SIRPα engager cells

Abstract

The disclosure provides compositions and methods for making genetically modified cells expressing a SIRPα engager protein. Also featured are methods of treatment using the cells.

Description

[0001] SIRPa ENGAGER CELLS

[0002] Cross-Reference to Related Applications

[0003] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 546,111 , filed October 27, 2023, and is incorporated herein by reference in its entirety

[0004] Background

[0005] Immune cells, such as natural killer (NK) cells are critical to the innate immune system. The role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected and cancerous cells. Typically, NK cells become activated by target cells downregulating major histocompatibility complex (MHO) as this is one major inhibitory NK cell signal NK cell activation triggers cytokine release resulting in lysis or apoptosis NK cells are unique, because they can recognize stressed cells as they upregulate other stimulatory NK cell signals and do not require prior exposure to certain cell epitopes. This makes them very fast responders. They can also quickly respond to antibody-laden cells because the binding of antibody Fc is a strong stimulatory NK cell signal. NK cells do not require major activation to kill cells that are missing “self” markers of MHC class I other than some cytokine exposure like IL-2 or IL-15. This role is especially important because harmful cells that have downregulated or missing MHC I markers cannot be detected and destroyed by other immune cells, such as T lymphocyte cells

[0006] Signal-regulatory-protein a (SIRPa) is a member of both the signal-regulatory-protein (SIRP) family and the immunoglobulin superfamily. SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signaling processes.

[0007] CD47 is a ligand for SIRPa. CD47 is a “marker-of-self” protein that can be overexpressed broadly across tumor types. It is emerging as a novel potent macrophage immune checkpoint for cancer immunotherapy. CD47 in tumor cells sends a “don’t-eat-me” signal that inhibits macrophage phagocytosis.

[0008] Immune cells, such as NK cells, express SIRPa upon stimulation and bind to CD47, thereby reducing their killing efficacy for CD47-expressing cells. Furthermore, innate immune cells, such as macrophages, phagocytose foreign cells lacking a CD47 signal. This presents challenges in cell therapy in which therapeutic cells may be rejected by the immune system, thereby reducing or eliminating their efficacy. Thus, cells expressing a SIRPa engager inhibit the activation of innate immune cells and increase immune evasiveness of engineered therapeutic cells, preventing their destruction.

[0009] Summary of the Invention

[0010] The present disclosure relates to compositions and methods relating to genetically modified cells expressing a SIRPa engager protein. This disclosure also provides a mammalian cell containing a heterologous gene, wherein the gene encodes a signal regulatory protein alpha (SIRPa) engager. This disclosure also provides a mammalian cell containing the heterologous gene inserted into an endogenous gene This disclosure also provides a mammalian cell containing 1, 2, 3, 4, 5, 6, 7, 8, or more copies of the heterologous gene. This disclosure also provides methods of editing mammalian cells to encode the heterologous gene, and, optionally, to disrupt one or more endogenous genes. This disclosure also provides expression cassettes for insertion into an endogenous gene in the cell. Compositions and methods described herein may be used to create immune evasive cells capable of escaping allogeneic cellular and IgG antibody mediated rejection.

[0011] In a first aspect, the disclosure provides a mammalian cell that includes a polynucleotide having a heterologous gene encoding a signal regulatory protein alpha (SIRPa) engager that binds SIRPa inserted into one or more sites within an endogenous p-2 microglobulin (B2M) locus, an endogenous class II transactivator (Cl ITA) locus, or an endogenous T cell receptor alpha subunit constant (TRAC) locus.

[0012] In some aspects, the heterologous gene is inserted into exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITAgene, exon 1 of the endogenous TRAC gene, or a combination thereof.

[0013] In some aspects, the mammalian cell includes 1, 2, 3, 4, 5, 6, 7, 8, or more copies of the heterologous gene.

[0014] In some aspects, the SIRPa engager includes a CD47 extracellular domain (ECD). In some aspects, the CD47 ECD includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1. In some aspects, the CD47 ECD includes the amino acid sequence of SEQ ID NO: 1 . In some aspects, the SIRPa engager includes a single chain variable fragment (scFv). In some aspects, the scFv includes heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 20-22 and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 23-25. In some aspects, the SIRPa engager lacks a functional CD47 intracellular domain (ICD) In some aspects, the SIRPa engager includes a hinge region. In some aspects, the hinge region is a CD8a hinge region.

[0015] In some aspects, the SIRPa engager includes a heterologous transmembrane domain (TMD). In some aspects, the heterologous TMD is selected from the group consisting of CD47, CD85f, CD349, CD284, CD261 , CD 172b, CD277, CD 186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61 , CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51 , CD141 , CD115, CD42b, CD221 , CD271 , CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331, CD222, CD20, CD79a, CD64, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227. CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CD1b, CD1c, CD154, CD6, CD52, CD132, CD32, CD66b, CD171, CD191, CD197, CD185, CD131, CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361, CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231 , CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD1581, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195. CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, OD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91, CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161, CD215, CD150, CD11d, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111, CD281 , CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158Z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD100, CD270, CD101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD114, CD180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201 , CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321 , CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51 , CD41 , CD29: CD18, CD61 , CD104, and PDGFR TMD.

[0016] In some aspects, the SIRPa engager includes a polypeptide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 5. In some aspects, the SIRPa engager includes the amino acid sequence of SEQ ID NO: 5.

[0017] In some aspects, the SIRPa engager includes a polypeptide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 29. In some aspects, the SIRPa engager includes the amino acid sequence of SEQ ID NO: 29

[0018] In some aspects, the heterologous gene is operably linked to an endogenous promoter at the insertion site.

[0019] In some aspects, the polynucleotide includes a heterologous promoter, an enhancer, and / or a polyadenylation sequence. In some aspects, the heterologous promoter is operably linked to the heterologous gene. In some aspects, the heterologous promoter is a constitutive promoter, an inducible promoter, a cell-type specific promoter, or a tissue-specific promoter. In some aspects, the heterologous promoter is selected from the group consisting of EF1a, UBC promoter, EFS promoter, CD11 b promoter, sp146 / 147 promoter, CD68 promoter, sp146 / gp9, CD47 promoter, PGK promoter, SV40 promoter, CMV promoter, human b-actin promoter, UAS promoter, AC5 promoter, CaMKIla promoter, GAL1 / GAL10 promoter, GDS promoter, TRE promoter, U6 promoter, H1 promoter, 7SK promoter, DHFR promoter, EGR 1 promoter, TBG promoter, EIF4A1 promoter, FerH promoter, FerL promoter, GAPDH promoter, HSPA5 promoter, HSPA4 promoter, UBB promoter, EBV promoter, MND promoter, LTR of MuMLV promoter, MMTV promoter, RSV promoter, Apolipoprotein E-Human Alpha 1 -Antitrypsin promoter, CAG promoter, CBA promoter, CK8 promoter, and Mu1a promoter

[0020] In some aspects, the enhancer is a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) or a Simian Virus 40 (SV40) enhancer. In some aspects, the polynucleotide includes a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of heterologous genes.

[0021] In some aspects, the polynucleotide includes a first heterologous gene encoding the SIRPa engager and a second heterologous gene encoding an Fc receptor. In some aspects, the Fc receptor is CD64, CD32, or CD16. In some aspects, the CD64 is a truncated (CD64t) that lacks a functional ICD. In some aspects, the CD64 is truncated (CD64t) and lacks a functional TMD In some aspects, the CD64t has at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 6. In some aspects, the CD64t has the amino acid sequence of SEQ ID NO: 6.

[0022] In some aspects, the polynucleotide encodes a self-cleaving linker between the plurality of heterologous genes. In some aspects, the self-cleaving linker is a 2A peptide. In some aspects, the 2A peptide is a Porcine teschovirus-1 2A (P2A), an Equine rhinitis A virus 2A (E2A), Foot-and-mouth disease virus 18 2A (F2A), or a Thosea asigna virus 2A (T2A) peptide.

[0023] In some aspects, the polynucleotide includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 7. In some aspects, the polynucleotide includes the sequence of SEQ ID NO: 7.

[0024] In some aspects, the heterologous gene is oriented 3’-5’ in relation to the endogenous gene. In some aspects, the heterologous gene is oriented 5’-3’ in relation to the endogenous gene. In some aspects, the mammalian cell is a human cell. In some aspects, the cell is a primary cell, a pluripotent cell, an embryonic stem cell, a somatic stem or progenitor cell, an immune cell, a cell differentiated from a pluripotent cell, or a cell differentiated from an embryonic stem cell. In some aspects, the pluripotent cell is a hypoimmune pluripotent cell or an induced pluripotent stem cell (iPSC) In some aspects, the cell is an iPSC or an embryonic stem cell (ESC) derived cell In some aspects, the immune cell is a T cell, a natural killer (NK) cell, or a macrophage. In some aspects, the immune cell is an innate immune cell. In some aspects, the cell includes reduced or eliminated human leukocyte antigen (HLA) class I (HLA-I), HLA class II (HLA-II), p-2 microglobulin (B2M), endogenous class II transactivator (CIITA), and / or endogenous T cell receptor alpha subunit constant (TRAC) expression.

[0025] In another aspect, the disclosure provides a method of editing a genome of a mammalian cell, including inserting a polynucleotide that includes a heterologous gene encoding a SIRPa engager into one or more sites within an endogenous B2M locus, an endogenous CIITA locus, or an endogenous TRAC locus.

[0026] In some aspects, the polynucleotide is inserted into exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITAgene, exon 1 of the endogenous TRAC gene, or a combination thereof.

[0027] In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, or more copies of the polynucleotide are inserted.

[0028] In some aspects, the method further includes introducing one or more single guide RNAs (sgRNA) into the cell that specifically hybridizes to a location within one or more sites including exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITA gene, exon 1 of the endogenous TRAC gene, or a combination thereof. In some aspects, 1, 2, 3, 4, or more sgRNAs are introduced into the cell.

[0029] In some aspects, the method further includes providing a nuclease that introduces a double stranded DNA break to create the insertion site. In some aspects, the nuclease is a Cas9 nuclease, a Cpf1 nuclease, a Cas12b nuclease, a CasX nuclease, a MAD7 nuclease, a LEG14 nuclease, a LEG95 nuclease, a LEG98 nuclease, or a LEG145 nuclease.

[0030] In some aspects, the method includes complexing the nuclease with the sgRNAto form a ribonucleoprotein (RNP) complex, wherein the RNP is then introduced into the cell to create the insertion site. In some aspects, the ratio of nuclease to sgRNA is 1 :1 to 1 :10 (e.g., 1 :1 , 1:2, 1:3, 1:4, 1 :5, 1:6, 1:7, 1:8, 1 :9, or 1 :10). In some aspects, the method includes a first incubation step, wherein the nuclease and sgRNA are incubated for between 1 to 120 (e.g., 10 to 90) minutes at a temperature of 15°C to 45 °C (e.g., 20 °C to 37 °C) to create the RNP complex. In some aspects, the method further includes a second incubation step. During the second incubation step, the RNP may be further complexed to the polynucleotide, e.g., at a concentration of from 0.5 pg to 400 pg per ml (e.g., 4 pg to 160 pg per ml). In some aspects, the second incubation step, wherein the RNP and polynucleotide are incubated for 1 to 100 (e.g., 5 to 30) minutes at a temperature of 15°C to 45 °C (e.g., 20 °C to 37 °C).

[0031] In some aspects, the method further includes delivering the RNP and polynucleotide complex into the cell. In some aspects, the RNP and polynucleotide complex is inserted via electroporation. In some aspects, the polynucleotide is on a plasmid. In some aspects, the heterologous gene is inserted into the insertion site by homology directed repair.

[0032] In some aspects, the mammalian cell is a human cell. In some aspects, the cell is a primary cell, a pluripotent cell, an embryonic stem cell, or an immune cell. In some aspects, the pluripotent cell is a hypoimmune pluripotent cell or an iPSC In some aspects, the cell is an iPSC or an ESC derived cell In some aspects, the immune cell is a T cell, an NK cell, or a macrophage. In some aspects, the immune cell is an innate immune cell. In some aspects, the cell includes reduced or eliminated HLA-I, HLA-II, B2M, CIITA, and / or TRAC expression.

[0033] In some aspects, the method further includes differentiating the pluripotent cell. In some aspects, the pluripotent cell is edited prior to differentiation. In some aspects, the pluripotent cell is differentiated prior to editing. In some aspects, the pluripotent cell is differentiated into a T cell, an NK cells, or a macrophage.

[0034] In some aspects, the SIRPa engager includes a CD47 ECD. In some aspects, the CD47 ECD includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1. In some aspects, the CD47 ECD includes the amino acid sequence of SEQ ID NO: 1. In some aspects, the SIRPa engager includes an scFv. In some aspects, the scFv includes heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 20- 22 and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 23-25. In some aspects, the SIRPa engager lacks a functional CD47 ICD In some aspects, the SIRPa engager includes a hinge region. In some aspects, the hinge region is a CD8a hinge region.

[0035] In some aspects, the SIRPa engager includes a heterologous TMD. In some aspects, the heterologous TMD is selected from the group consisting of CD47, CD85f, CD349, CD284, CD261 , CD 172b, CD277, CD 186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221 , CD271 , CD55, CD243, OD98, CD10, CD41 , CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331 , CD222, CD20, CD79a, CD64, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227. CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, OD252, CD89, CD124, CD181 , CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CD1 b, CD1c, CD154, CD6, CD52, CD132, CD32, CD66b, CD171 , CD191 , CD197, CD185, CD131 , CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361 , CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231 , CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD1581, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195. CD196, CDWI98, CDWI99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91 , CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161 , CD215, CD150, CD11d, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111 , CD281 , CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD300f, CD92, CD351 , CD112, CD100, CD270, CD101 , CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD114, CD180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201 , CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11 , CD51 , CD41 , CD29: CD18, CD61 , CD104, and PDGFR TMD.

[0036] In some aspects, the SIRPa engager includes a polypeptide having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to ID NO: 5. In some aspects, the SIRPa engager includes a polypeptide sequence having the sequence of SEQ ID NO: 5.

[0037] In some aspects, the SIRPa engager includes a polypeptide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 29. In some aspects, the SIRPa engager includes the amino acid sequence of SEQ ID NO: 29.

