture, indicating functional bioactivity. The co-culture of B2M- / - iPSC-derived macrophages with allogeneic PBMC-derived T cells did not upregulate T-cell activation markers CD25 or CD69, as assessed via flow cytometry, suggesting a lack of host-vs-graft-type immunoreactivity of the B2M- / - iPSC-derived macrophages.

[0396] B2M-knockout (B2M- / -) iPSCs efficiently differentiate into macrophages that do not express MHC-I molecules while maintaining expression of MHC-II, preserving their ability to present antigens to T and B cells. When co-cultured with PBMC-derived T cells, wildtype iPSC- -130-WSGR Docket No.61057-724.601 derived macrophages, but not B2M- / - iPSC-derived macrophages, upregulate T cell early activation marker CD69 (2.52- and 0.88-fold, respectively), suggesting that the B2M- / - iPSC- derived macrophages do not cause T cell-mediated alloreactivity. B2M- / - iPSC-derived macrophages transfected with mRNA encoding the immunostimulatory cytokine IL-12 and co- cultured with PBMC-T cells showed greater cell lysis of MDA-MB-231 breast adenocarcinoma cells (12% vs 2%; p=0.01) and SK-OV-3 ovarian adenocarcinoma cells (62% vs.33%; p=0.03) compared to mock transfected macrophages. When co-cultured with IL-12 mRNA transfected B2M- / - iPSC-derived macrophages, ROR1-CAR T cells showed greater cell lysis of MDA-MB- 231 cells (46%) compared to the ROR1-CAR T cells alone (29%, p=0.001) or with mock transfected macrophages (39%, p=0.05).

[0397] The IL-12 family of cytokines (IL-12, IL-23, IL-27, and IL-35) are all heterodimers. The active IL-12 heterodimer IL-12p70 is composed of IL-12α and IL-12β subunits. Diagrams of the IL-12 fusion and Il-12 P2A constructs are shown in FIG.7.

[0398] THP-1 cells (1.5x106cells per well) were treated with 5ng / mL PMA transfection reagent for 48 hours and then transfected with 1.5 ug of mRNA complexed with ToRNAdo™. After 24 hours the supernatant was collected and analyzed using an IL-12p70 ELISA. As shown in FIG. 8, the IL-12 P2A variant shows approximately 2 fold more IL-12p70 in supernatant compared to the IL-12 fusion construct.

[0399] 1x106iPSC-Macrophages (iMacrophages) per well were transfected with 1 µg of mRNA using ToRNAdo™.4 hours later, 500k PBMC-T cells were added to the iMacrophage co-culture. 48 hours later, the supernatant was collected and analyzed using an IL-12p70 and IFNγ ELISA. As shown in FIG. 9, IL-12 P2A construct showed greater IL-12p70 concentration in the supernatant (p=0.0154). However, as shown in FIG.10, both constructs elicited comparable levels of IFNγ from the T cells (p=0.433).

[0400] When IL-12 mRNA transfected iPSC-derived macrophages were co-cultured with PBMC- derived T-cells and a GFP-expressing ovarian adenocarcinoma cell line SK-OV-3 at a 10:1 effector to target ratio for 72 hours, cancer cell killing was significantly greater when compared to non-transfected iPSC-derived macrophages, as assessed by real-time fluorescence imaging (p=0.03) and as shown in FIG.11.

[0401] FIG.12 shows a schematic of the co-culture of IL-12 B2M- / -iPSC-derived macrophages with PBMC T cells. The supernatant was saved for ELISA for IL-12p70 and IFNγ. As shown in FIG.13, significant fold change was observed for the IL12 fusion construct transfected cells for T cell activation marker CD69 after co-culture with wildtype and B2M- / -iPSC-derived macrophages. Fold change is relative to PBMC T cell alone. HLA-DR expression is shown in FIG. 14. It was shown that B2M- / -iPSC-derived macrophages did not express HLA-DR in -131-WSGR Docket No.61057-724.601 response to IFNγ. IL-12 expression stimulated the T cells to express IFNγ.

[0402] FIG. 15 shows a schematic of experiments done to test the cytotoxicity of IL-12 B2M- / -iPSC-derived Macrophage and ROR1-CAR PBMC T cells against MDA-MB-231 cancer cells. 2.5x105PBMC-T cells and / or B2M- / -iPSC-derived Macrophages with 1x105MDA-MB-231 cells were tested per well of a 24 well plate. As shown in FIG.16, there was significantly more cancer cell lysis in co-culture with IL12 fusion construct transfected macrophages than with mock transfected or IL12 P2A transfected macrophages. All values were normalized to cancer cell lysis detected in the MDA-MB-231 only well. Data shown is the mean of 2 replicate triplicate well. Error bars represent SEM.

[0403] FIG.17 shows a schematic of a V2-ROR1-CAR (top) and construct (bottom). As shown in FIG.18, IL12 fusion construct transfected macrophages displayed the highest percentage lysis of MDA-MB-231 cells when MDA-MB-231 cells were co-cultured with B2M- / -iPSC-derived Macrophages and ROR1-CAR-T cells following mRNA transfection. All values were normalized to the cell lysis detected in the MD-MBA-231 cell only well. Data is a mean of 2 technical triplicate wells. Error bars represent SEM. Example 3: Engineered Transgene-Expressing T Cells

[0404] In this Example, T cells were simultaneously engineered to express a CAR construct as well as knockout of T-cell receptor alpha and beta (TCRab).

[0405] To generate mRNA-edited T cells, primary T Cells were isolated from donor PBMCs by CD3+ magnetic bead selection and cryopreserved. The CD3+ T cells were then thawed and activated with CD3 / CD28 or alternatively with CD3 / CD28 / CD2 antibodies in the presence of IL2, IL7, and / or IL15 for 3 days. The cells were then electroporated with 31600v / 10ms pulses with mRNA encoding for example TRACe1 gene editing protein (500ng for each target sequence, 1,000ng in total) and / or a DNA repair template (500ng of DNA). The cells recovered in the same culture media for 3 days, and then gene editing was quantified by flow cytometry and / or PCR and gel electrophoresis. T cells displaying loss of TCRab indicated successful knockout of the target gene, while insertion of the DNA-encoding transgene was indicated by GFP+ / fluorescent indicator-stained cells, depending on the target transgene. Genes targeted using the method disclosed herein include B2Me2, CIITAe1, CD52e1, and TRACe1. Assessment of successful knockdown of these target genes via flow cytometry is shown in FIG. 19. Surprisingly, simultaneous editing of two target genes CD52e1 and TRACe1 was achieved with a single electroporation as shown in FIG.20. Flow cytometry assessment indicated that functional protein expression for both target genes was achieved.