[0038] In some aspects, the polynucleotide further includes a heterologous promoter, an enhancer, and / or a polyadenylation sequence In some aspects, the heterologous promoter is operably linked to the heterologous gene. In some aspects, the heterologous promoter is a constitutive promoter, an inducible promoter, a cell-type specific promoter, or a tissue specific promoter In some aspects, the heterologous promoter is selected from the group consisting of EF1a, UBC promoter, EFS promoter, CD11 b promoter, sp146 / 147 promoter, CD68 promoter, sp146 / gp9, CD47 promoter, PGK promoter, SV40 promoter, CMV promoter, human b-actin promoter, UAS promoter, AC5 promoter, CaMKIla promoter, GAL1 / GAL10 promoter, GDS promoter, TRE promoter, U6 promoter, H1 promoter, 7SK promoter, DHFR promoter, EGR 1 promoter, TBG promoter, EIF4A1 promoter, FerH promoter, FerL promoter, GAPDH promoter, HSPA5 promoter, HSPA4 promoter, UBB promoter, EBV promoter, MND promoter, LTR of MuMLV promoter, MMTV promoter, RSV promoter, Apolipoprotein E-Human Alpha 1 -Antitrypsin promoter, CAG promoter, CBA promoter, CK8 promoter, and Mu1a promoter.

[0039] In some aspects, the enhancer is a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) or Simian Virus 40 (SV40) enhancer.

[0040] In some aspects, the polynucleotide includes a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of heterologous genes. In some aspects, the polynucleotide includes a first heterologous gene encoding the SIRPa engager and a second heterologous gene encoding an Fc receptor In some aspects, the Fc receptor is CD64, CD32, or CD16. In some aspects, the CD64 is CD64t that lacks a functional ICD and / or TMD. In some aspects, the CD64t includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 6. In some aspects, the CD64t includes the amino acid sequence of SEQ ID NO: 6.

[0041] In some aspects, the polynucleotide includes a polynucleotide encoding a self-cleaving linker between the plurality of heterologous genes. In some aspects, the self-cleaving linker is a 2A peptide. In some aspects, the 2A peptide is a P2A, E2A, F2A, or T2A peptide.

[0042] In some aspects, the polynucleotide includes a sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 7.

[0043] In another aspect, the disclosure provides an expression cassette that includes a polynucleotide arranged in the following 5’ -3’ order, a 5’ homology arm homologous to an endogenous sequence upstream of the insertion site, a nucleic acid encoding a SIRPa engager, and a 3’ homology arm homologous to an endogenous sequence downstream of the insertion site, the expression cassette is configured to be inserted into an endogenous B2M gene, an endogenous CIITA gene, or an endogenous TRAC gene.

[0044] In another aspect, the disclosure provides an expression cassette including a polynucleotide arranged in the following 5’-3’ order, a 3’ homology arm homologous to an endogenous sequence upstream of the insertion site, a nucleic acid encoding a SIRPa engager, and a 5’ homology arm homologous to an endogenous sequence downstream of the insertion site, where the expression cassette is configured to be inserted into an endogenous B2M gene, an endogenous CIITA gene, or an endogenous TRAC gene.

[0045] In some aspects, the insertion site is within exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITAgene, or exon 1 of the endogenous TRAC gene

[0046] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least an 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 9, 11 , 13, or 15. In some aspects, the 5’ homology arm includes a polynucleotide sequence having at least 85% (e.g. , at least 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 8, 10, 12, or 14.

[0047] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 9. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 8.

[0048] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 11. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 10.

[0049] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 13. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 12.

[0050] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 15. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 14.

[0051] In some aspects, the SIRPa engager includes a CD47 ECD. In some aspects, the CD47 ECD includes an amino acid sequence having at least an 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 1 . In some aspects, the CD47 ECD includes the amino acid sequence of SEQ ID NO: 1 In some aspects, the SIRPa engager includes an scFv In some aspects, the scFv includes heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 20- 22 and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 23-25. In some aspects, the SIRPa engager lacks a functional CD47 ICD. In some aspects, the SIRPa engager includes a hinge region. In some aspects, the hinge region is a CD8a hinge region.

[0052] In some aspects, the SIRPa engager includes a heterologous TMD. In some aspects, the heterologous TMD is selected from the group consisting of CD47, CD85f, CD349, CD284, CD261 , CD 172b, CD277, CD 186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61 , CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221 , CD271 , CD55, CD243, CD98, CD10, CD41 , CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331 , CD222, CD20, CD79a, CD64, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227. CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181 , CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CD1 b, CD1c, CD154, CD6, CD52, CD132, CD32, CD66b, CD171 , CD191 , CD197, CD185, CD131 , CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361 , CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231 , CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD1581, CD178, CD295, CD151, CD97, CD183, CD39, CD239, CD193, CD194, CD195. CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91 , CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161, CD215, CD150, CD11d, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111, CD281 , CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD100, CD270, CD101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD114, CD180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201 , CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51, CD41 , CD29: CD18, CD61, CD104, and PDGFR TMD.

[0053] In some aspects, the SIRPa engager includes a polypeptide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 5. In some aspects, the SIRPa includes the amino acid sequence of SEQ ID NO: 5.

[0054] In some aspects, the expression cassette further includes a heterologous promoter, an enhancer, and / or a polyadenylation sequence. In some aspects, the heterologous promoter is operably linked to the heterologous gene. In some aspects, the heterologous promoter is a constitutive promoter, an inducible promoter, a cell-type specific promoter, or a tissue-specific promoter. In some aspects, the heterologous promoter is selected from the group consisting of EF1a, UBC promoter, EFS promoter, CD11b promoter, sp146 / 147 promoter, CD68 promoter, sp146 / gp9, CD47 promoter, PGK promoter, SV40 promoter, CMV promoter, human b-actin promoter, UAS promoter, AC5 promoter, CaMKIla promoter, GAL1 / GAL10 promoter, GDS promoter, TRE promoter, U6 promoter, H1 promoter, 7SK promoter, DHFR promoter, EGR 1 promoter, TBG promoter, EIF4A1 promoter, FerH promoter, FerL promoter, GAPDH promoter, HSPA5 promoter, HSPA4 promoter, UBB promoter, EBV promoter, MND promoter, LTR of MuMLV promoter, MMTV promoter, RSV promoter, Apolipoprotein E-Human Alpha 1-Antitrypsin promoter, CAG promoter, CBA promoter, CK8 promoter, and Mu1a promoter.

[0055] In some aspects, the enhancer is a WPRE or SV40 enhancer.

[0056] In some aspects, the expression cassette includes a plurality e g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of heterologous genes. In some aspects, the expression cassette includes a first heterologous gene encoding the SIRPa engager and a second heterologous gene encoding an Fc receptor. In some aspects, the Fc receptor is CD64, CD32, or CD16. In some aspects, the CD64 is CD64t that lacks a functional ICD or TMD. In some aspects, the CD64t includes an amino acid sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to of SEQ ID NO: 6. In some aspects, the CD64t includes the amino acid sequence of SEQ ID NO: 6.

[0057] In some aspects, the expression cassette includes a polynucleotide encoding a self-cleaving linker between the plurality of heterologous genes In some aspects, the self-cleaving linker is a 2A peptide. In some aspects, the 2A peptide is a P2A, E2A, F2A, or T2A peptide.

[0058] In some aspects, the expression cassette includes a polynucleotide sequence having at least an 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 7.

[0059] In another aspect, the disclosure provides a pharmaceutical composition that includes the cell of any of the above aspects and a pharmaceutically acceptable carrier.

[0060] The invention provides the modified cells as disclosed herein further comprising a suicide gene that is activated by a trigger that causes the modified cell to die In some aspects, the suicide gene is a herpes simplex virus thymidine kinase gene (HSV-tk) and the trigger is ganciclovir. In a preferred aspect, the HSV-tk gene encodes a protein comprising at least a 90% sequence identity to SEQ ID NO: 26. In another preferred aspect, the HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO: 26. In another aspect, the suicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) and the trigger is 5-fluorocytosine (5-FC). In a preferred aspect, the EC-CD gene encodes a protein comprising at least a 90% sequence identity to SEQ ID NO: 27. In another preferred aspect, the EC-CD gene encodes a protein comprising the sequence of SEQ ID NO: 27. In another aspect, the suicide gene encodes an inducible Caspase protein and the trigger is a chemical inducer of dimerization (CID) In a preferred spect, the suicide gene encodes an inducible Caspase protein comprising at least a 90% sequence identity to SEQ ID NO: 28. In another preferred aspect, the suicide gene encodes an inducible Caspase protein comprising the sequence of SEQ ID NO: 28 In another aspect, the CID is AP1903.

[0061] In another aspect, the disclosure provides a method of treating a disease in a subject by administering a cell or pharmaceutical composition including the cell as described herein. The method may include transplanting the cell or pharmaceutical composition of any of the above aspects into the subject. In some aspects, the disease is Type 1 diabetes, a cardiac disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease. In some aspects, the cancer is a blood cancer (e.g., leukemia, lymphoma, or myeloma)

[0062] In another aspect, the invention provides cells with heterologous genes as disclosed herein for transplanting into a subject for treating a disease In some aspects, the disease is Type 1 diabetes, a cardiac disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease. In other aspects, the cancer is a blood cancer. In preferred aspects, the blood cancer is leukemia, lymphoma, or myeloma.

[0063] In another aspect, the invention provides a kit, comprising the pharmaceutical compositions as disclosed herein and instructions for their use

[0064] In another aspect, the invention provides a polypeptide including a SIRPa engager and a CD64 (e.g., CD64t lacking a functional ICD and / or TMD). In some aspects, the polypeptide includes a self-cleaving peptide between the SIRPa engager and the CD64. The self-cleaving peptide may be a 2A peptide. The polypeptide may include the structure from the amino terminus to the carboxy terminus a SIRPa engager-2A-CD64 or a CD64-2A- SIRPa engager.

[0065] In another aspect, the invention provides a polynucleotide (e.g., an expression cassette) encoding the polypeptide of the foregoing aspect.

[0066] Definitions

[0067] Terms as set forth hereinafter are generally to be understood by their plain meaning unless indicated otherwise.

[0068] In this application, unless otherwise clear from context, (i) the term “a” may be understood to mean “at least one”; (ii) the term “or” may be understood to mean “and / or”; and (iii) the terms “including” and “comprising” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps.

[0069] The term “about" refers to an amount that is ± 10% of the recited value and may be ± 5% of the recited value or ± 2% of the recited value.

[0070] As used interchangeably herein, in the context of a gene of interest, the term “disrupt” and “knock-out” refer to preventing the formation of a functional gene product. Disruption, or knocking out, the gene prevents expression of a functional factor encoded by the gene (e.g., RNAor protein) and may be achieved, for example, by way of an insertion, deletion, or substitution of one or more bases in a sequence encoded by the gene and / or a promoter and / or an enhancer that is necessary for expression of the gene in a subject. The disrupted gene may be disrupted by, e.g., removal of at least a portion of the gene from a genome of the subject, alteration of the gene to prevent expression of a functional factor encoded by the gene (e.g., protein or RNA), an interfering RNA, or expression of a dominant negative factor by an exogenous gene

[0071] As used interchangeably herein, in the context of a gene of interest, the term “insertion” and “knock-in” (KI) refer to allowing the formation of a functional gene product. Insertion, or knocking-in, the gene allows for expression of a functional factor encoded by the gene (e.g., protein or RNA) and may be achieved, for example, by introducing a gene, transgene, or expression cassette into the genome.

[0072] As used herein, the term “endogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell).

[0073] As used herein, the term “exogenous” describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is not found naturally in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, a tissue, or a cell, such as a human cell). Exogenous materials include those that are provided from an external source to an organism or to cultured matter extracted therefrom.

[0074] As defined herein, the term “nucleic acid” and “polynucleotide” are interchangeable and refer to RNA, DNA, or derivatives thereof that are linear or branched, single or double stranded, or a hybrid thereof, regardless of length (e g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, or more nucleic acids). The term also encompasses RNA / DNA hybrids. Nucleotides are typically linked in a nucleic acid by phosphodiester bonds, although the term “nucleic acid” also encompasses nucleic acid analogs having other types of linkages or backbones (e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoroamidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or backbones, among others). The nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequence. A nucleic acid can contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-canonical bases (including, e.g., hypoxanthine, xanthine, 7-methylguanine, 5,6- dihydrouracil, 5-methylcytosine, and 5 hydroxymethylcytosine).

[0075] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide can be a single molecule or may be a multi- molecular complex such as a dimer, trimer, or tetramer. They can also include single chain or multichain polypeptides such as antibodies or insulin and can be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0076] As used herein, “promoter” refers to a recognition site on DNA that is bound by an RNA polymerase. The polymerase drives transcription of the polynucleotide. Exemplary promoters suitable for use with the compositions and methods described herein are described, for example, in Sandelin et al., Nature Reviews Genetics 8:424, 2007, the disclosure of which is incorporated herein by reference as it pertains to nucleic acid regulatory elements. Additionally, the term “promoter” may refer to synthetic promoters, which are regulatory DNA sequences that do not occur naturally in biological systems. Synthetic promoters contain parts of naturally occurring promoters combined with polynucleotide sequences that do not occur in nature and can be optimized to express recombinant DNA using a variety of polynucleotides, vectors, and target cell types

[0077] As used herein, “percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are well-known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Using well-recognized and conventional methods, the appropriate parameters can be determined for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows:

[0078] 100 multiplied by (the fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0079] As used herein, the term “pluripotent cell” refers to a cell that has the unlimited capacity to divide, self-renew and differentiate into cells of early primary germ cell layers, namely mesoderm, endoderm, and ectoderm. Examples of pluripotent cells are embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). “Multipotent cells” can develop into more than one differentiated cell type but are more limited than pluripotent cells. Examples for multipotent cells are hematopoietic stem cells such as CD34+ cells or mesenchymal stromal cells (MSCs) Hematopoietic stem cells (HSCs) are multipotent primitive cells that can develop into all types of blood cells, including myeloid-lineage and lymphoid-lineage cells Hematopoietic lineages include granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g., megakaryoblasts, platelet producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g , NK cells, B-cells and T- cells)..

[0080] As used interchangeably herein, the terms “polyA” and “polyA sequence” refer to an untranslated, contiguous region of a nucleic acid molecule of at least 5 nucleotides in length and consisting of adenosine residues In some aspects, a polyA sequence is at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides in length. In some aspects, a polyA sequence is located 3’ to (e.g., downstream of) an open reading frame (e g., an open reading frame encoding a polypeptide), and the polyA sequence is 3’ to a termination element (e.g., a stop codon) such that the polyA is not translated. In some aspects, a polyA sequence is located 3’ to a termination element and a 3' untranslated region.

[0081] As used herein, the term “regulatory element” includes promoters, enhancers, cleavage sequences, insulators, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the gene(s) Such regulatory sequences are described, for example, in Perdew et al., Regulation of Gene Expression (Humana Press, New York, NY, 2014), incorporated by reference in its entirety.