[0406] Two example homologous DNA templates for use with a gene editing protein according to some embodiments of the present disclosure targeting the TRAC locus are shown in FIG.21. -132-WSGR Docket No.61057-724.601 Either GFP or a CD19 CAR were inserted into the TRAC locus of primary T cells. FIG.22 shows efficient on-target genomic integration of each construct 3 and 7 days after electroporation across 3 PBMC donors. A pJeT promoter and human growth hormone poly-A (hGHpA) signal were used.

[0407] Next, a repair template construct encoding epidermal growth factor receptor (EGFR) was used to knockdown the TRAC locus in T cells while overexpressing EGFR. mRNA encoding a gene editing protein according to some embodiments of the present disclosure targeting TRACe1, when co-delivered via electroporation with a homologous DNA repair template encoding the EGFR protein, efficiently knocked down TCRab expression while overexpressing the EGFR transgene. Different combinations of promoters and polyA sequences resulted in different expression levels, with the JeT promoter-hGHpA poly-A signal combination construct displaying the highest level of expression while P2A-drived expression resulted in a lower level of EGFR expression (FIG. 23). EGFR was selected for its cell surface expression to assist in analyzing surface level saturation of expressed constructs. In some embodiments, EGFR construct may be preferred in order to provide a safety switch for easily targeting and killing engineered cells with a commercially available antibody drug treatment. In some embodiments, EGFR construct may be preferred to allow for easy sorting of engineered cells. In some embodiments, a full length EGFR construct may be used. In some embodiments, a truncated EGFR construct may be used.

[0408] Next, it was assessed whether simultaneous knockdown of two target genes CD52e1 and TRACe1 along with expression of an EGFR transgene could be achieved with one electroporation. mRNA encoding a gene editing protein according to some embodiments of the present disclosure targeting CD52e1 and TRACe1 was co-delivered to T cells alongside a homologous DNA repair template encoding the EGFR protein. Surprisingly, efficient knockdown of functional protein expression for both target genes as well as EGFR expression was observed when assessed via flow cytometry as shown in FIGs.24A-B.

[0409] Next, a repair template construct encoding a CD19 CAR protein was used to knockdown the TRAC locus. mRNA encoding a gene editing protein according to some embodiments of the present disclosure targeting TRACe1, when co-delivered via electroporation with a homologous dsDNA repair template encoding a CD19 CAR, efficiently knocked down TCRab expressionwhile overexpressing the CD19 CAR transgene (FIG. 25). For each condition shown, 3x106 Tcells (100μL tips) were electroporated with 5μg each gene editing protein encoding mRNA and 5μg dsDNA repair template, if applicable.

[0410] Next, engineered CD19 CAR T cell cytotoxicity toward RAJI B cells was assessed. First, T cells were electroporated with mRNA encoding a gene editing protein according to some embodiments of the present disclosure targeting TRACe1 as described in the paragraph above -133-WSGR Docket No.61057-724.601 along with a dsDNA repair template encoding a CD19-CAR. After 72 hours, engineered T cells were co-cultured with RAJI cancer cells for 24 hours at a 10:1 effector-to-target ratio. Flow cytometry was used to determine the ratio of live to dead cancer cells. As shown in FIG. 26, TRAC knockdown, CD19-CAR expressing T cells efficiently lysed CD19+ RAJI B cells when compared to B cells alone or TRAC-knockdown T cells. Example 4: iPSC-Derived Hypoimmune Macrophages Engineered for Sustained Transgene Expression

[0411] In this Example, an mRNA-engineered iPSC line was developed to have constitutive IL- 12 overexpression under the control of an engineered chromatin opening sequence (ECOS). The ECOS construct used herein also comprised the human EF1a promoter as well as the human GH poly adenylation sequence. In some embodiments, the ECOS used was selected from a CBX3- ECOS, an A2ECOS, a SRF-ECOS, an ECOS-455, an ECOS-63, or a biologically active fragment thereof. In some embodiments, the ECOS-63 construct was preferred. Some IL-12 overexpressing iPSCs were also engineered to comprise a B2M-HLA-E construct as described in Example 1, resulting in hypoimmune B2M-HLA-E / IL12 iPSCs. Some IL-12 overexpressing iPSCs were separately engineered to comprise a bi-allelic knockout of the beta-2-microglobulin gene (B2M- / -). In some embodiments, the IL12 subunits were either joined by a self-cleaving peptide (IL-12 P2A) or a flexible linker (IL-12 fusion). In some embodiments, the IL-12 subunits were joined by a bovine elastin motif (BEM) flexible linker. In some embodiments, the highest levels of IL-12 expression and highest bioactivity were observed with the BEM linker as compared to a self- cleaving peptide linker.

[0412] iPSCs were engineered from mRNA-reprogrammed fibroblasts that were electroporated with two 1120v / 20ms pulses with AAVS1 comprising mRNA encoding a gene editing protein according to some embodiments of the present disclosure (600 ng per pulse) as well as ssDNA encoding an ECOS-EF1a-IL12-hGHpA repair template (500 ng).

[0413] iPSCs modified with a B2M-HLA-E clone were used as starting material for generation of an IL12-overexpressing cell line. ELISA was used to confirm expression and secretion of the IL- 12p70 protein (FIG. 27). Edited iPSCs were clonally isolated to identify a biallelically-edited IL12-overexpressing clone. After plating in a 96-well plate and 2 week recovery, the supernatant was harvested and assessed via ELISA for IL-12p70 to identify overexpressing clones. A WT / IL12 colony and B2M-HLA-E / IL12 colony E6 (FIG. 28) were subsequently expanded. iPSCs were also cryopreserved for future use.

[0414] WT or modified iPSCs were differentiated into macrophages in a small-scale vertical- flywheel bioreactor system. The system was feeder-cell free and scalable to large culture vessels. A cartoon schematic of the bioreactor is shown in FIG.29. Briefly, the iPSCs were thawed from -134-WSGR Docket No.61057-724.601 cryopreservation and 4x10(6) cells were plated in 100 mL of pre-warmed iPSC culture media. In some embodiments, iPSC culture media comprised mTeSR Plus Basal Media. After 72 hour recovery, the cells were induced toward CD34+ cells via hematopoietic differentiation for 12 days. Following CD34+ differentiation, the cells were then placed in myeloid induction medium for 14 days. In some embodiments, myeloid induction medium comprised Glutamax and Macrophage Colony Stimulating Factor (M-CSF). Subsequently, macrophages were harvested by straining culture supernatant and adding fresh myeloid induction media. The culture was continuously cultured for more than 150 days with weekly harvests.

[0415] Harvested macrophages, either derived from iPSCs or primary human PBMCs, were analyzed using flow cytometry for key myeloid markers as well as other lineage markers. Macrophages canonically express myeloid markers CD14 and CD64 as well as the pan-leukocyte marker CD45. Macrophages do not express the granulocyte marker CD66b or pluripotency markers TRA-1-60 and TRA-1-81. iPSCs differentiated into macrophages displayed robust expression of CD14, CD45, and CD64 while expressing no detectable levels of CD66b, TRA-1- 60, or TRA-1-81 (FIG.30). Similarly, PBMC CD14+ monocyte-derived macrophages displayed consistent expression of key identity markers CD14, CD45, and CD64, with minimally detectable CD66b, TRA-1-60, or TRA-1-81 expression (representative results of at least 2 PBMC donors) (FIG. 31). Brightfield microscopy revealed similar morphology between iPSC-derived macrophages (iMacrophages or iMacs) and PBMC CD14+ monocyte-derived macrophages (FIG. 32). Differentiation yield was tracked across 4 independent iPSC lines (FIG.33).

[0416] Sustained IL12 expression after differentiation was confirmed via ELISA. As shown in FIG. 34, WT and B2M-HLA-E IL12 overexpressing iPSCs as well as the engineered iPSC- derived macrophages (iMacs) differentiated from these iPSCs secreted detectable levels of IL12 into their supernatant. Example 5: Engineered Macrophages Demonstrate Enhanced SKOV3 Cell Killing

[0417] In this Example, engineered macrophages of Example 4 were further characterized and tested for efficacy.

[0418] To test the ability of engineered macrophages to phagocytose tumor cells, 1x10(6) harvested macrophages, either derived from iPSCs or from primary human PBMCs, were first plated in 6 well plates with RPMI1640 + 10% FBS + 100ng / mL M-CSF. 4 days later, 1x10(6) ovarian adenocarcinoma SK-OV-3 cells, labelled with a cell permeable fluorescent dye, were added to the macrophage culture. The culture was then supplemented with a human anti-HER2 antibody, or a human IgG control antibody, at 5µg / mL. The cells were cultured for 2 hours at 37C. The cells were then lifted with TrypLE and a Cell Scraper before staining for CD14+ cells. The cells were assessed via flow cytometry to quantify phagocytosis, which is defined as the -135-WSGR Docket No.61057-724.601 proportion of macrophages (CD14+) that were also positive for the cancer cell fluorescent dye (FIG. 35). A representative image of an iPSC-derived macrophage (GFP+) engulfing two opsonized SKOV3 cells after a 2 hour co-culture is shown in FIG.36. iPSC-derived macrophages outperformed PBMC-derived macrophages in phagocytosis of HER2-opsonized SKOV3 cells (34% vs 20%, p=0.018) (FIG.37).

[0419] To test engineered macrophages for tumor infiltration, engineered GFP-expressing macrophages were injected intravenously via tail vein injection into mice. 72 hours later, tissue was collected from a subcutaneous SKOV3 adenocarcinoma tumor. Immunohistochemistry revealed infiltration of engineered macrophages into the tumor site (FIG.38) as well as persistent GFP expression when stained with a GFP-specific antibody. As shown in FIG. 38, GFP expression was cytosolic, consistent with in vitro results, and accumulation of engineered macrophages was found at the outside of the tumor with infiltration of cells moving internally up to 1000 µm.

[0420] To test engineered macrophages for T cell alloreactivity, T cells were first isolated from PBMCs by CD3+ magnetic bead selection and co-cultured with iPSC-derived macrophages. 72 hours later, the culture was stained for CD4, CD8, and CD69 and assessed via flow cytometry. The median-fluorescence intensity of CD69 across both CD4+ and CD8+ T cell populations was compared to T cells cultured alone to quantify upregulation of the activation marker. Both B2M- / - and B2M-HLA-E iMacrophages exhibited significantly lower upregulation of the early activation marker CD69 when compared to wildtype iMacrophages across both CD4+ (p=0.00004 and p=0.011, respectively) and CD8+ (p=0.00003 and p=0.0167, respectively) T cells across 3 independent donors (FIG.39).

[0421] To test engineered macrophages for cytotoxicity against tumor spheroids, first, ovarian adenocarcinoma spheroids are generated by plating SKOV3 cells overexpressing GFP in low- attachment, round-bottom 96-well plates and incubated for 96 hours at 37C. Following spheroid formation, PBMC-derived T cells isolated as described above alone or alongside iMacrophages were added to the spheroid culture. The co-culture was maintained in a high-content imaging system taking Z-stack images every 12 hours across the brightfield and GFP fluorescent channels for 96-156 hours. Lysis of the GFP SKOV3 spheroids was quantified by the median fluorescent intensity of the maximally projected image across the Z-stack for each time point. SKOV3 spheroids treated with T cells alongside IL12 overexpressing macrophages consistently outperformed T cells alone across numerous PBMC donors (*: p<0.05, **: p<0.01, ***: p<0.001) (FIG. 40). As shown in FIG. 41, engineered macrophages enhanced lysis of GFP+ SKOV3 spheroids when compared to T cells alone, with the IL12 expressing macrophages showing the greatest effect. As shown in FIG. 42, IL12 expressing macrophages enhanced lysis of GFP+ -136-WSGR Docket No.61057-724.601 SKOV3 spheroids when compared to T cells alone or when supplemented with increasing titrations of recombinant IL12p70 protein. As shown in FIG. 43, IL12 expressing macrophages enhanced lysis of GFP+ SKOV3 spheroids when compared to T cells alone, including when supplemented with the immunosuppressive recombinant protein IL-4 (2 ng) known to be present in ovarian cancer microenvironments at high levels. As shown in FIG. 44, IL12 expressing macrophages enhanced lysis of GFP+ SKOV3 spheroids when compared to T cells alone, with results across 3 independent PBMC donors shown. Example 6: Intratumoral Delivery of Therapeutic Payload