[0082] As used herein, the term “transgene” refers to a recombinant nucleic acid (e.g., DNA or cDNA) encoding a gene product (e g , a gene product described herein) The gene product may be an RNA, peptide, or protein. In addition to the coding region for the gene product, the transgene may include or be operably linked to one or more elements to facilitate or enhance expression. Examples include a promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements. Aspects of the disclosure may utilize any known suitable promoter, enhancer(s), destabilizing domain(s), response element(s), reporter element(s), insulator element(s), polyadenylation signal(s), and / or other functional elements.

[0083] As used herein, the term “expression cassette” refers to a polynucleotide generated recombinantly or synthetically that contains a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a target cell. The expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and optionally, one or more of a promoter, an enhancer, and a polyadenylation sequence. In certain aspects, the expression cassette of the invention includes polynucleotide sequences that encode a SIRPa engager, as described herein

[0084] As used herein, the term “transfection” refers to any of a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell. Examples include electroporation, lipofection, calcium- phosphate precipitation, DEAE- dextran transfection, Nucleofection, squeezeporation, sonoporation, optical transfection, Magnetofection, impalefection, and the like.

[0085] Descriptions of the Drawings

[0086] FIG. 1A is a schematic drawing of the site-specific insertion of an exemplary SIRPa engager (SIRPaE) cassette. The SIRPa engager coding sequence is operably linked to the EF1a promoter, followed by a stop codon (“Stop”), a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE), and a polyadenylation sequence (pA) The cassette is shown inserted into exon 1 of the B2M locus via Cas9 and single guide RNA (sg 1). SIRPaE used had the following structure: CD8a signal peptide - myc tag - VH - (GGGGS)s linker - VL - CD8a hinge - PDGFR TMD (SEQ ID NO: 29). Experiments employed a myc-tagged SIRPaE construct (myc tag not shown in FIG. 1 A)

[0087] FIG. 1B are graphs showing the knock-in (KI) efficiency of the SIRPaE transgene into the B2M locus in the presence or absence of the DNA-dependent protein kinase inhibitor AZD7648. Unedited human wild-type (WT) iPSCs transduced with a glypican-3 (GPC3)-reactive T cell receptor (TOR) served as starting material. Flow cytometry analyses were performed using antibodies against HLA-ABC and the G4S linker, which is part of the SIRPaE molecular structure. The knock-in (KI) efficiency was 7.1%. Using AZD7648, the KI was improved to 24.9%.

[0088] FIGS. 2A and 2B are graphs showing the percentage of human iPSCs positive for GPC3 TOR (shown by a CD19 tag), establishing the differentiation capacity of SIRPa engager iPSCs into CD8ap T cells. (FIG. 2A) and SIRPaE (shown by a myc-tag) (FIG. 2B) prior to iPSC differentiation. Human iPSCs that had previously been transduced to express a GPC3-reactive TCR together with a CD19 tag were used as parental cells FIG. 2A shows the iPSC populations that are positive for the constitutively expressed, pluripotent cell marker SSEA-4 and the CD19-tagged GPC3 TCR. FIG. 2B shows the percentage of iPSCs expressing the myc-tagged SIRPaE and CD64, which is constitutively expressed on monocytes and macrophages. The SIRPaE transgene with a myc-tag had been transduced using lentiviral particles.

[0089] FIG. 3 are graphs showing the differentiation capacity of SIRPaE-transduced GPC3-TCR+iPSCs (lower panel) into hematopoietic progenitor cells (CD45+CD7+) assessed and compared to its parental IPSO line (upper panel). Cells of interest were identified, shown by the first plots on the left, based on size (forward scatter or FSC) and granularity (side scatter or SSC). Cell viability was assessed using the DNA binding dye propidium iodide (PI), detectable within the phycoerythrin (PE) channel (second plots from the left). Cell differentiation was further assessed by the presence of: CD45 and CD7 (markers of hematopoietic progenitor cells) (middle plots), the T-cell marker CD8a and CD19-tagged GPC3-TCR (second plots from the right), and the differentiation marker CD7 and T cell marker CD5 (plots on the right). The SIRPaE-transduced iPSCs were differentiated into hematopoietic progenitor cells (CD45+CD7+) at high efficiency (97.6%) and 94.2% expressed the TCR-tagged with CD19.

[0090] FIG. 4 is a set of graphs showing the differentiation of SIRPaE-transduced iPSCs into CD3+TCRap+T cells (lower panel). These were assessed and compared to its parental iPSC line (upper panel). Dendritic cell marker CD1a and T cell marker CD5 were assessed in the left-hand plots. Differentiation into T cells was further assessed by the presence of CD3 and TCR a and p chains (second plots from the left), and CD4 and CD8 to determine the type of T cell. 93.6% of cells differentiated from SIRPaE-transduced IPSCs were positive for CD8a and CD8p.

[0091] FIGS. 5A and 5B show the site-specific insertion of the SIRPaE and truncated CD64 (CD64t) sequences into the B2M locus. FIG. 5A is a schematic showing an exemplary donor template design for the cassette. An EF1a promoter is operably linked to a SIRPaE, followed by a self-cleaving 2A sequence, a CD64t, a stop codon, a WPRE regulatory element and a polyadenylation sequence. The cassette is shown knocked-into the B2M locus using Cas9 and single guide RNA (sg 1) In FIG. 5B, edited human iPSCs with or without the SIRPaE, CD64t cassette knocked into the B2M locus (B2M- B2M-KO, respectively) were sorted for HLA l-depleted iPSCs with (B2M-KOSIRPa‘ B2M-KO) transgene expression FIG. 5B is a set of flow cytometry histograms of B2M-KO, B2M-KOSIRPa’E CD64t, and unedited WT iPSCs. The stained samples are labeled against isotype controls. Antibodies against B2M, CD64, and myc-tagged SIRPaE (myc-tag not shown) were used. The WT iPSCs showed B2M expression, which was successfully knocked out in the two edited iPSCs The B2M-KOSIRPa'E CD64tshowed high expression of both transgenes

[0092] FIG. 6 is a set of flow cytometry graphs showing the stability of transgene expression, SIRPaE and CD64t, in the B2M locus in iPSCs over time. The upper panel shows flow plots with antibodies against CD64 and HLA-ABC or B2M. The lower panel shows CD64 against the myc- tagged SIRPaE protein. The data show some fluctuation, but overall stable expression of both CD64t and the SIRPaE over 49 days post KI.

[0093] FIG. 7 is set of graphs showing in vitro cytotoxicity assays over time with activated NK cells or macrophages as effector cells The effector cells were used in a 1 :1 effector-to-target cell (E:T) ratio The upper panel shows, from left to right, the cell index for B2M-KO iPSCs without added effector cells, rapid killing with added effector NK cells, and rapid killing with added effector macrophages. The lower panel shows, from left to right, the cell index for B2M-KOSIRPchE CD64tiPSCs without added effector cells, and a maintained stable cell index level in the presence of NK cells or macrophages. This shows that the SIRPaE protein protected the cells from both innate immune cell populations.

[0094] FIGS. 8A and 8B are graphs showing lgG1 -mediated target cell killing over time. Unedited (WT) and B2M-KOSIRP“'E CD64tiPSCs were assessed, from left to right, with the iPSCs alone, NK cells added at a 1 : 1 E:T ratio, and NK cells at a 1 : 1 E:T ratio with increasing concentrations of a human lgG1 antibody against the constitutively expressed stem cell antigen SSEA4. WT IPSCs were rapidly killed at antibody concentrations of 0.01 pg / ml and above. In contrast, the B2M-KOSIRPa'E CD64tiPSCs were protected from antibody-mediated cellular cytotoxicity (ADCC) and showed stable cell index curves throughout the antibody concentration range (FIG. 8A). FIG. 8B shows complementdependent cytotoxicity (CDC) assays with the same anti-SSEA4 lgG1 antibody. Unedited (WT) and B2M-KOSIRPa'E CD64tiPSCs were assessed, from left to right, with the iPSCs alone, added complement, and complement along with increasing concentrations of anti-SSEA4 WT iPSCs were again rapidly killed with increasing anti-SSEA4 antibody concentrations, while B2M-KOSIRPa’E CD64tiPSCs remained protected.

[0095] FIG. 9 is a set of flow cytometry plots showing iPSCs edited to disrupt CIITA, T Cell Receptor Beta Constant 1 (TRBC1 ), and T Cell Receptor Beta Constant 2 (TRBC2). They were further edited to express (1 ) SIRPaE and CD64t transgenes knocked into the B2M locus and (2) a GPC3-reactive TCR and a CD19 transgene knocked into the TRAC locus. CD64-negative and CD64-positive populations were sorted, as seen on the Y-axis assessing Brilliant Violet™ 421 (BV421 ) stained CD64. Both populations were HLA class I and class II depleted, as seen on the X-axis assessing phycoerythrincyanine? (PE-Cy7) stained B2M.

[0096] FIGS. 10A and 10B are graphs showing in vitro cytotoxicity assays over time of target cells as described in FIG. 9, with activated NK cells or macrophages as effector cells. From left to right, each panel first shows target cells alone, and then added effector cells in increasing effector-to-target cell (E:T) ratios. In FIG. 10A, the upper panel in shows the cell index for the B2M7CD64' iPSCs that did not express CD64t or SIRPaE. These target cells were rapidly killed with NK cells at an E:T ratio of 1 :5 or above. The lower panel shows B2M7CD64+iPSCs that were fully protected against NK cell killing. FIG. 10B shows cytotoxicity assays with macrophages (MAC) The B2M7CD64’ iPSCs were again killed at E:T ratios of 1 :5 or above (upper panel), while B2M7CD64+iPSCs were protected from MACs (lower panel).

[0097] FIGS. 11 A and 11 B are graphs showing antibody-mediated cellular cytotoxicity (ADCC) assays over time of target cells as described in FIG. 9. From left to right, each panel first shows target cells alone, with added NK cells at an E:T ratio of 1 : 1 , and then with NK cells at an E:T ratio of 1 :1 and increasing concentrations of an anti-SSEA4 lgG1 antibody (FIG. 11 A) or an anti-CD19 lgG1 antibody (FIG. 11 B). Using the anti-SSEA4 lgG1 antibody, the B2M7CD64+iPSCs maintained stable cell indices for all tested antibody concentrations (FIG. 11 A) Using a human anti-CD19 lgG1 antibody, ADCC assays showed that B2M7CD64+iPSCs were protected and showed stable cell indices (FIG. 11 B). FIGS. 12A and 12B are graphs showing complement dependent cytotoxicity (CDC) assays over time of target cells as described in FIG. 9. From left to right, each panel first shows target cells alone, target cells with complement, and then target cells with added complement and increasing concentrations of either an anti-SSEA4 (FIG. 12A) or an anti-CD19 (FIG. 12B) IgG 1 antibody. The upper panels of FIG. 12A and FIG. 12B show B2M7CD64" IPSCs that are susceptible to antibody- mediated complement killing via anti-SSEA4 and anti-CD19 antibodies, respectively. The lower panels of FIG. 12A and FIG. 12B show the B2M7CD64+iPSCs, which are protected against CDC killing with both antibodies across the range tested.

[0098] FIGS. 13A and 13B are schematic drawings showing an exemplary donor template and different insertion sites. The donor template is a cassette that includes an EF1a promoter operably linked to a SIRPaE sequence, followed by a self-cleaving 2A sequence, a CD64t sequence, a stop codon, a WPRE regulatory element, and a polyadenylation sequence (FIG. 13A). Two insertion sites are located in the B2M locus, one site is located in the CIITA locus, and one site is located in the TRAC locus (FIG. 13B). The sites are targeted via the nucleases Cas9 or MAD7, and sgRNAs sg1, sg2, sg3 and cr8.

[0099] FIGS. 14A-14D are graphs showing the knock-in (KI) efficiencies of the donor template (as described in FIG. 13A) at four insertion sites: the B2M locus site 1 , targeted with Cas9 nuclease and sg1 sgRNA (FIG. 14A), the B2M locus site 2, targeted with MAD7 nuclease and cr8 sgRNA (FIG. 14B), CIITA locus, targeted with Cas9 nuclease and sg2 sgRNA (FIG. 14C), and the TRAC locus, targeted with Cas9 nuclease and sg3 sgRNA (FIG. 14D). The flow cytometry plots show CD64 expression as marker for the donor template, HLA-ABC as a proxy for B2M, and side scatter (SSC) as a measure of granularity. From left to right, the panels show cells with the indicated locus knocked out (KO), the donor template knocked in (KI), and KI cells with either the DNA-dependent protein kinase inhibitor AZD7648 or CTS™ Xenon™ genome editing (GE) buffer to increase KI efficiency The donor template was successfully knocked into both sites at the B2M locus (FIG. 14A, FIG. 14B). Insertion into the CIITA locus had low efficiency rates (FIG. 14C), and KI efficiency at the TRAC locus was modest (FIG. 14D).

[0100] FIGS. 15A and 15B are graphs exemplifying expression of SIRPa on IL-2 stimulated primary human NK cells (top plot) and macrophages (bottom plot), as assessed by cell size (FSC) and presence of SIRPa (FIG. 15A). Percentages of NK cells and macrophages positive for SIRPa were determined from the mean of 5 healthy human donors (FIG. 15B).

[0101] FIGS. 16A and 16B are graphs of cytotoxicity assays over time showing the susceptibility to killing by effector NK cells and macrophages at increasing E:T ratios. Fig. 16A shows B2M'ACIITA'" double knock-out (DKO) iPSC-derived endothelial cells (iECs). FIG. 16B shows DKO iECs expressing the SIRPa engager. The SIRPa engager was transduced into DKO iECs using lentiviral particles From left to right, the panels show DKO or DKOSIRPaEiECs alone or with increasing E:T ratios of either NK cells or macrophages The DKO iECs were killed by the effector cells at increasing E:T ratios (FIG. 16A), while the DKOSIRPaEiECs were protected from killing (FIG. 16B). When compared to the results shown in FIGS. 7 and 10, the CRISPR-engineered SIRPa engager was as effective at protecting the cells from innate immune cell killing as the lentiviral-engineered SIRPa engager cells. FIGS. 17A-17E are graphs showing the susceptibility of DKO iECs (FIG. 17A) and DKOCD64tcells (FIG. 17B) to killing by NK cells at a 1 :1 E:T ratio, over time DKOCD64t slRPaEiECs expressing the SIRPa engager were protected at increasing E:T ratios (FIG. 17C). In FIG. 17D, antibody-mediated cellular cytotoxicity (ADCC) of DKOSIRPaEand of DKOCD84t’SIRPaEiECs was assessed by adding increasing E:T ratios of NK cells to target cells, along with an anti-MHC class l-related sequence A (MICA) antibody added at a constant concentration of 1 pg / ml. In FIG. 17E, both ADCC and complement dependent cytotoxicity (CDC) of DKOSIRPaEand of DKOCD64t'SIRPaEiECs was assess by adding serum to target cells, along with increasing E:T ratios of NK cells and a constant concentration of the anti-MICA antibody. Only iECs expressing both the SIRPa engager and CD64t were protected from NK cell killing, ADCC, and CDC.