[0422] In this Example, a therapeutic payload is delivered directly to a tumor in vivo. The therapeutic payload comprises a nucleic acid molecule. The nucleic acid molecule is a DNA molecule encoding a cytokine. The cytokine is interleukin-12 (IL-12). The nucleic acid molecule comprises an engineered chromatin opening sequence (ECOS). The ECOS comprises ECOS-63 (SEQ ID NO: 28). The nucleic acid molecule comprises an elongation factor-1 alpha (Ef1α) promoter. The nucleic acid molecule encodes a human growth hormone polyadenylation (hGHpA) signal. The nucleic acid molecule is delivered via intratumoral injection of a viral vector. The nucleic acid molecule is delivered simultaneously with a synthetic RNA molecule encoding a gene editing protein of the present disclosure. The nucleic acid molecule and synthetic RNA molecule contact a myeloid cell in vivo. The contacting results in the myeloid cell internalizing the nucleic acid molecule and synthetic RNA molecule and overexpressing the therapeutic payload. The overexpression of the therapeutic payload causes the myeloid cell to exhibit enhanced cancer cell killing and more efficiently infiltrate the solid tumor. NUMBERED EMBODIMENTS

[0423] Embodiment 1. A method of manufacturing an engineered induced mesenchymal stem cell (EiMSC), comprising: contacting a stem cell with one or more RNA molecules encoding one or more gene editing proteins, thereby resulting in expression of the one or more gene editing proteins that results in disruption of a B2M gene in the stem cell and generation of a gene-edited stem cell; and differentiating the gene-edited stem cell into the EiMSC that has its B2M gene disrupted.

[0424] Embodiment 2. The method of embodiment 1, wherein the EiMSC is characterized by low or reduced inflammation.

[0425] Embodiment 3. The method of embodiment 1 or 2, wherein the EiMSC is characterized by low or reduced immunogenicity.

[0426] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the EiMSC is self- renewing.

[0427] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the EiMSC is -137-WSGR Docket No.61057-724.601 multipotent.

[0428] Embodiment 6. The method of any one of embodiments 1 to 5, wherein the EiMSC reduces the proliferation, quantity, and / or activity of an immune cell, optionally a T cell or an NK cell.

[0429] Embodiment 7. The method of any one of embodiments 1 to 6, wherein the EiMSC is suitable for in vitro expansion without substantial loss of immunosuppressive properties.

[0430] Embodiment 8. The method of any one of embodiments 1 to 7, wherein the method disrupts both alleles of the B2M gene.

[0431] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the EiMSC exhibits enhanced immunosuppressive activity and / or stealthing features as compared to a corresponding MSC that does not have its B2M gene disrupted.

[0432] Embodiment 10. The method of embodiment 9, wherein the corresponding MSC is generated by otherwise the same method but does not have its B2M gene disrupted.

[0433] Embodiment 11. The method of any one of embodiments 1 to 10, wherein the EiMSC exhibits enhanced immunosuppressive activity and / or stealthing features as compared to a bone marrow-derived MSC (bmMSC).

[0434] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the EiMSC expresses one or more of CD73, CD90, and CD105.

[0435] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the EiMSC substantially does not express one or more of CD14, CD34 and CD45.

[0436] Embodiment 14. The method of any one of embodiments 1 to 13, wherein the EiMSC is CD90+, CD73+, CD105+, TRA-1-60-, TRA-1-80-, and CD34-, as assayed by flow cytometry.

[0437] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the EiMSC is capable trilineage differentiation via adipogenesis, osteogenesis, and chondrogenesis.

[0438] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the EiMSC expresses an increased amount of Indoleamine 2,3-dioxygenase 1 (IDO1) upon IFNγ stimulation as compared to a corresponding MSC that does not have its B2M gene disrupted.

[0439] Embodiment 17. The method of embodiment 16, wherein the amount of IDO1 expressed by the EiMSC upon IFNγ stimulation is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher than that of the corresponding MSC upon IFNγ stimulation.

[0440] Embodiment 18. The method of any one of embodiments 1 to 17, wherein the EiMSC does not express HLA-I or HLA-II.

[0441] Embodiment 19. The method of any one of embodiments 1 to 18, wherein the EiMSC exerts enhanced inhibitory activity on proliferation of peripheral blood mononuclear cells (PBMCs) that are exposed to immunostimulation when the EiMSC is co-cultured with the PBMCs, as compared to that of a corresponding MSC that does have its B2M gene disrupted. -138-WSGR Docket No.61057-724.601

[0442] Embodiment 20. The method of embodiment 19, wherein the corresponding MSC is generated by otherwise the same method but does not have its B2M gene disrupted.

[0443] Embodiment 21. The method of embodiment 19 or 20, wherein the EiMSC induces at least 5%, 8%, 10%, 12%, or 15% greater suppression of the proliferation of PBMCs as compared to that of the corresponding MSC.

[0444] Embodiment 22. The method of any one of embodiments 1 to 21, wherein the EiMSC exerts enhanced inhibitory activity on proliferation of peripheral blood mononuclear cells (PBMCs) that are exposed to immunostimulation when the EiMSC is co-cultured with the PBMCs, as compared to that of a bmMSC.

[0445] Embodiment 23. The method of embodiment 22, wherein the EiMSC induces at least 50%, 60%, 70%, 80%, 90%, 100%, or 120% greater suppression of the proliferation of the PBMCs as compared to that of the bmMSC.

[0446] Embodiment 24. The method of any one of embodiments 1 to 23, wherein the stem cell is an induced pluripotent stem cell (iPSC).

[0447] Embodiment 25. The method of embodiment 24, wherein the iPSC is derived from a human.

[0448] Embodiment 26. The method of embodiment 24, wherein the iPSC is derived from a subject who is not intended to receive a therapy comprising administration of the EiMSC.

[0449] Embodiment 27. The method of embodiment 24, wherein the iPSC is allogeneic to a patient intended to receive the therapy comprising administration of the EiMSC.

[0450] Embodiment 28. The method of embodiment 24, wherein the iPSC is from a master cell bank.

[0451] Embodiment 29. The method of any one of embodiments 1 to 28, wherein the RNA molecule is a synthetic RNA molecule.

[0452] Embodiment 30. The method of embodiment 29, wherein the synthetic RNA molecule is an mRNA molecule comprising one or more non-canonical nucleotides that substantially avoid cellular toxicity.

[0453] Embodiment 31. The method of embodiment 29 or 30, wherein the synthetic RNA molecule is an mRNA molecule comprising one or more non-canonical nucleotides selected from 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5- methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5- hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5- ethoxypseudouridine, 5-hydroxymethyluridine, 5-hydroxymethylpseudouridine, 5- carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5- azauridine, 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methylpseudouridine, 5- -139-WSGR Docket No.61057-724.601 aminopseudouridine, 5-hydroxypseudouridine, 4-thio-5-azauridine, 4-thiopseudouridine, 4-thio- 5-methyluridine, 4-thio-5-aminouridine, 4-thio-5-hydroxyuridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methylpseudouridine, 4- thio-5-aminopseudouridine, 4-thio-5-hydroxypseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5- methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytydine, 5-methyl-5-azacytidine, 5-amino- 5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylpseudoisocytidine, 5-aminopseudoisocytidine, 5-hydroxypseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine, 2-thio-5- azacytidine, 2-thiopseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2- thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5- hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5- hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2- thio-5-hydroxypseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4- methylpseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl- 5-hydroxycytidine, N4-methyl-5-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4- methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methylpseudoisocytidine, N4-methyl-5- aminopseudoisocytidine, N4-methyl-5-hydroxypseudoisocytidine, N4-amino-5-azacytidine, N4- aminopseudoisocytidine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5- hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-amino-5-azacytidine, N4- amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5- aminopseudoisocytidine, N4-amino-5-hydroxypseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxypseudoisocytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4- hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5- azacytidine, N4-hydroxy-5-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4- hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxypseudoisocytidine, 2-thio-N4- methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5- hydroxycytidine, 2-thio-N4-methyl-5-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5- azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5- methylpseudoisocytidine, 2-thio-N4-methyl-5-aminopseudoisocytidine, 2-thio-N4-methyl-5- hydroxypseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-aminopseudoisocytidine, 2-thio-N4-amino-5-methylcytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5- hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-amino-5- azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5- methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5- -140-WSGR Docket No.61057-724.601 hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4- hydroxypseudoisocytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, 2-thio-N4-hydroxy-5-hydroxycytidine, 2-thio-N4-hydroxy-5-methyl-5-azacytidine, 2-thio-N4- hydroxy-5-amino-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-5-azacytidine, 2-thio-N4- hydroxy-5-methylpseudoisocytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytidine, 2-thio-N4- hydroxy-5-hydroxypseudoisocytidine, N6-methyladenosine, N6-aminoadenosine, N6- hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl- 8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7- deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6- hydroxyadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-azaadenosine, N6-hydroxy-7- deaza-8-azaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-azaguanosine, 6-thio-7- deazaguanosine, 6-thio-8-azaguanosine, 7-deaza-8-azaguanosine, and 6-thio-7-deaza-8- azaguanosine.

[0454] Embodiment 32. The method of any one of embodiments 29 to 31, wherein the synthetic RNA molecule is in vitro transcribed.

[0455] Embodiment 33. The method of any one of embodiments 1 to 32, wherein the method further comprises reprogramming a somatic cell into the stem cell or wherein the stem cell was reprogrammed from a somatic cell.

[0456] Embodiment 34. The method of embodiment 33, wherein the reprogramming is non-viral.

[0457] Embodiment 35. The method of embodiment 33 or 34, wherein the reprogramming comprises transfecting a somatic cell with a nucleic acid that expresses is one or more of Oct4, Sox2, Klf4, c-Myc, l-Myc, Tert, Nanog, and Lin28.

[0458] Embodiment 36. The method of any one of embodiments 1 to 35, wherein the method further comprises formulating the EiMSC in a composition for therapeutic use.

[0459] Embodiment 37. The method of embodiment 36, wherein the composition is suitable for use in the treatment of amyotrophic lateral sclerosis (ALS), spinal cord injury, degenerative disc disease, coronary artery disease, acute myocardial infarction, alcoholic liver cirrhosis, hepatitis C virus (HCV)-induced cirrhosis, multiple sclerosis (MS), osteoarthritis (OA), osteoarthritis of the knee, kidney allograft, critical limb ischemia, ischemic cardiomyopathy, Crohn’s disease, idiopathic pulmonary fibrosis, anal fistula, spinal cord injury, systemic lupus erythematosus (SLE), acute respiratory distress syndrome (ARDS), acute graft-versus-host disease (aGvHD), preterm bronchopulmonary dysplasia (BPD), autism nonischemic heart failure, and / or Type 2 diabetes mellitus.

[0460] Embodiment 38. The method of embodiment 36, wherein the composition is suitable for use in the treatment of an infectious disease, optionally an infection with a pathogen, optionally a -141-WSGR Docket No.61057-724.601 bacterium, virus, fungus, or parasite.

[0461] Embodiment 39. The method of embodiment 38, wherein the pathogen is a virus.

[0462] Embodiment 40. The method of embodiment 39, wherein the virus is: an influenza virus, optionally selected from a Type A, a Type B, a Type C, and a Type D influenza virus, or a member of the Coronaviridae family, optionally selected from a betacoronavirus, optionally selected from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, Middle East Respiratory Syndrome—Corona Virus (MERS-CoV), HCoV-HKU1, and HCoV-OC43, or an alphacoronavirus, optionally selected fromHCoV-NL63 and HCoV-229E.

[0463] Embodiment 41. An EiMSC produced by the method of any one of embodiments 1 to 35.

[0464] Embodiment 42. A population of cells comprising EiMSCs produced by the method of any one of embodiments 1 to 35.

[0465] Embodiment 43. An EiMSC comprising a disruption in a B2M gene, wherein the EiMSC exhibits enhanced immunosuppressive activity as compared to a corresponding MSC that does not have its B2M gene disrupted, and / or as compared to a bone marrow-derived MSC (bmMSC).

[0466] Embodiment 44. The EiMSC of embodiment 43, wherein the corresponding MSC is generated by otherwise the same method but does not have its B2M gene disrupted.

[0467] Embodiment 45. The EiMSC of embodiment 43 or 44, wherein the EiMSC is characterized by low or reduced inflammation.

[0468] Embodiment 46. The EiMSC of any one of embodiments 43 to 45, wherein the EiMSC is characterized by low or reduced immunogenicity.

[0469] Embodiment 47. The EiMSC of any one of embodiments 43 to 46, wherein the EiMSC is self-renewing.