[0102] FIGS. 18A-18H are schematic drawings and graphs showing DKOCD64t’SIRPaEiEC survival during harsh allogeneic immune conditions. Cells expressed CD52, making them susceptible to the highly cytotoxic anti-CD52 lgG1 antibody alemtuzumab. Susceptibility of WTCD52(FIG. 18A) and DKOCD64t'SIRPaE-0052cells (FIG. 18B) to hostile in vitro immune conditions including antibody-mediated cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) was assessed From top left to bottom right, WTCD52and DKOCD64t’SIRPaE’CD52iECs were assessed alone, or challenged with NK cells; NK cells and alemtuzumab; NK cells, alemtuzumab, and serum; macrophages; macrophages and alemtuzumab; primed allogeneic peripheral blood mononuclear cells (PBMCs); PBMCs and alemtuzumab; PBMCs, alemtuzumab, and IL-2; PBMCs, alemtuzumab, IL-2, and serum; serum alone; and serum and alemtuzumab. Only DKOCD64t slRPaE CD52cells were protected from all allogeneic immune cell and IgG attacks. In FIGS. 18C-18H, WTCD52cells, DKOCD64ti SIRPaE-CD52cells, and DKOCD64t’ SIRPOEce||s weretested in a stringent, allogeneic, NSG-SGM3 (NOD.Cg-PrkdcscidI I2rgtm1 WjlTg(CMV-IL3,CSF2,KITLG)1 Eav / MloySzJ) humanized mouse model Mice were additionally injected with one dose of NK cells, and three doses of either an anti-CD52 antibody (alemtuzumab) (FIGS. 18C-18E) or an anti-MICA antibody (FIGS. 18F-18H). Retention of cell grafts over time was assessed via bioluminescence imaging (BLI). In the presence of supplemental NK cells and alemtuzumab, the WTOD52grafts vanished quickly (FIG. 18D), while the DKOCD64ti SIRPaE’CD52grafts were retained throughout the 28 days tested (FIG. 18E). In the presence of supplemental NK cells and the anti- MICA antibody, the WTCD52grafts vanished over the course of two weeks (FIG. 18G), while the DKOCD64t’SIRPaEgrafts were again retained throughout the 28 days tested (FIG. 18H).

[0103] FIG.19 shows that double knock-out (DKO) iPSC-derived ap T cells (IT) and DKO iT- expressing SIRPa engager and CD64t resist killing by NK cells and macrophages at increasing E:T ratios The SIRPa engager and CD64t were introduced into the iT cells via gamma retroviral vector transductions. DKO cells expressing both the SIRPa engager and CD64t were protected from innate immune cell killing. In contrast, the DKO cells lacking the SIRPa engager and CD64t were killed.

[0104] Detailed Description

[0105] The present disclosure provides compositions, polynucleotides, and methods relating to genomic insertion and cellular expression of a Signal-regulatory-protein a (SIRPa) engager that binds SIRPa. This disclosure also provides methods of inserting one or more copies of the SIRPa engager transgene into a mammalian cell, e g., at a plurality of insertion sites. Compositions and cells described herein may be hypoimmune or resist the innate immune response when transplanted into a subject. Cells described herein may be used to treat or prevent a disease, disorder, or condition in a subject.

[0106] Signal-regulatory-protein a (SIRPa) is a member of both the signal-regulatory-protein (SIRP) family and the immunoglobulin superfamily. SIRP family members are receptor-type transmembrane glycoproteins known to be involved in the negative regulation of receptor tyrosine kinase-coupled signaling processes. CD47 is a ligand for SIRPa. CD47 is a “marker-of-self” protein that can be overexpressed broadly across tumor types. It is emerging as a novel potent macrophage immune checkpoint for cancer immunotherapy. CD47 in tumor cells sends a “don’t-eat-me” signal that inhibits macrophage phagocytosis.

[0107] Immune cells, such as NK cells, express SIRPa upon stimulation and bind to CD47, thereby reducing their killing efficacy for CD47-expressing cells. Furthermore, innate immune cells, such as NK cells, trigger cytotoxicity of foreign cells lacking a CD47 signal or innate immune cells, such as macrophages, trigger phagocytosis of foreign cells lacking a CD47 signal This presents challenges in cell therapy in which therapeutic cells may be rejected by the immune system, thereby reducing or eliminating their efficacy. Thus, cells expressing a SIRPa engager increase immune cell evasiveness to prevent destruction.

[0108] The present invention provides the insertion of a SIRPa engager into one or more specific loci in the cell, e.g , a p-2 microglobulin (B2M) locus, an endogenous class II transactivator (CIITA) locus, or an endogenous T cell receptor alpha subunit constant (TRAC) locus. This results in efficient targeted integration and stable cell surface expression of the SIRPa engager. Such cells may be particularly useful for cellular therapies for regenerative medicine applications and the treatment of a wide range of diseases such as cancer.

[0109] Insertion Sites

[0110] Described herein are mammalian cells that include a polynucleotide having a heterologous gene encoding a SIRPa engager that binds SIRPa. The SIRPa engager is inserted into one or more sites within an endogenous B2M locus, an endogenous CIITA locus, or an endogenous TRAC locus. By inserting the gene into one or more of these loci, the target cell is engineered to both express the heterologous gene (e.g., a SIRPa engager) while simultaneously knocking out the endogenous gene at one of these loci

[0111] In some aspects, the heterologous gene is inserted into exon 1 of the endogenous B2M gene. In some aspects, the heterologous gene is inserted into exon 2 of the endogenous B2M gene. In some aspects, the heterologous gene is inserted into exon 3 of the endogenous CIITAgene. In some aspects, the heterologous gene is inserted into exon 1 of the endogenous TRAC gene. In some aspects, the heterologous gene is inserted into one or more of exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITAgene, exon 1 of the endogenous TRAC gene. SIRPa Engagers

[0112] CD47 expression on engineered cells provide protection against innate immune cell killing and phagocytosis. However, upon binding of its ligand SIRPa, CD47 can initiate downstream signaling in the engineered cell with potentially unwanted perturbations of its physiology. To only achieve protection against immune cell killing, some aspects of the invention separate the extracellular SIRPa-binding function from intracellular signaling in the engineered cell.

[0113] The Invention provides a SIRPa engager fusion protein that is expressed on an engineered cell and designed to bind to SIRPa on an immune cell in an agonistic manner that activates SIRPa signaling.

[0114] The SIRPa engager fusion protein described herein contains an extracellular domain (ECD) and a transmembrane domain (TMD). In some aspects, the fusion protein may further include an intracellular domain (ICD).

[0115] In some aspects, the ECD may include the CD47 ECD. In some aspects, the ECD may further include the CD47 immunoglobulin superfamily (IgSF) domain, complementarity-determining regions (CDRs) of an anti-SIRPa antibody or antigen-biding fragment thereof, such as a single chain variable fragment (scFv) of an anti-SIRPa antibody. The anti-SIRPa antibody or antigen-biding fragment (e.g. , scFv) thereof may include heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 20-22 and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 23-25 In some aspects, the ECD includes one, two, or three anti-SIRPa CDRs. In some aspects, one or more residues of a sequence are altered to modify binding to achieve a more favored on-rate of binding, a more favored off-rate of binding, or both, such that an optimized binding is achieved.

[0116] In some aspects, the ECD contains linker regions or hinges connecting the sequences provided with either the TMD or with each other. In other aspects, modifications are made within one or more of the linker regions or hinge regions so long as these modifications do not eliminate the binding affinity of the fusion protein with SIRPa. In some aspects, the hinge region is a CD8a hinge region.

[0117] In some aspects, an ECD has a contiguous sequence of at least about 10 amino acids as set forth in SEQ ID NO: 1, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, up to the complete provided region. ECDs also include sequences that differ by up to 1 , 2, 3, 4, 5, 6 or more amino acids as compared to the ammo acids sequence set forth in SEQ ID NO: 1. In other aspects, an ECD has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1 .

[0118] Generally, the transmembrane domain (TMD) of the SIRPa engager fusion protein is not limited to a specific TMD sequence Preferably, the TMD allows stable anchorage of the fusion protein in the membrane of a cell expressing the fusion protein It may further allow binding of the ECD to SIRPa. In some aspects, the fusion protein contains an ICD and binding to SIRPa allows signaling via the ICD This might be beneficial for the engineered cell if such signaling enhances the intrinsic function of this cell. Enhanced functions can, for example, be achieved through enhanced adhesion via the activation of integrins. In other aspects, the fusion protein does not contain an ICD, but rather, is truncated after the TMD. In the latter case, binding of the fusion protein to SIRPa does not result in intracellular signaling in the engineered cell. In some aspects, the SIRPa engager contains a nonfunctional ICD.

[0119] In some aspects, the TMD is selected from the group consisting of CD47, OD85f, CD349, CD284, CD261, CD 172b, CD277, CD 186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61, CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141, CD115, CD42b, CD221 , CD271, CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11 b, CD107a, CD331 , CD222, CD20, CD79a, CD64, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227. CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181 , CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121 b, CD34, CD38, CD30, CD1b, CD1c, CD154, CD6, CD52, CD132, CD32, CD66b, CD171, CD191 , CD197, CD185, CD131 , CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361 , CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231 , CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD1581 , CD178, CD295, CD151 , CD97, CD183, CD39, CD239, CD193, CD194, CD195. CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241 , CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91, CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161 , CD215, CD150, CD11d, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111 , CD281, CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD100, CD270, CD101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD114, CD180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201 , CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321, CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11 , CD51 , CD41, CD29: CD18, CD61, CD104, and PDGFR.

[0120] In some aspects, a polynucleotide encoding a SIRPa engager may also encode additional heterologous genes. An additional heterologous gene may be encoded in the polynucleotide upstream or downstream of the SIRPa engager. One or more regulatory elements may separate the SIRPa engager and one or more heterologous genes. In some aspects, the SIRPa engager may be located directly adjacent to an additional heterologous gene. In some aspects, the SIRPa engager and one or more heterologous genes may be operably linked. In some aspects, the SIRPa engager and one or more heterologous genes are unlinked. In some aspects, the polynucleotide is multicistronic. In some aspects, each heterologous gene includes a separate promoter. In some aspects, a single promoter drives expression of the plurality of heterologous genes.

[0121] Additional heterologous genes may be expressed to protect against antibody dependent cellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC). This additional heterologous gene may be an antibody Fc receptor. In some aspects, the Fc receptor is an Fey receptor. In some aspects, the Fey receptor is CD64, CD32, or CD16. The Fey may be modified. For example, a modified, truncated CD64 (CD64t) may be used to capture cytotoxic IgG antibodies and protect cells from both ADCC and CDC. A truncated CD64 may lack a functional ICD and / or TMD. A truncated CD64 may be as effective as full length CD64. In some aspects, a heterologous gene encoding CD64t may be included in the polynucleotide encoding a SIRPa engager. In some aspects, CD64t has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6.

[0122] Polynucleotides

[0123] The cells described herein include a polynucleotide encoding one or more heterologous genes (e.g., a SIRPa engager). The polynucleotide may be present in an expression cassette that promotes the expression of the one or more heterologous genes. An expression cassette may be present as single stranded DNA, double stranded DNA, or incorporated into a vector, e.g., a plasmid. Generally, an expression cassette will include at least one or more heterologous genes (e.g., SIRPa engager, CD64t), optionally one or more regulatory elements (e.g., promoter, enhancer, polyA, selfcleaving linker), optionally a 5’ homology arm, and optionally a 3’ homology arm. In some aspects, an expression cassette will include the donor template DNA for insertion into the genome

[0124] In some aspects, the expression cassette may be oriented such that when inserted into the genome, it is in the opposite orientation as the endogenous gene. For example, the schematic in FIG. 1A shows an expression cassette inserted into exon 1 of the B2M locus where the expression cassette is in the reverse orientation to the orientation of the B2M locus. In this example, the expression cassette may include from 5’ to 3’: a 3’ homology arm, an EF1a promoter, SIRPa engager, WPRE, polyA, and a 5' homology arm, such that the expression cassette will integrate into the correct location (e g., B2M exon 1 ) in the correct orientation (e g., reverse orientation of the endogenous gene). In some aspects, an expression cassette may be oriented such that it is in the same orientation as the endogenous gene in which it is inserted. In this aspect, the expression cassette may include from 5’ to 3’: a 5' homology arm, an EF1a promoter, SIRPa engager, WPRE, polyA, and a 3’ homology arm, such that the expression cassette will integrate into the correct location (e.g., B2M exon 1 ) in the same orientation as the endogenous gene. An exemplary expression cassette may have at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the polynucleotide sequence of SEQ ID NO: 7. In some aspects, a 5’ homology arm has a contiguous sequence of at least about 10 nucleotides as set forth in any one of SEQ ID NOs: 8, 10, 12, or 14, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, up to the complete provided sequence. 5’ homology arms may also include sequences that differ by up to 1 , 2, 3, 4, 5, 6 or more nucleotides as compared to the polynucleotide sequence set forth in any one of SEQ ID NOs: 8, 10, 12, or 14. In other aspects, a 5’ homology arm has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the polynucleotide sequence of any one or more of SEQ ID NOs: 8, 10, 12, or 14.

[0125] In some aspects, a 3’ homology arm has a contiguous sequence of at least about 10 nucleotides as set forth in any one of SEQ ID NOs: 9, 11, 13, or 15, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, up to the complete provided sequence. 3’ homology arms may also include sequences that differ by up to 1 , 2, 3, 4, 5, 6 or more nucleotides as compared to the polynucleotide sequence set forth in any one of SEQ ID NOs: 9, 11 , 13, or 15. In other aspects, a 3’ homology arm has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the polynucleotide sequence of any one or more of SEQ ID NOs: 9, 11 , 13, or 15.

[0126] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g. , at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 9. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 8.

[0127] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 11. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e g , at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 10.

[0128] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 13. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 12.

[0129] In some aspects, the 3’ homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 15. In some aspects, the 5' homology arm includes a polynucleotide sequence having at least 85% (e.g., at least 90%, 95%, 97%, 99%, or 100%) sequence identity to SEQ ID NO: 14.