[0470] Embodiment 48. The EiMSC of any one of embodiments 43 to 47, wherein the EiMSC is multipotent.

[0471] Embodiment 49. The EiMSC of any one of embodiments 43 to 48, wherein the EiMSC reduces the proliferation, quantity, and / or activity of an immune cell, optionally a T cell or an NK cell.

[0472] Embodiment 50. The EiMSC of any one of embodiments 43 to 49, wherein the EiMSC is suitable for in vitro expansion without substantial loss of immunosuppressive properties.

[0473] Embodiment 51. The EiMSC of any one of embodiments 43 to 49, wherein the EiMSC comprises a disruption in both alleles of the B2M gene.

[0474] Embodiment 52. The EiMSC of any one of embodiments 43 to 51, wherein the EiMSC exhibits enhanced immunosuppressive activity and / or stealthing features as compared to a corresponding MSC that does not have its B2M gene disrupted.

[0475] Embodiment 53. The EiMSC of embodiment 52, wherein the corresponding MSC is -142-WSGR Docket No.61057-724.601 generated by otherwise the same method but does not have its B2M gene disrupted.

[0476] Embodiment 54. The EiMSC of any one of embodiments 43 to 53, wherein the EiMSC exhibits enhanced immunosuppressive activity and / or stealthing features as compared to a bone marrow-derived MSC (bmMSC).

[0477] Embodiment 55. The EiMSC of any one of embodiments 43 to 54, wherein the EiMSC expresses one or more of CD73, CD90, and CD105.

[0478] Embodiment 56. The EiMSC of any one of embodiments 43 to 55, wherein the EiMSC substantially does not express one or more of CD14, CD34 and CD45.

[0479] Embodiment 57. The EiMSC of any one of embodiments 43 to 56, wherein the EiMSC is CD90+, CD73+, CD105+, TRA-1-60-, TRA-1-80-, and CD34-, as assayed by flow cytometry.

[0480] Embodiment 58. The EiMSC of any one of embodiments 43 to 57, wherein the EiMSC is capable of trilineage differentiation via adipogenesis, osteogenesis, and chondrogenesis.

[0481] Embodiment 59. The EiMSC of any one of embodiments 43 to 58, wherein the EiMSC expresses an increased amount of Indoleamine 2,3-dioxygenase 1 (IDO1) upon IFNγ stimulation as compared to the corresponding MSC that does not have its B2M gene disrupted.

[0482] Embodiment 60. The EiMSC of embodiment 59, wherein the amount of IDO1 expressed in the EiMSC upon IFNγ stimulation is at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, or 10-fold higher than that of the corresponding MSC upon IFNγ stimulation.

[0483] Embodiment 61. The EiMSC of any one of embodiments 43 to 60, wherein the EiMSC does not express HLA-I or HLA-II.

[0484] Embodiment 62. The EiMSC of any one of embodiments 43 to 61, wherein the EiMSC exerts enhanced inhibitory activity on proliferation of peripheral blood mononuclear cells (PBMCs) that are exposed to immunostimulation when the EiMSC is co-cultured with the PBMCs, as compared to that of the corresponding MSC that does not have its B2M gene disrupted.

[0485] Embodiment 63. The EiMSC of embodiment 62, wherein the EiMSC induces at least 5%, 8%, 10%, 12%, or 15% greater suppression on the proliferation of PBMCs as compared to that of the corresponding MSC.

[0486] Embodiment 64. The EiMSC of any one of embodiments 43 to 63, wherein the EiMSC exerts enhanced inhibitory activity on proliferation of peripheral blood mononuclear cells (PBMCs) that are exposed to immunostimulation when the EiMSC is co-cultured with the PBMCs, as compared to that of the bmMSC.

[0487] Embodiment 65. The EiMSC of embodiment 64, wherein the EiMSC induces at least 50%, 60%, 70%, 80%, 90%, 100%, or 120% greater suppression of the proliferation of PBMCs as compared to that of the bmMSC.

[0488] Embodiment 66. A population of cells comprising the EiMSCs of any one of embodiments -143-WSGR Docket No.61057-724.601 43 to 65.

[0489] Embodiment 67. A nucleic acid molecule comprising a sequence encoding a fusion protein that comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, and an HLA-G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the nucleic acid molecule has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

[0490] Embodiment 68. The nucleic acid molecule of embodiment 67, wherein the coding sequence for the linker comprises the sequence of any one of SEQ ID NOs: 13-15.

[0491] Embodiment 69. The nucleic acid of embodiment 67...

Claims

WSGR Docket No.61057-724.601 CLAIMS What is claimed is:

1. A nucleic acid molecule comprising a sequence encoding a fusion protein that comprises: (a) a B2M polypeptide; (b) an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, and an HLA-G polypeptide; and (c) a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the nucleic acid molecule has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

2. The nucleic acid molecule of claim 1, wherein the HLA polypeptide comprises HLA-E polypeptide.

3. The nucleic acid molecule of claim 2, wherein the HLA-E polypeptide comprises the sequence of SEQ ID NO:

5.

4. A method of engineering a cell, the method comprising inserting into the genome of the cell a sequence encoding a fusion protein, thereby producing a gene-edited cell, wherein the fusion protein comprises: (a) a B2M polypeptide; (b) an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA- B, an HLA-C, an HLA-E, an HLA-F, and an HLA-G polypeptide; and (c) a linker sequence (G4S)4 that links the B2M polypeptide and the HLApolypeptide, and wherein the coding sequence for the linker in the sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

5. The method of claim 4, wherein the HLA polypeptide comprises HLA-E.

6. The method of claim 4 or claim 5, wherein the gene-edited cell expresses the fusion protein upon interferon-γ (IFNγ) stimulation.

7. The method of any one of claims 4 to 6, wherein the cell is an iPSC, and wherein the method comprises producing a population of gene-edited iPSCs.

8. The method of claim 7, wherein at least 50%, 60%, 70%, 80%, or 90% of the iPSCs in the population express the fusion protein upon IFNγ stimulation.

9. The method of claim 7 or 8, wherein the percentage of the iPSCs in the population that expresses the fusion protein upon IFNγ stimulation is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, or 8-fold higher than the percentage of cells in a -172-WSGR Docket No.61057-724.601 corresponding population of iPSCs that are otherwise the same but comprise a coding sequence for a fusion protein comprising a linker with higher than 70%, 80%, or 90% GC content.

10. A cell comprising an exogenous sequence encoding a fusion protein that comprises: (a) a B2M polypeptide; (b) an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA- B, an HLA-C, an HLA-E, an HLA-F, and an HLA-G polypeptide; and (c) a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the exogenous sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

11. The cell of claim 10, wherein the HLA polypeptide comprises HLA-E polypeptide.

12. The cell of claim 10 or 11, wherein the cell is an induced pluripotent stem cell (iPSC).

13. The cell of claim 10 or 11, wherein the cell is a macrophage or a lymphocyte.

14. The cell of any one of claims 10 to 13, wherein the cell expresses the HLA polypeptide upon interferon-γ (IFNγ) stimulation.

15. A method of manufacturing a therapeutic macrophage, the method comprising contacting the macrophage with a nucleic acid molecule to result in overexpression of an IL-12p70 dimer by the macrophage, thereby producing the therapeutic macrophage overexpressing the IL12p70 dimer.

16. The method of claim 15, wherein the macrophage is differentiated from an induced pluripotent stem cell (iPSC).

17. The method of claim 16, wherein the macrophage is differentiated from the iPSC in vitro.

18. The method of any one of claims 15 to 17, wherein the method further comprises differentiating a stem cell into the macrophage in vitro.

19. The method of claim 18, wherein the stem cell is a hematopoietic stem cell (HSC).

20. The method of claim 18, wherein the stem cell is an induced pluripotent stem cell (iPSC).

21. The method of claim 20, wherein the method further comprises reprogramming a somatic cell into the iPSC.

22. The method of claim 21, wherein the reprogramming comprises introducing into the somatic cell one or more synthetic RNA molecules encoding one or more reprogramming factors, resulting in the somatic cell expressing the one or more reprogramming factors. -173-WSGR Docket No.61057-724.601 23. The method of any one of claims 15 to 22, wherein the IL-12p70 dimer comprises IL- 12α and IL-12β, and wherein the IL-12α and IL-12β are fused via a peptide linker.

24. The method of claim 23, wherein the peptide linker comprises a 2A sequence or a 2A- like sequence.

25. The method of any one of claims 15 to 24, wherein PBMC-T cells, when co-cultured with the therapeutic macrophage, induce at least 1.2-fold, 1.5-fold, 1.8-fold, 1.9-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold, or 10-fold greater cell lysis of cancer cells that are in contact with the PBMC-T cells as compared to when co-cultured with the corresponding macrophage.

26. The method of any one of claims 15 to 25, wherein T cells that express a chimeric antigen receptor (CAR), when co-cultured with the therapeutic macrophage, induce at least 1.2-fold, 1.5-fold, 1.8-fold, 1.9-fold, or 2-fold greater cell lysis of cancer cells that are in contact with the T cells as compared to when the T cells are contacted to the cancer cells alone without co-culture with the macrophage that is otherwise the same but lacks overexpression of the IL-12p70 dimer.

27. The method of any one of claims 15 to 26, wherein T cells that express a chimeric antigen receptor (CAR), when co-cultured with the therapeutic macrophage, induce at least 5%, 8%, 10%, 12%, 15%, 16%, 17%, 18%, or 20% greater cell lysis of cancer cells that are in contact with the T cells as compared to when co-cultured with the corresponding macrophage.

28. The method of claim 26 or 27, wherein the CAR specifically binds to ROR1.

29. A therapeutic macrophage overexpressing an IL12p70 dimer and comprising a disruption in a genomic B2M gene.

30. The therapeutic macrophage of claim 29, wherein the therapeutic macrophage is differentiated from an induced pluripotent stem cell (iPSC).

31. The therapeutic macrophage of claim 30, wherein the iPSC is reprogrammed from a somatic cell.

32. The therapeutic macrophage of any one of claims 29 to 31, wherein the therapeutic macrophage comprises a disruption in both alleles of the B2M gene.

33. A nucleic acid molecule comprising a sequence encoding a fusion protein that comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, and an HLA-G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, wherein the coding sequence for the linker in the nucleic acid molecule has a GC content of at most 70%, 65%, 64%, 63%, or 62%. -174-WSGR Docket No.61057-724.601 34. A method of engineering a cell, the method comprising inserting into the genome of the cell a sequence encoding a fusion protein, thereby producing a gene-edited cell, wherein the fusion protein comprises: a B2M polypeptide; an HLA polypeptide selected from the group consisting of: an HLA-A, an HLA-B, an HLA-C, an HLA-E, an HLA-F, and an HLA-G polypeptide; and a linker sequence (G4S)4 that links the B2M polypeptide and the HLA polypeptide, and wherein the coding sequence for the linker in the sequence encoding the fusion protein has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

35. A method of generating a therapeutic myeloid cell, the method comprising contacting a stem cell with a nucleic acid molecule to result in persistent overexpression of a cytokine by the stem cell, thereby producing an engineered stem cell overexpressing the cytokine; and differentiating the engineered stem cell into the therapeutic myeloid cell overexpressing the cytokine.

36. The method of claim 35, wherein the cytokine comprises IL-12.

37. The method of claim 36, wherein the IL-12 comprises an IL-12p70 dimer.

38. The method of claim 37, wherein the IL-12p70 dimer is encoded by a sequence comprising at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 59 or SEQ ID NO:

61.

39. The method of claim 37 or claim 38, wherein the IL-12p70 dimer comprises a peptide linker.

40. The method of claim 39, wherein the peptide linker is a flexible linker.

41. The method of claim 40, wherein the flexible linker is a bovine elastin motif (BEM) linker.

42. The method of any one of claims 35 to 41, wherein the nucleic acid molecule comprises an engineered chromatin opening sequence (ECOS).

43. The method of any one of claims 35 to 42, wherein the therapeutic myeloid cell comprises a disruption in a B2M gene in its genome.

44. The method of claim 43, wherein the disruption comprises insertion of a B2M-HLA-E construct.

45. The method of claim 44, wherein the B2M-HLA-E construct comprises a linker.

46. The method of claim 45, wherein the linker comprises a (G4S)4 linker sequence.

47. The method of claim 46, wherein the coding sequence for the (G4S)4linker sequence has a GC content of at most 70%, 65%, 64%, 63%, or 62%.

48. The method of any one of claims 35 to 47, wherein the nucleic acid molecule encodes a poly-adenylation signal. -175-WSGR Docket No.61057-724.601 49. The method of claim 48, wherein the poly-adenylation signal comprises a human growth hormone poly-adenylation (hGHpA) signal.