[0130] In some aspects, the expression cassette includes a polynucleotide arranged in the following 5’-3’ order, a 5’ homology arm homologous to an endogenous sequence upstream of the insertion site, a SIRPa engager, and a 3’ homology arm homologous to an endogenous sequence downstream of the insertion site, the expression cassette is configured to be inserted into an endogenous B2M gene, an endogenous CIITA gene, or an endogenous TRAC gene

[0131] In some aspects, the expression cassette includes a polynucleotide arranged in the following 5’-3’ order: a 3’ homology arm homologous to an endogenous sequence upstream of the insertion site, a SIRPa engager, and a 5’ homology arm homologous to an endogenous sequence downstream of the insertion site. Here, the expression cassette is configured to be inserted into an endogenous B2M gene, an endogenous CIITA gene, or an endogenous TRAC gene

[0132] SIRPa Engager Cells

[0133] The SIRPa engagers described herein may include any one or more of the elements described herein. In some aspects, the SIRPa engager includes any feature, or any combination of features as disclosed in International Patent Publication No. WO 2022 / 125439, which is hereby incorporated by reference herein in its entirety.

[0134] The invention provides cells that have an increased SIRPa engagement function that resist innate immunity when transplanted into a subject when compared to a parental cell having an unmodified SIRPa engagement function. Cells expressing the SIRPa engager may be primary cells, pluripotent cells, embryonic stem cells, or immune cells. In some aspects, the SIRPa engager cells are hypoimmune cells. In other aspects, the SIRPa engager cells are differentiated somatic cells. In other aspects, the SIRPa engager cells are hypoimmune pluripotent cells. In some aspects, the SIRPa engager cells have been derived or differentiated from pluripotent cells. In some aspects, the SIRPa engager cells are immune cells. Immune cells may be from a myeloid lineage, e.g., macrophages, or a lymphoid lineage, e.g., T cells or NK cells. Differentiated SIRPa engager cells may be endothelial cells, cardiomyocytes, hepatocytes, dopaminergic neurons, pancreatic islet cells, retinal pigment endothelium cells, and other cell types used for transplantation and medical therapies. In some aspects, SIRPa engager cells may include chimeric antigen receptor (CAR) cells, such as CAR-T cells, NK cells, and CAR-NK cells or T cell receptor (TCR) cells, such as TCR-T cells or TCR- NK cells.

[0135] Cells expressing the SIRPa engager may have reduced function and / or expression of one or more genes In some aspects, human leukocyte antigen (HLA) class I (HLA-I), HLA class II (HLA-II), B2M, CIITA, and / or TRAC expression may be reduced. In some aspects, HLA-I, HLA-II, B2M, CIITA, and / or TRAC expression may be eliminated.

[0136] In some aspects, SIRPa engager cells may be edited. In some aspects, the SIRPa engager cells may be pluripotent cells that are edited prior to differentiation. In some aspects, the SIRPa engager cells may be pluripotent cells that are differentiated prior to editing.

[0137] Regulatory Elements

[0138] In some aspects, a polynucleotide or expression cassette including the same includes one or more regulatory elements, e.g., one or more sequences that modify expression of an expression sequence within the polynucleotide

[0139] A regulatory element may include a sequence that is located adjacent to an expression sequence that encodes an expression product. A regulatory element may be operably linked to the adjacent sequence A regulatory element may increase the amount of product expressed as compared to an amount of the expressed product when no regulatory element is present. A regulatory element may be used to increase the expression of one or more heterologous genes encoded by a polynucleotide. Likewise, a regulatory element may be used to decrease the expression of one or more heterologous genes encoded by a polynucleotide. In some aspects, a regulatory element may be used to increase expression of a heterologous gene and another regulatory element may be used to decrease expression of another heterologous gene encoded on the same polynucleotide. Hence, one regulatory element can enhance the expression of one or more expression sequences. Multiple regulatory elements can also be used, for example, to differentially regulate expression of different expression sequences.

[0140] In some aspects, a regulatory element is a cleavage sequence In some aspects, the cleavage sequence may be a self-cleaving linker. A polynucleotide may include the sequence encoding the self-cleaving linker. In some aspects, a plurality of heterologous genes may be separated by a 2A self-cleaving linker. For example, a polynucleotide may encode a SIRPa engager, a 2A sequence, and a CD64t. In some aspects, the 2A peptide is a Porcine teschovirus-1 2A (P2A), an Equine rhinitis A virus 2A (E2A), Foot-and-mouth disease virus 18 2A (F2A), or a Thosea asigna virus 2A (T2A) peptide

[0141] In some aspects, a regulatory element is a promoter Recognition and binding of a polynucleotide by mammalian RNA polymerase is important for gene expression. As such, one may include sequence elements within the polynucleotide that exhibit a high affinity for transcription factors that recruit RNA polymerase and promote the assembly of the transcription complex at the transcription initiation site. Such sequence elements include, e g., a mammalian promoter, the sequence of which can be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase. Promoter sequences are typically located upstream of the translation start site (e.g., within two kilobases upstream of the translation start site). Examples of mammalian promoters have been described in Smith et al , Mol. Sys. Biol., 3:73, 2007, the disclosure of which is incorporated herein by reference in its entirety The promoter used in the methods and compositions described herein can be a ubiquitous promoter or a cell type-specific promoter (e.g., a promoter that induces or increases expression of a polynucleotide in one or more specific cell types, such as hair cells or supporting cells). Ubiquitous promoters include the CAG promoter, cytomegalovirus (CMV) promoter, smCBA promoter (described in Haire et al , Invest. Opthalmol. Vis. Sol. 47:3745-3753, 2006, incorporated by reference herein in its entirety), dihydrofolate reductase (DHFR) promoter, human [3-actin promoter, phosphoglycerate I kinase (PGK) promoter, EF1a promoter, apolipoprotein E-human a1-antitrypsin promoter (HaaT), CK8 promoter, murine U1 promoter (MU1 a), early growth response 1 (EGR1 ) promoter, thyroxine binding globulin (TBG) promoter, chicken p-actin (CBA) promoter, hybrid CMV enhancer / chicken -actin promoter, SV40 early promoter, eukaryotic translation initiation factor 4A1 (EIF4A1) promoter, ferritin heavy (FerH) promoter, ferritin light (FerL) promoter, glyceraldehyde-3-phospohate dehydrogenase (GAPDH) promoter, heat shock protein family A member 5 (HSPA5) gene, heat shock protein family A member 4 (HSPA4) promoter, and ubiquitin B (UBB) promoter. Alternatively, promoters derived from viral genomes can also be used for the stable expression of polynucleotides in primate (e g , human) cells Examples of functional viral promoters that can be used for the expression of polynucleotides in primate (e.g., human) cells include adenovirus late promoter, vaccinia virus 7.5K promoter, thymidine kinase (tk) promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, LTR promoter of Moloney murine leukemia virus (MuMLV), MND promoter, promoter of Moloney virus, Epstein barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter. A pol II promoter, such as a ubiquitous promoter or a cell type-specific promoter, can be used to express any protein-coding transgene described herein. A pol III promoter, including ubiquitous pol III promoters U6, H1, and 7SK, can be used to express a polynucleotide that is an shRNA or an siRNA.

[0142] Other DNAsequence elements that may be included in polynucleotides (e.g., polynucleotides containing a promoter operably linked to a polynucleotide that can be transcribed to produce a desired expression product) for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce a conformational change in the polynucleotide containing the gene of interest such that the DNA adopts a three-dimensional orientation that is favorable for binding of transcription factors and RNA polymerase at the transcription initiation site. Thus, polynucleotides for use in the compositions and methods described herein include those that contain a polynucleotide of interest and a polynucleotide that can be transcribed to produce a miRNA target sequence and additionally include a mammalian enhancer sequence. Many enhancer sequences are now known from mammalian genes, and examples include enhancers from the genes that encode mammalian globin, elastase, albumin, a- fetoprotein, and insulin. Enhancers for use in the compositions and methods described herein also include those that are derived from the genetic material of a virus capable of infecting a eukaryotic cell Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Additional enhancer sequences that induce activation of eukaryotic gene transcription include the CMV enhancer and RSV enhancer. An enhancer may be included in a polynucleotide encoding a SIRPa engager, for example, at a position 5’ or 3’ to the gene

[0143] An enhancer, as described herein, may include a Woodchuck Hepatitis Virus (WHV) post- transcriptional regulatory element (WPRE). The WPRE acts at the transcriptional level, by promoting nuclear export of transcripts and / or by increasing the efficiency of polyadenylation of the nascent transcript, thus increasing the total amount of mRNA in the cells. The addition of the WPRE may result in a substantial improvement in the level of transgene expression from several different promoters, both in vitro and in vivo.

[0144] Suicide Genes

[0145] The invention provides the modified cells as disclosed herein further comprising a suicide gene that is activated by a trigger that causes the modified cell to die In some aspects, the suicide gene is a herpes simplex virus thymidine kinase gene (HSV-tk) and the trigger is ganciclovir. In a preferred aspect, the HSV-tk gene encodes a protein comprising at least a 90% sequence identity to SEQ ID NO: 26. In another preferred aspect, the HSV-tk gene encodes a protein comprising the sequence of SEQ ID NO: 26 In another aspect, the suicide gene is an Escherichia coli cytosine deaminase gene (EC-CD) and the trigger is 5-fluorocytosine (5-FC). In a preferred aspect, the EC-CD gene encodes a protein comprising at least a 90% sequence identity to SEQ ID NO: 27. In another preferred aspect, the EC-CD gene encodes a protein comprising the sequence of SEQ ID NO: 27. In another aspect, the suicide gene encodes an inducible Caspase protein and the trigger is a chemical inducer of dimerization (CID) In a preferred spect, the suicide gene encodes an inducible Caspase protein comprising at least a 90% sequence identity to SEQ ID NO: 28. In another preferred aspect, the suicide gene encodes an inducible Caspase protein comprising the sequence of SEQ ID NO: 28. In another aspect, the CID is AP1903.

[0146] Expression Cassettes

[0147] The invention described herein may include an expression cassette. An expression cassette may encode and / or promote the expression of one or more heterologous genes (e.g., a SIRPa engager). An expression cassette may be present as single stranded DNA, double stranded DNA, or incorporated into a vector, e.g., a plasmid or a viral vector. Generally, an expression cassette will include at least one or more heterologous genes (e.g., SIRPa engager, CD64t), optionally one or more regulatory elements (e.g., promoter, enhancer, polyA, self-cleaving linker), optionally a 5’ homology arm, and optionally a 3’ homology arm. In some aspects, an expression cassette will include a donor template DNA for insertion into the genome. In some aspects, the expression cassette may be transcribed and translated into a polypeptide. In some aspects, the expression cassette may be transcribed and translated into one or more polypeptides.

[0148] The expression cassette may include one or more heterologous genes, in which at least one heterologous gene is a SIRPa engager, as described above. In some aspects, the expression cassette additionally includes an antibody Fc receptor. In some aspects, the Fc receptor is an Fey receptor, in which the Fey receptor is CD64, CD32, or CD16. The Fey may be modified. For example, a modified, truncated CD64 (CD64t) may lack a functional ICD and / or TMD. A truncated CD64 may be as effective as full length CD64 In some aspects, a heterologous gene encoding CD64t may be included in the expression cassette encoding a SIRPa engager. In some aspects, CD64t has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 6.

[0149] In some aspects, the expression cassette may include one or more sequences, such that two or more proteins may result from one continuous RNA For example, one continuous RNA may encode two heterologous genes separated by a 2A self-cleaving linker, resulting in two separate proteins. In some aspects, the expression cassette may include 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences that encode 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 separate proteins. In some aspects, the expression cassette includes a sequence encoding a SIRPa engager and a CD64t, separated by a 2A cleavage sequence.

[0150] In some aspects, the expression cassette includes additional regulatory elements. Additional regulatory elements may include a cleavage sequence, an enhancer, and / or a promoter. In some aspects, the expression cassette contains a promoter, such that the promoter is operably linked to one or more heterologous genes encoded in the expression cassette In some aspects, the expression cassette does not include a promoter. In this aspect, expression of the one or more heterologous genes encoded in the expression cassette may be driven by an endogenous promoter. For example, the expression cassette may be inserted into the genome such that a promoter upstream of the insertion site is able to drive expression of the elements encoded in the expression cassette.

[0151] In some aspects, the expression cassette is configured to insert into an insertion site in the genome of a target cell. The expression cassette may be inserted into any location within the genome, including a safe harbor locus, a gene, a locus, a coding sequence, a non-coding sequence, or an untranslated region. In some aspects, the expression cassette includes a 5’ homology arm and a 3’ homology arm to facilitate integration into the genome. The 5’ homology arm and 3’ homology arm may contain a polynucleotide sequence sufficiently similar to the polynucleotide sequences located 5’ and 3' to an insertion site of interest, such that the expression cassette may be integrated via homologous recombination.

[0152] In some aspects, the expression cassette encodes, from 5’ to 3’: a promoter, a heterologous gene, an enhancer, and a polyadenylation sequence. In some aspects, the expression cassette encodes, from 5' to 3’: a heterologous gene, an enhancer, and a polyadenylation sequence. In some aspects, the expression cassette encodes, from 5’ to 3’: a first heterologous gene, a second heterologous gene, an enhancer, and a polyadenylation sequence. In some aspects, the expression cassette encodes, from 5’ to 3’: a promoter, a first heterologous gene, a second heterologous gene, an enhancer, and a polyadenylation sequence. In some aspects, the first or second heterologous gene is a SIRPa engager. In some aspects, the first or second heterologous gene is a CD64t.

[0153] In some aspects, the expression cassette encodes, from 5’ to 3’: an EF1a promoter, a SIRPa engager, a 2A self-cleavage sequence, a OD64t, a WPRE enhancer, and a polyadenylation sequence In some aspects, the expression cassette encodes, from 5’ to 3’: a 5’ homology arm, an EF1a promoter, a SIRPa engager, a 2A self-cleavage sequence, a CD64t, a WPRE enhancer, a polyadenylation sequence, and a 3’ homology arm

[0154] In some aspects, the expression cassette encodes, from 3’ to 5’: an EF1a promoter, a SIRPa engager, a 2A self-cleavage sequence, a CD64t, a WPRE enhancer, and a polyadenylation sequence In some aspects, the expression cassette encodes, from 3’ to 5’: a 3’ homology arm, an EF1a promoter, a SIRPa engager, a 2A self-cleavage sequence, a OD64t, a WPRE enhancer, a polyadenylation sequence, and a 5’ homology arm.