50. The method of any one of claims 35 to 49, wherein the method further comprises contacting the stem cell with a nucleic acid molecule encoding a transgene.

51. The method of claim 50, wherein the transgene comprises a chimeric antigen receptor (CAR).

52. The method of claim 51, wherein the CAR specifically binds to CD19.

53. The method of any one of claims 35 to 52, wherein the contacting comprises transfecting the stem cell with one or more synthetic RNA molecules encoding one or more gene- editing proteins, thereby resulting in the stem cell expressing the one or more gene- editing proteins.

54. The method of any one of claims 35 to 53, wherein the nucleic acid molecule comprises an EF1a promoter.

55. The method of any one of claims 49 to 54, wherein the nucleic acid molecule comprises a ECOS-EF1a-IL12-hGHpA repair template.

56. The method of claim 55, wherein the ECOS-EF1a-IL12-hGHpA repair template comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 58 or SEQ ID NO:

60.

57. The method of any one of claims 35 to 56, wherein the therapeutic myeloid cell comprises a therapeutic macrophage.

58. The method of claim 57, wherein the therapeutic macrophage phagocytoses 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% more SK-OV-3 cancer cells, when co-cultured with the PBMC-derived macrophage, as compared to a PBMC-derived macrophage.

59. The method of claim 57 or 58, wherein the therapeutic macrophage infiltrates a spheroid solid tumor model comprising SK-OV-3 cells, when contacted with the spheroid solid tumor model, by 100 µm, 150 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900 µm, 950 µm, or 1000 µm.

60. The method of any one of claims 57 to 59, wherein the therapeutic macrophage induces 25%, 50%, 75%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275%, or 300% less CD69 upregulation in CD4+ or CD8+ T cells, when co-incubated with the CD4+ or CD8+ T cells and the wildtype macrophage, as compared to a wildtype macrophage.

61. A method for intratumoral therapeutic delivery, the method comprising: contacting a tumor tissue with a nucleic acid molecule that encodes a cytokine.

62. The method of claim 61, wherein the cytokine comprises IL-12. -176-WSGR Docket No.61057-724.601 63. The method of claim 62, wherein the IL-12 comprises an IL-12p70 dimer.

64. The method of claim 63, wherein the IL-12p70 dimer is encoded by a sequence comprising at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 59 or SEQ ID NO:

61.

65. The method of claim 63 or 64, wherein the IL-12p70 dimer comprises a peptide linker.

66. The method of claim 65, wherein the peptide linker is a flexible linker.

67. The method of claim 66, wherein the flexible linker is a bovine elastin motif (BEM) linker.

68. The method of any one of claims 61 to 67, wherein the nucleic acid molecule comprises a sequence encoding an ECOS-EF1a-IL12-hGHpA construct.

69. The method of claim 68, wherein the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 58 or SEQ ID NO:

60.

70. A therapeutic myeloid cell persistently overexpressing a cytokine, wherein the therapeutic myeloid cell comprises a genomic insertion.

71. The therapeutic myeloid cell of claim 70, wherein the cytokine comprises IL-12.

72. The therapeutic myeloid cell of claim 71, wherein the IL-12 comprises an IL-12p70 dimer.

73. The therapeutic myeloid cell of claim 72, wherein the IL-12p70 dimer is encoded by a sequence comprising at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 59 or SEQ ID NO:

61.

74. The therapeutic myeloid cell of claim 72 or 73, wherein the IL-12p70 dimer comprises a peptide linker.

75. The therapeutic myeloid cell of claim 74, wherein the peptide linker is a flexible linker.

76. The therapeutic myeloid cell of claim 75, wherein the flexible linker is a bovine elastin motif (BEM) linker.

77. The therapeutic myeloid cell of any one of claims 70 to 76, wherein the genomic insertion comprises a sequence encoded by a repair template.

78. The therapeutic myeloid cell of claim 77, wherein the repair template comprises an engineered chromatin opening sequence (ECOS).

79. The therapeutic myeloid cell of claim 78, wherein the repair template comprises a sequence encoding an ECOS-EF1a-IL12-hGHpA construct.

80. The therapeutic myeloid cell of claim 79, wherein the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to SEQ ID NO: 58 or SEQ ID NO:

60. -177-WSGR Docket No.61057-724.601 81. The therapeutic myeloid cell of any one of claims 70 to 80, wherein the therapeutic myeloid cell is a therapeutic macrophage.

82. The therapeutic myeloid cell of any one of claims 70 to 81, wherein the therapeutic myeloid cell is differentiated from an engineered stem cell.

83. The method of claim 82, wherein the engineered stem cell is a pluripotent stem cell.

84. The therapeutic myeloid cell of claim 83, wherein the pluripotent stem cell is an induced pluripotent stem cell.

85. The therapeutic myeloid cell of any one of claims 82 to 84, wherein the therapeutic myeloid cell is differentiated from the engineered stem cell in vitro.

86. A pharmaceutical composition comprising a nucleic acid molecule encoding a cytokine, wherein the cytokine comprises a bovine elastin motif (BEM) linker.

87. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of claim 86.

88. A method of generating an engineered immune cell comprising contacting an immune cell with a nucleic acid molecule, wherein the nucleic acid molecule comprises a sequence encoding a chimeric antigen receptor (CAR), thereby generating the engineered immune cell.

89. The method of claim 88, wherein the CAR specifically binds to CD19.

90. The method of claim 89, wherein the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NO: 55- 57.

91. The method of any one of claims 88 to 90, wherein the method further comprises contacting the immune cell with one or more synthetic RNA molecules encoding one or more gene editing proteins.

92. The method of claim 91, wherein the one or more gene editing proteins target the TRAC locus and the CD52 locus.

93. The method of any one of claims 88 to 92, wherein the engineered immune cell overexpresses EGFR.

94. The method of any one of claims 91 to 93, wherein a single administration of the nucleic acid molecule and the one or more synthetic RNA molecules simultaneously results in a disruption in the genome of the engineered immune cell at the TRAC locus and the CD52 locus.

95. The method of claim 94, wherein the administration comprises electroporation.

96. The method of any one of claims 88 to 95, wherein the engineered immune cell lyses at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% more RAJI cancer -178-WSGR Docket No.61057-724.601 cells as compared to an immune cell which does not comprise the nucleic acid molecule when co-cultured with the RAJI cancer cells. -179-

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