[0155] In some aspects, the expression cassette may be non-virally delivered to a cell. This may include contacting the cell with the expression cassette, or with the expression cassette integrated into a vector, e.g., a plasmid. A nuclease and / or a guide RNA may be used to facilitate integration of the expression cassette into the genome. In some aspects, the expression cassette may be inserted into the genome using CRISPR, as described below.

[0156] In some aspects, the expression cassette may be delivered to a cell via a viral vector. The viral vector may encode the expression cassette. The viral vector may also encode additional elements to aid in delivery of the expression cassette to the cell and / or insertion of the expression cassette into the cell’s genome These additional elements may include a nuclease, a guide RNA, and / or any other additional elements. The viral vector that contains the expression cassette may be an adeno-associated virus (AAV), an adenovirus, a parvovirus, a coronavirus, a rhabdovirus, a paramyxovirus, a picornavirus, an alphavirus, a herpes virus, a poxvirus, and a Retroviridae family virus. In some aspects, the viral vector that encodes the expression cassette is an integrationdeficient lentiviral vector (IDLV).

[0157] SIRPa engager-CD64 peptide

[0158] The invention also provides a polypeptide including a SIRPa engager and a CD64 (e.g. , CD64t lacking a functional ICD and / or TMD). In some aspects, the polypeptide includes a selfcleaving peptide between the SIRPa engager and the CD64. The self-cleaving peptide may be a 2A peptide. The polypeptide may include the structure from the amino terminus to the carboxy terminus SIRPa engager-2A-CD64 or CD64-2A- SIRPa engager. Also featured is a polynucleotide (e.g., an expression cassette) expressing the polypeptide.

[0159] Gene Editing

[0160] The invention described herein may include one or more components of a gene editing system. Exemplary gene editing systems are the clustered regulatory interspaced short palindromic repeat (CRISPR) system, zinc finger nucleases (ZFNs), and Transcription Activator-Like Effectorbased Nucleases (TALEN)

[0161] In a typical CRISPR / Cas system, an endonuclease is directed to a target nucleotide sequence (e.g., a site in the genome that is to be sequence-edited) by sequence-specific, non-coding guide RNAs that target single- or double-stranded DNA sequences. Three classes (l-lll) of CRISPR systems have been identified. The class II CRISPR systems use a single Cas endonuclease (rather than multiple Cas proteins). One class II CRISPR system includes a type II Cas endonuclease such as Cas9, a CRISPR RNA (crRNA), and a trans-activating crRNA (tracrRNA). The crRNA contains a guide RNA, which is typically an about 20-nucleotide RNA sequence that corresponds to a target DNA sequence The crRNA also contains a region that binds to the tracrRNAto form a partially doublestranded structure which is cleaved by RNase III, resulting in a crRNA / tracrRNA hybrid. The RNAs serve as guides to direct Cas proteins to silence specific DNA / RNA sequences, depending on the spacer sequence. See, e.g., Horvath et al., Science 327:167-170, 2010; Makarova et al., Biology Direct 1:7, 2006; Pennisi, Science 341 :833-836, 2013, which are herein incorporated by reference. The target DNA sequence must generally be adjacent to a protospacer adjacent motif (PAM) that is specific for a given Cas endonuclease; however, PAM sequences appear throughout a given genome. Some endonucleases, e.g., Cas9 endonucleases, are associated with G-rich PAM sites, and perform blunt-end cleaving of the target DNA at a location 3 nucleotides upstream from (5’ from) the PAM site. Other Cas nucleases include, for example, a Cas12b nuclease or a CasX nuclease.

[0162] Another class II CRISPR system includes the type V endonuclease Cpf1 , which is smaller than Cas9; examples include AsCpfl (from Acidaminococcus sp.) and LbCpfl (from Lachnospiraceae sp.). Cpf1 -associated CRISPR arrays are processed into mature crRNAs without the requirement of a tracrRNA; in other words a Cpf1 system requires only the Cpf1 nuclease and a crRNAto cleave the target DNA sequence. Cpf1 endonucleases, are associated with T-rich PAM sites, e g., 5 -TTN. Cpf1 can also recognize a 5’-CTA PAM motif. Cpf1 cleaves the target DNA by introducing an offset or staggered double-strand break with a 4- or 5-nucleotide 5’ overhang, for example, cleaving a target DNAwith a 5-nucleotide offset or staggered cut located 18 nucleotides downstream from (3’ from) from the PAM site on the coding strand and 23 nucleotides downstream from the PAM site on the complimentary strand; the 5-nucleotide overhang that results from such offset cleavage allows more precise genome editing by DNA insertion by homologous recombination than by insertion at blunt-end cleaved DNA.

[0163] For the purposes of gene editing, CRISPR arrays can be designed to contain one or multiple guide RNA sequences corresponding to a desired target DNA sequence; see, for example, Cong et al., Science 339:819-823, 2013; Ran et al., Nature Protocols 8:2281-2308, 2013. At least about 16 or 17 nucleotides of gRNA sequence are required by Cas9 for DNA cleavage to occur. In practice, guide RNA sequences are generally designed to have a length of between 17-24 nucleotides (e.g., 19, 20, or 21 nucleotides) and complementarity to the targeted gene or nucleic acid sequence. Custom gRNA generators and algorithms are available commercially for use in the design of effective guide RNAs. Gene editing has also been achieved using a chimeric single guide RNA (sgRNA), an engineered (synthetic) single RNA molecule that mimics a naturally occurring crRNA-tracrRNA complex and contains both a tracrRNA (for binding the nuclease) and at least one crRNA (to guide the nuclease to the sequence targeted for editing). Chemically modified sgRNAs have also been demonstrated to be effective in genome editing; see, for example, Hendel et al , Nature Biotechnol. 985-991 , 2015

[0164] CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications US 2016 / 0138008 A1 and US 2015 / 0344912 A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641 , 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871 ,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616 The foregoing are incorporated by reference in their entirety.

[0165] In some instances, the desired genome modification involves homologous recombination, wherein one or more double-stranded DNA breaks in the target nucleotide sequence is generated by the RNA-guided nuclease and guide RNA(s), followed by repair of the break(s) using a homologous recombination mechanism (homology-directed repair (HDR)). In such instances, a donor template that encodes the desired nucleotide sequence to be inserted or knocked-in at the double-stranded break is provided to the cell or subject; examples of suitable templates include single-stranded DNA templates and double-stranded DNA templates (e.g., linked to the polypeptide described herein). In general, a donor template encoding a nucleotide change over a region of less than about 50 nucleotides is provided in the form of single-stranded DNA; larger donor templates (e.g., more than 100 nucleotides) are often provided as double-stranded DNA plasmids. In some instances, the donor template is provided to the cell or subject in a quantity that is sufficient to achieve the desired homology-directed repair but that does not persist in the cell or subject after a given period of time (e.g., after one or more cell division cycles). In some instances, a donor template has a nucleotide sequence for a heterologous gene that differs from the target nucleotide sequence (e.g., a homologous endogenous genomic region) by at least 1 , at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, or more nucleotides This heterologous gene sequence is flanked by homology arms or regions of high sequence identity with the targeted nucleotide sequence; in some instances, the regions of high identity include at least 10, at least 50, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 600, at least 750, or at least 1000 nucleotides on each side of the heterologous gene sequence. In some instances where the donor template is in the form of a single-stranded DNA, the core sequence is flanked by homology arms including at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 100 nucleotides on each side of the heterologous gene sequence. In instances, where the donor template is in the form of a double-stranded DNA, the heterologous gene sequence is flanked by homology arms including at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 nucleotides on each side of the heterologous gene sequence. In one instance, two separate double-strand breaks are introduced into the cell or subject’s target nucleotide sequence with a double nickase Cas9 (see Ran et al., Cell 154:1380-1389, 2013), followed by delivery of the donor template.

[0166] In some instances, the method includes providing a composition that includes a gRNAand a targeted nuclease, e.g , a Cas9, e.g., a wild type Cas9, MAD7, LEG14, LEG95, LEG98, LEG145, or a nucleic acid encoding such a nuclease. The choice of nuclease and gRNA(s) is determined by location of the targeted mutation. In instances, the editing system includes a gRNA for use in a CRISPR system for gene editing. In some instances, the editing system includes a zinc finger nuclease (ZFN), or a mRNA encoding a ZFN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNAsequence) of a gene in the cell. In some instances, the editing system includes a TALEN, or an mRNA encoding a TALEN, that targets (e.g., cleaves) a nucleic acid sequence (e.g., DNA sequence) in a gene in the cell.

[0167] For example, the gRNA can be used in a CRISPR system to engineer an alteration in a gene in the cell. Exemplary alterations include insertions (e.g., knock ins), deletions (e.g., knock outs), translocations, inversions, single point mutations, or other mutations. The alteration can be introduced in the gene in a cell, e.g , in vitro, ex vivo, or in vivo In some examples, the alteration allows for expression and / or activity (e.g., knock in or insertion) of a heterologous gene in the cell. In other examples, the alteration decreases the level and / or activity of (e.g., knocks down or knocks out) an endogenous gene in the cell. In some instances, the CRISPR system is used to edit (e.g., to add or delete a base pair) a target gene in the cell. In other instances, the CRISPR system is used to introduce a premature stop codon, e.g., thereby decreasing the expression of a target gene. In yet other instances, the CRISPR system is used to turn off a target gene in a reversible manner, e.g., similarly to RNA interference. In some instances, the CRISPR system is used to direct Cas to a promoter of a gene, thereby blocking an RNA polymerase sterically.

[0168] In some instances, one sgRNA and one nuclease is used to edit one target gene in the cell. In other instances, multiple sgRNAs and / or nucleases may be used to target multiple endogenous genes in the cell. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sgRNAs that each directed to a unique genomic location may be used in conjunction with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more nucleases to edit 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unique locations in the cell genome.

[0169] In some aspects, sgRNAs may target the B2M locus, the CIITA locus, and / or the TRAC locus More specifically, sgRNAs may target exon 1 of the B2M locus, exon 2 of the B2M locus, exon 3 of the CIITA locus, and / or exon 1 of the TRAC locus (FIG. 13B). sgRNAs may target the endogenous genes at the sequences of any one of SEQ ID NOs: 16-19 In some aspects, the method includes complexing the nuclease with the sgRNAto form a ribonucleoprotein (RNP) complex, wherein the RNP is then introduced into the cell to create the insertion site. In some aspects, the ratio of nuclease to sgRNA is 1 :1 to 1 :10 (e.g., 1 :1 , 1:2, 1:3, 1:4, 1 :5, 1:6, 1:7, 1:8, 1 :9, or 1 :10). In some aspects, the method includes a first incubation step, wherein the nuclease and sgRNA are incubated for between 1 to 120 (e.g., 10 to 90) minutes at a temperature of 15°C to 45 °C (e.g., 20 °C to 37 °C) to create the RNP complex. In some aspects, the method further includes a second incubation step. During the second incubation step, the RNP may be further complexed to the polynucleotide, e.g., at a concentration of 0.5 pg to 400 pg (e.g., 4 pg to 160 pg). In some aspects, the second incubation step, wherein the RNP and polynucleotide are incubated for 1 to 100 (e.g., 5 to 30) minutes at a temperature of 15°C to 45 °C (e.g., 20 °C to 37 °C).

[0170] In some aspects, the first incubation step and / or the second incubation step is performed at a temperature of 15 °C to 45 °C (e.g., 15 °C to 45 °C, 20 °C to 45 °C, 25 °C to 45 °C, 30 °C to 45 °C, 40 °C to 45 °C, 15 °C to 40 °C, 15 °C to 35 °C, 15 °C to 30 °C, 15 °C to 25 °C, 15 °C to 20; 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, or 45 °C).

[0171] In some aspects, the RNP is complexed to the polynucleotide at a concentration of 0.5 pg to 400 pg (e.g., 1 pg to 400 pg, e.g., 1 pg to 200 pg, e.g., 4 pg to 160 pg).

[0172] In some aspects, the method further includes delivering the RNP and polynucleotide complex into the cell, e g., via transfection In some aspects, the RNP and polynucleotide complex is inserted via electroporation. In some aspects, the polynucleotide is on a plasmid. In some aspects, the heterologous gene is inserted into the insertion site by homology directed repair.

[0173] Methods of Treatment

[0174] Also featured herein is a method of using the SIRPa engager cells for treating a disease in a subject. The SIRPa engager cells may be transplanted into the subject. In some aspects, the disease is Type 1 diabetes, a cardiac disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease. In some aspects, the cancer is a blood cancer (e.g., leukemia, lymphoma, or myeloma). In some aspects, the cancer is a solid cancer.

[0175] The cells may be formulated as a pharmaceutical composition with one or more pharmaceutically acceptable carriers. As used herein, a “pharmaceutically acceptable carrier” or “therapeutic effective carrier” is aqueous or nonaqueous (solid), for example alcoholic or oleaginous, or a mixture thereof, and can contain a surfactant, emollient, lubricant, stabilizer, dye, perfume, preservative, acid or base for adjustment of pH, a solvent, emulsifier, gelling agent, moisturizer, stabilizer, wetting agent, time release agent, humectant, or other component commonly included in a particular form of pharmaceutical composition. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, and oils such as olive oil. A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize or to increase the absorption of specific inhibitor, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.

[0176] The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer’s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.

[0177] Table 1 . Nucleotide and Amino Acid Sequences

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186] Examples

[0187] The following examples provide those of ordinary skill in the art with a description of how the compositions and methods described herein may be used, made, and evaluated. They are intended to be exemplary and are not intended to limit the scope of the disclosure or invention.

[0188] Example 1. Generation of cells expressing a SIRPa engager

[0189] An exemplary non-viral method for making pluripotent cells, e.g., embryonic stem cells (ESCs), or induced pluripotent stem cells (iPSCs), that express a functional SIRPa engager is by way of electroporation, e.g., nucleofection Using molecular biology procedures described herein and known in the art, plasmid DNA containing a polynucleotide encoding a SIRPa engager can be produced. For example, a nucleic acid encoding a SIRPa engager may be amplified from a human cell line using PCR-based techniques known in the art Likewise, a nucleic acid encoding a SIRPa engager may be synthesized, for example using solid-phase synthesis procedures. The nucleic acid can then be ligated into a plasmid of interest, for example, using suitable restriction endonuclease- mediated cleavage and ligation protocols. After the plasmid DNA is engineered, the plasmid can be introduced to the cells via electroporation. Single or double stranded DNA amplified or synthesized, without ligation into a plasmid, may also be utilized during electroporation.

[0190] In this example, HLA-I was depleted from iPSCs by knocking-out the B2M locus, and simultaneously inserting a SIRPa engager into the B2M locus to protect the HLA-I depleted target cells from innate immune killing. Unedited human WT iPSCs transduced with a glypican-3 (GPC3)- reactive T cell receptor (TCR) were utilized. Guide RNA sgl (100 pmol), targeting exon 1 of the B2M locus, was mixed with Cas9 (20 pmol), and incubated at room temperature for 10 minutes to make a ribonucleoprotein (RNP) before adding 0.5-1 pg of donor template. A schematic of an exemplary expression cassette encoding the SIRPa engager introduced into the B2M locus is shown in FIG. 1A. The RNP plus donor DNA were mixed with the GPC3-TCR iPSCs and electroporated. After disruption of the insertion site, the donor DNA was incorporated into the genome by homology direct repair (HDR). The efficiency of HLA-I knock-out and donor DNA knock-in was assessed via flow cytometry using anti-G4S (to assess SIRPaE expression) and anti-HLA-ABC (to assess B2M inactivation) antibodies (FIG. 1 B) The knock-in efficiency of the SIRPa engager donor was 7.1% but rose to 24.9% when the DNA protein kinase (DNA-PK) inhibitor AZD7648 was used.

[0191] Example 2. Differentiation of SIRPa engager iPSCs into CD8a(3 T cells

[0192] This example establishes the differentiation capacity of SIRPa engager iPSCs into CD8a[3 T cells. Human iPSCs previously transduced with a GPC3-reactive TCR and a CD19 tag were used as parental cells. The donor DNA template encoding a myc-tagged SIRPa engager was transduced into the parental cells using lentiviral particles As seen in FIG. 2A, 88 7% of the iPSCs were positive for the pluripotent cell marker SSEA-4 and were CD19 positive (upper right quadrant), thus expressing the GPC3-reactive TCR. 77.9% of iPSCs were positive for the myc-tag, thus expressing the SIRPa engager (FIG. 2B, upper left quadrant). To assess differentiation capacity of the iPSCs, the transduced cells were differentiated into hematopoietic progenitor cells (CD45+CD7+) and compared to the parental iPSCs Cells of interest were identified based on size and granularity using forward scatter (FSC) and side scatter (SSC) (first plots on the left), and viability was assessed using the DNA binding dye propidium iodide (PI), detectable within the phycoerythrin (PE) channel (second plots from the left). Cell differentiation was further assessed by the presence of CD45 and CD7 (markers of hematopoietic progenitor cells) (middle plots), the T-cell marker CD8a and CD19-tagged GPC3-TCR (second plots from the right), and the differentiation marker CD7 and T cell marker CD5 (plots on the right). FIG. 3 shows that the SIRPa engager cells were differentiated into hematopoietic progenitor cells (CD45+CD7+) at a high efficiency (97.6%), similar to differentiation of the parental cells.

[0193] Further differentiation of the iPSCs into CD3+TCRap+T cells was also assessed and compared to its parental iPSC line (FIG. 4). Dendritic cell marker CD1a and T cell marker CD5 were assessed in the left-hand plots. Differentiation into T cells was further assessed by the presence of CD3 and T CR a and p chains (second plots from the left), and assessment of CD4 and CD8 determined the type of T cell. iPSC differentiation into T cells was very high, with the majority of parental and SIRPa engager cells being CD8ap+(FIG. 4).

[0194] Example 3. SIRPa engagers protect cells from NK cell and macrophage killing

[0195] Human peripheral blood macrophages and IL-2 activated NK cells were assessed based on cell size (forward scatter or FSC) and for their expression of a SIRPa immune checkpoint. Percentages of NK cells and macrophages positive for SIRPa were determined from the mean of 5 healthy human donors As seen in FIGS. 15A and 15B, SIRPa was robustly expressed on the majority of NK cells and macrophages

[0196] Design of the SIRPa engager protein

[0197] Having established the presence of SIRPa on the surface of NK cells and macrophages, an immune cell checkpoint engager was generated against SIRPa. It included an antibody single-chain variable fragment (scFv) attached to a CD8a hinge region and a platelet derived growth factor receptor (PDGFR) transmembrane domain. The SIRPa engager lacked an intracellular domain (ICD) to avoid retrograde signaling. The SIRPa engager sequence was cloned into a lentiviral particle and transduced into a B2M’ACIIT double knock-out (DKO) iPSC-derived endothelial cell (iEC)

[0198] SIRPa engager inhibits killing by innate immune cells

[0199] The functionality of the SIRPa engager was assessed using highly sensitive in vitro impedance cytotoxicity assays with IL-2 activated NK cells or macrophages as effector cells in different effector-to-target ratios (E:T ratios), where the target cells are the DKO and DKOSIRPaEiECs. The target cells were plated on electrode plates and the cell index was normalized at steady state In this assay, any cytotoxicity that disturbs the target cell covering of the electrodes leads to a decrease of impedance and the cell index drops. Target cells were also transduced to express firefly luciferase so that cytotoxicity could additionally be quantified through the drop of their ATP-dependent bioluminescence signals after the addition of effector cells. From left to right, the panels show DKO or DKOSIRPaEiECs alone, and then with increasing E:T ratios of either NK cells or macrophages. DKO iECs were killed increasingly rapidly with increasing E:T ratios of NK cell or macrophage effector cells (FIG. 16A). The SIRPa engager-expressing DKO cells were fully protected from innate immune cell killing (FIG. 16B).

[0200] SIRPa engager with CD64t protects against cytotoxic IgG

[0201] Allogeneic cell therapeutics may face antibody-mediated rejection when they are administered for long-term survival and in more immunocompetent patients Expression of truncated CD64 (CD64t), a modified FcyRI receptor, captures cytotoxic IgG antibodies and protects cells from both antibody-dependent cellular cytotoxicity (ADCC) as well as from complement-dependent cytotoxicity (CDC) (See WO2022 / 212393, incorporated by reference herein in its entirety.) As seen in FIG. 17A and 17B, CD64t alone did not protect against NK cell killing at a 1 : 1 E:T ratio. To combine the antibody protection of CD64t and the cellular immune evasion of the SIRPa engager, the DKOSIRPaEiECs were transduced with lentiviral particles carrying a CD64t transgene. The potential impact of CD64t on SIRPa engager function to protect the cells from NK cell killing was assessed in FIG. 17C, and the DKOSIRPaE CD64tiECs retained their resistance to NK cell killing in vitro at increasing E:T ratios. A human IgG 1 antibody against the constitutively expressed MHC class l-related sequence A (MICA) was added at a constant concentration of 1 g / ml to induce ADCC. As seen in FIG. 17D, DKOSIRPaEiECs were killed increasingly quickly with increasing NK cell E:T ratios (upper panels), but DKOSIRPaE-CD64tiECs were fully protected against antibody-mediated NK cell killing (lower panels). To evaluate CDC, an anti-MICA antibody at a constant concentration of 1 pg / ml, and serum were both added to the assay As seen in FIG. 17E, DKOSIRPaE’CD64tiECs remained protected against CDC, whereas DKOSIRPaEiECs cells underwent killing at increasing NK cell E:T ratios. Thus, CD64t expression was confirmed to protect engineered cells from cytotoxic IgG without interfering with the SIRPa engager function.

[0202] Non-viral knock-in of SIRPa engager and CD64t into the B2M locus in iPSCs are protective against innate immune cells, ADCC, and CDC

[0203] Next, the feasibility of engineering immune evasive cells using non-viral editing was assessed. As iPSCs do not express HLA class II unless stimulated, only the B2M locus (and not CIITA) was targeted with Cas9, a guide RNA, and a donor DNA template encoding both the SIRPa engager and CD64t, as shown in an exemplary expression cassette in FIG. 5A. The donor DNA was introduced into exon 1 of the B2M locus as described in Example 1. Expression of B2M, CD64t, and SIRPa engager in the B2M-KO, B2M-KOCD64t slRPaE, and WT iPSCs were assessed. As seen in FIG. 5B, The WT iPSCs showed B2M expression. B2M was successfully knocked out in the two edited iPSCs The B2M-KOCD64t-SIRPaEcells showed high expression of both CD64t and the SIRPa engager (FIG. 5B, middle row). Additionally, as seen in FIG. 6, expression of both the SIRPa engager and CD64t was stable over time, as evaluated over the course of 49 days. The B2M-K0 iPSCs were rapidly killed by both activated NK cells and macrophages, while the B2M-KOSIRPaE CD64tiPSCs were protected from killing (FIG. 7). In FIGS. 8A and 8B, protection against ADCC and CDC was evaluated Unedited (WT) and B2M-KOSIRPa'E CD64tiPSCs were assessed, from left to right, with the iPSCs alone, NK cells added at a 1 : 1 E:T ratio, and NK cells at a 1 :1 E:T ratio with increasing concentrations of a human lgG1 antibody against the constitutively expressed stem cell antigen SSEA4. WT IPSCs were killed with different concentrations of stem cell antigen (SSEA4) lgG1 antibody, whereas B2M-KOSIRPc,E CD64tiPSCs were protected from antibody- mediated killing (FIG. 8A). FIG. 8B shows complement-dependent cytotoxicity (CDC) assays with the same anti-SSEA4 IgG 1 antibody. Unedited (WT) and B2M-KOSIRPa'E CD64tiPSCs were assessed, from left to right, with the iPSCs alone, added complement, and complement along with increasing concentrations of anti-SSEA4. WT iPSCs were again rapidly killed with increasing anti-SSEA4 antibody concentrations, while B2M-KOSIRP°'E CD64tiPSCs remained protected. These data show the engineering of iPSCs with effective immune evasion features and demonstrate phenotypic continuity between virally and non-virally edited cells.

[0204] Protection of DKOSIRPaE CDe4tiECs against allogeneic cellular and antibody responses

[0205] The potential survival of DKOSIRPaE’CD64tiEC cells under the harshest allogeneic immune conditions was tested in maximally stringent immune environments. Engineered allogeneic cell therapeutics will eventually have to evade all of a patient’s adaptive and innate immune attacks to allow for persistent efficiency The FDA-approved anti-CD52 lgG1 antibody alemtuzumab (Campath) is well-known for its high cytotoxicity and was chosen for in vitro testing because it was considered the highest bar to reach. Target cells were transduced with lentiviral particles expressing CD52 to confer susceptibility to alemtuzumab, as CD52 is not constitutively expressed on endothelial cells The WTCD52iECs were challenged with NK cells and macrophages and showed expedited killing whenever anti-CD52 lgG1 was added (FIG. 18A). To assess potential T cell allograft rejection, fresh PBMCs from an HLA-A2-negative donor were primed in vitro with the HLA-A2+WTCD52iECs for 14 days. CD8+T cells were then sorted and used for adaptive T cell cytotoxicity assays. A typical in vitro T cell allo-rejection curve was observed, which is slower than that for innate killing (FIG. 18A). When anti-CD52 IgG 1 was added, T cell ADCC killed the WTCD52targets faster. Additionally, the WTCD52iECs were susceptible to CDC killing, and were killed expeditiously when incubated in serum supplemented with anti-CD52 lgG1 (FIG. 18A). In comparison, the DKOSIRPaE CD64tiECs were protected from all allogeneic cellular immune responses as well as from ADCC and CDC (FIG. 18B).

[0206] The cells were next analyzed in a stringent allogeneic in vivo model. Humanized NSG-SGM3 (NOD.Cg-PrkdcscidH2rgtm1wj' Tg(CMV-IL3,CSF2,KITLG)1 Eav / MloySzJ) mice were reconstituted with allogeneic CD34+hematopoietic stem cells and showed >40% of human CD45+cells among the overall CD45+population and >3% human CD3+cells among human CD45+cells. The humanized mice were additionally supplemented with one million human NK cells and three 1 mg doses of anti- CD52 lgG1 antibody (FIG. 18C). The NK cells and the first dose of alemtuzumab was mixed and injected subcutaneously together with 5 x 104firefly luciferase* target cells to maximize the cytotoxic potential of the antibody. All cell transplants were followed by bioluminescence imaging (BLI). The WTCD52grafts vanished in just one day, most likely attributed to the highly cytotoxic nature of the anti- 0052 lgG1 and NK cells (FIG. 18D). Conversely, the DKOSIRPaE’CD64tgrafts showed unobstructed survival, thus confirming their full protection from all allogeneic cellular and antibody responses (FIG. 18E). Immune evasiveness was also tested using a humanized lgG1 antibody against MICA. 5 x 104firefly luciferase+WT or DKOSIRPaE’CD64tiECs were mixed with 1 x 106human NK cells and 1 mg anti- MICA human lgG1 and subcutaneously injected into humanized mice (FIG. 18F). Two days later, a second dose of the anti-MICA human lgG1 was injected into the same site. The WT grafts vanished over the course of approximately two weeks (FIG. 18G). Again, the DKOSIRPaE CD64tgrafts were protected from cellular allo-immunity as well as from antibody-mediated killing (FIG. 18H).

[0207] Conclusion

[0208] These results present an immune editing platform that comprehensively protects engineered cells from all immune cells and IgG Utilizing a SIRPa engager allowed for utilization of inhibitory innate immune checkpoints and demonstrated an inhibitory function of NK cells and macrophages. Importantly, the foregoing experiments demonstrate that a SIRPa engager can be combined with truncated CD64 to generate fully immune evasive cells capable of escaping all allogeneic cellular and IgG antibody mediated rejection, both when introduced to cells via a lentiviral vector and when using non-viral insertion techniques.

[0209] Example 4. SIRPa engager and CD64t in serially edited cells

[0210] In this example, the SIRPa engager and CD64t were evaluated in iPSCs that included additional gene edits that would be practical for an allogeneic T cell product Human iPSCs underwent serial knockout (KO) editing of the CIITA, TRBC1 , and TRBC2 loci, and single cell cloning. A GPC3- reactive TOR and truncated CD19 transgene were then knocked into the TRAC locus of the triple KO clone. The exemplary expression cassette shown in FIG. 5A was then knocked into the B2M locus. The edited cells were then sorted into B2M7CD64' and B2M7CD64* populations (FIG. 9) and both populations were HLA class I and class II depleted.

[0211] In vitro cytotoxicity assays were performed with activated NK cells and macrophages at increasing effector to target cell (E:T) ratios as described in Example 3. As seen in the top panels of FIG. 10A, B2M CD64' cells (i.e. , lacking SIRPa engager and CD64t), were rapidly killed by NK cells at an E:T ratio of 1 :5 and above. B2M7CD64+cells (i.e., encoding SIRPa engager and CD64t), were fully protected against NK cell killing (FIG. 10A, lower panels). The B2M7CD64’ and B2M7CD64+cells responded the same way to macrophages, with B2M 7CD64 cells being killed at E:T ratios of 1 :5 and above, while B2M7CD64+cells were protected from killing (FIG. 10B).

[0212] The serially edited cells were next evaluated in an ADCC assay using an anti-SSEA4 lgG1 antibody as described in Example 3 FIG. 11 A shows that B2M7CD64+iPSCs maintained stable cell indices for all tested antibody concentrations. The same results were seen in 11 B when a human anti- CD19 lgG1 antibody was utilized. Protection against CDC was assessed using complement and either an anti-SSEA4 IgG 1 antibody (FIG. 12A), or an anti-CD19 antibody (FIG. 12B), as described in Example 3. For both antibodies tested, the top panels demonstrate that B2M7CD64' iPSCs were susceptible to antibody- mediated complement killing, while the lower panels demonstrate that B2M7CD64+iPSCs were protected against antibody killing.

[0213] Example 5. Site specific insertion of SIRPa engagers at multiple genomic locations

[0214] In this example, expression levels of SIRPa engager and CD64t were explored in several genomic locations. This editing strategy allows for insertion of up to 8 copies of an expression cassette into a single cell (e.g., four separate insertion sites, with two copies of each gene per cell).

[0215] An exemplary expression cassette encoding an EF1a promoter, SIRPa engager, 2A selfcleaving peptide, CD64t, WPRE, and poly A (FIG. 13A) was inserted into site-specific locations in the B2M locus, CIITA locus, and TRAC locus (FIG. 13B) using the editing methods described in Example 1. The knock-in (KI) efficiencies of the expression cassette in exons 1 and 2 of the B2M locus are shown as the CD64+ HLA-ABC- quadrant (FIGS. 14A and 14B). Addition of the DNA-PK inhibitor AZD7648 and CTS™ Xenon™ genome editing (GE) buffer increased the knock-in efficiency Knock-in efficiency was further assessed for the CIITA and TRAC loci (FIGS. 14C and 14D). These results illustrate the efficacy of targeted insertion at each of the tested loci.

[0216] SIRPa is expressed on NK cells and macrophages. FIGS. 15A and 15B are graphs exemplifying expression of SIRPa on IL-2 stimulated primary human NK cells (top plot) and macrophages (bottom plot), as assessed by cell size (FSC) and presence of SIRPa (FIG. 15A). Percentages of NK cells and macrophages positive for SIRPa were determined from the mean of 5 healthy human donors (FIG. 15B).

[0217] Example 6. SIRPa engager protects DKO Cells from Innate Cell Killing

[0218] Cytotoxicity assays over time showed the susceptibility to killing by effector NK cells and macrophages at increasing E:T ratios. Fig. 16A shows B2M+CIITA-'' double knock-out (DKO) iPSC- derived endothelial cells (iECs). FIG. 16B shows DKO iECs expressing the SIRPa engager. The SIRPa engager was transduced into DKO iECs using lentiviral particles. From left to right, the panels show DKO or DKOSIRPaEiECs alone or with increasing E:T ratios of either NK cells or macrophages. The DKO iECs were killed by the effector cells at increasing E:T ratios (FIG. 16A), while the DKOSIRPaEiECs were protected from killing (FIG. 16B). When compared to the results shown in Figs. 7 and 10, the CRISPR-engineered SIRPa engager was as effective at protecting the cells from innate immune cell killing as the lentiviral-engineered SIRPa engager cells.

[0219] FIG.19 shows that double knock-out (DKO) iPSC-derived a T cells (IT) and DKO iT- expressing SIRPa engager and CD64t resist killing by NK cells and macrophages at increasing E:T ratios The SIRPa engager and CD64t were introduced into the iT cells via gamma retroviral vector transductions. DKO cells expressing both the SIRPa engager and CD64t were protected from innate immune cell killing. In contrast, the DKO cells lacking the SIRPa engager and CD64t were killed. Example 7. CD64t protects the DKO cells from ADCC and CDC

[0220] CD64t protects the DKO cells from antibody-mediated cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). FIGS. 17A-17E are graphs showing the susceptibility of DKO iECs (FIG. 17A) and DKOCD64tcells (FIG. 17B) to killing by NK cells at a 1 : 1 E:T ratio, over time. DKOCDS4t’SIRPaEiECs expressing the SIRPa engager were protected at increasing E:T ratios (FIG. 17C). In FIG. 17D, antibody-mediated cellular cytotoxicity (ADCC) of DKOSIRPaEand of DKOCD64t'SIRPaEiECs was assessed by adding increasing E:T ratios of NK cells to target cells, along with an anti-MHC class l-related sequence A (MICA) antibody added at a constant concentration of 1 g / ml. In FIG. 17E, both ADCC and complement dependent cytotoxicity (CDC) of DKOSIRPaEand of DKOCD64t’SIRPaEiECs was assess by adding serum to target cells, along with increasing E:T ratios of NK cells and a constant concentration of the anti-MICA antibody. Only iECs expressing both the SIRPa engager and CD64t were protected from NK cell killing, ADCC, and CDC.

[0221] Example 8. SIRPa engager plus CD64t protects DKO cells following allogeneic transplantation

[0222] DKOCD64t’SIRPaEiEC survive during harsh allogeneic immune conditions. FIGS. 18A-18H are schematic drawings and graphs showing DKOCD64t’SIRPaEiEC survival during following allogeneic transplantation. Cells expressed CD52, making them susceptible to the highly cytotoxic anti-CD52 lgG1 antibody alemtuzumab. Susceptibility of WTCD52(FIG. 18A) and DKOCD64t’SIRPaE.CD52cells (FIG. 18B) to hostile in vitro immune conditions including antibody-mediated cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) was assessed. From top left to bottom right, WTCD52and DKOCD64t'SIRPaE’CD52iECs were assessed alone, or challenged with NK cells; NK cells and alemtuzumab; NK cells, alemtuzumab, and serum; macrophages; macrophages and alemtuzumab; primed allogeneic peripheral blood mononuclear cells (PBMCs); PBMCs and alemtuzumab; PBMCs, alemtuzumab, and IL-2; PBMCs, alemtuzumab, IL-2, and serum; serum alone; and serum and alemtuzumab. Only DKOCD64t-SIRPaE-CD52cells were protected from all allogeneic immune cell and IgG attacks. In FIGS. 18C-18H, WTCD52cells, DKOCD64 SIRPaE-CD52cells, and DKOCD64t SIRPaEcells were transplanted into a stringent, allogeneic, NSG-SGM3 (NOD.Cg-Prkdcscidll2rgtm1WjlTg(CMV- IL3,CSF2,KITLG)1 Eav / MloySzJ) humanized mouse model. Mice were additionally injected with one dose of NK cells, and three doses of either an anti-CD52 antibody (alemtuzumab) (FIGS. 18C-18E) or an anti-MICA antibody (FIGS. 18F-18H). Retention of cell grafts over time was assessed via bioluminescence imaging (BLI). In the presence of supplemental NK cells and alemtuzumab, the WTCD52grafts vanished quickly (FIG. 18D), while the DKOCD64t’SIRPaE’CD52grafts were retained throughout the 28 days tested (FIG. 18E). In the presence of supplemental NK cells and the anti- MICA antibody, the WTCD52grafts vanished over the course of two weeks (FIG. 18G), while the DKOCD64t' siRPaE grafts Were again retained throughout the 28 days tested (FIG. 18H).

[0223] Other Embodiments

[0224] Various modifications and variations of the described compositions, methods, and uses of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention known in the art are intended to be within the scope of the invention. All publications, patents, and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0225] All other embodiments are in the claims.

Claims

Claims1. A mammalian cell comprising a polynucleotide comprising a heterologous gene inserted into one or more sites within:(a) an endogenous p-2 microglobulin (B2M) locus;(b) an endogenous class II transactivator (CIITA) locus; or(c) an endogenous T cell receptor alpha subunit constant (TRAC) locus; wherein the heterologous gene encodes a signal regulatory protein alpha (SIRPa) engager that binds SIRPa.

2. The cell of claim 1 , wherein the heterologous gene is inserted into exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITAgene, exon 1 of the endogenous TRAC gene, or a combination thereof.

3. The cell of claim 1 or 2, wherein the cell comprises 2, 3, 4, 5, 6, 7, 8, or more copies of the heterologous gene.

4. The cell of any one of claims 1-3, wherein the SIRPa engager comprises a CD47 extracellular domain (ECD).

5. The cell of any one of claim 4, wherein the CD47 ECD comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1.6 The cell of any one of claims 1-5, wherein the SIRPa engager lacks a functional CD47 intracellular domain (ICD).

7. The cell of any one of claims 1-3, wherein the SIRPa engager comprises a single chain variable fragment (scFv).

8. The cell of claim 7, wherein the scFv comprises heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 20-22 and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 23-25.

9. The cell of any one of claims 1-8, wherein the SIRPa engager comprises a hinge region.

10. The cell of claim 9, wherein the hinge region comprises CD8a hinge region.11 The cell of any one of claims 1-10, wherein the SIRPa engager comprises a heterologous transmembrane domain (TMD).

12. The cell of claim 11, wherein the heterologous TMD is selected from the group consisting of CD47, CD85f, CD349, CD284, CD261 , CD 172b, CD277, CD 186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61 , CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51 , CD141 , CD115, CD42b, CD221, CD271, CD55, CD243, CD98, CD10, CD41 , CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11 b, CD107a, CD331 , CD222, CD20, CD79a, CD64, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227. CD44, CD62P, CD104, CD43, CD140a, CD31 , CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181 , CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121 b, CD34, CD38, CD30, CD1b, CD1c, CD154, CD6, CD52, CD132, CD32, CD66b, CD171 , CD191 , CD197, CD185, CD131 , CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361 , CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231 , CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD1581, CD178, CD295, CD151 , CD97, CD183, CD39, CD239, CD193, CD194, CD195. CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241 , CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91 , CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161 , CD215, CD150, CD11d, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111 , CD281 , CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158Z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD100, CD270, CD101 , CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD114, CD180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319, CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201 , CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321 , CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51 , CD41 , CD29: CD18, CD61 , CD104, and PDGFR TMD.

13. The cell of any one of claims 1-12, wherein the SIRPa engager comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 5.

14. The cell of any one of claims 1-13, wherein the heterologous gene is operably linked to an endogenous promoter at the insertion site15. The cell of any one of claims 1-14, wherein the polynucleotide comprises a heterologous promoter, an enhancer, and / or a polyadenylation sequence.

16. The cell of claim 15, wherein the heterologous promoter is operably linked to the heterologous gene.

17. The cell of claim 15 or 16, wherein the heterologous promoter is a constitutive promoter, an inducible promoter, a cell-type specific promoter, or a tissue-specific promoter.

18. The cell of any one of claims 15-17, wherein the heterologous promoter is selected from the group consisting of EF1a, UBC promoter, EFS promoter, CD11b promoter, sp146 / 147 promoter, CD68 promoter, sp146 / gp9, CD47 promoter, PGK promoter, SV40 promoter, CMV promoter, human b-actin promoter, UAS promoter, AC5 promoter, CaMKIla promoter, GAL1 / GAL10 promoter, GDS promoter, TRE promoter, U6 promoter, H1 promoter, 7SK promoter, DHFR promoter, EGR 1 promoter, TBG promoter, EIF4A1 promoter, FerH promoter, FerL promoter, GAPDH promoter, HSPA5 promoter, HSPA4 promoter, UBB promoter, EBV promoter, MMTV promoter, RSV promoter, Apolipoprotein E- Human Alpha 1 -Antitrypsin promoter, CAG promoter, CBA promoter, CK8 promoter, and mU1a promoter.

19. The cell of any one of claims 15-18, wherein the enhancer is a Woodchuck Hepatitis Virus (WHV) post-transcriptional regulatory element (WPRE) or a Simian Virus 40 (SV40) enhancer.

20. The cell of any one of claims 1-19, wherein the polynucleotide comprises a plurality of heterologous genes.

21. The cell of claim 20, wherein the polynucleotide comprises a first heterologous gene encoding the SIRPa engager and a second heterologous gene encoding an Fc receptor.

22. The cell of claim 21 , wherein the Fc receptor is CD64, CD32, or CD16.

23. The cell of claim 22, wherein the CD64 is a truncated (CD64t) that lacks a functional ICD.

24. The cell of claim 22 or 23, wherein the CD64 is a truncated (CD64t) that lacks a functional TMD.

25. The cell of claim 24, wherein the CD64t comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 6.

26. The cell of any one of claims 20-25, wherein the polynucleotide encodes a self-cleaving linker between the plurality of heterologous genes27. The cell of claim 26, wherein the self-cleaving linker is a 2A peptide.

28. The cell of claim 27, wherein the 2A peptide is a Porcine teschovirus-1 2A (P2A), an Equine rhinitis A virus 2A (E2A), Foot-and-mouth disease virus 182A (F2A), or a Thosea asigna virus 2A (T2A) peptide.

29. The cell of any one of claims 1-28, wherein the polynucleotide comprises a sequence having at least 85% sequence identity to SEQ ID NO:

730. The cell of any one of claims 1-29, wherein the heterologous gene is oriented 3’-5’ in relation to the endogenous gene.

31. The cell of any one of claims 1-30, wherein the heterologous gene is oriented 5’-3’ in relation to the endogenous gene.

32. The cell of any one of claims 1-31, wherein the mammalian cell is a human cell.

33. The cell of any one of claims 1-32, wherein the cell is a primary cell, a pluripotent cell, an embryonic stem cell, a somatic stem or progenitor cell, an immune cell, a cell differentiated from a pluripotent cell, or a cell differentiated from an embryonic stem cell.

34. The cell of claim 33, wherein the pluripotent cell is a hypoimmune pluripotent cell or an induced pluripotent stem cell (iPSC).35 The cell of any one of claims 1-33, wherein the cell is an iPSC or an embryonic stem cell (ESC) derived cell.

36. The cell of any one of claim 33-35, wherein the immune cell is a T cell, natural killer (NK) cell, or macrophage.

37. The cell of any one of claims 33-36, wherein the immune cell is an innate immune cell.

38. The cell of any one of claims 1-37, wherein the cell comprises reduced or eliminated human leukocyte antigen (HLA) class I (HLA-I), HLA class II (HLA-II), B2M, CIITA, and / or TRAC expression.

39. A method of editing a genome of a mammalian cell, the method comprising inserting a polynucleotide comprising a heterologous gene encoding a SIRPa engager into one or more sites within:(a) an endogenous B2M locus;(b) an endogenous CIITA locus; or(c) an endogenous TRAC locus.

40. The method of claim 39, wherein the polynucleotide is inserted into exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITA gene, exon 1 of the endogenous TRAC gene, or a combination thereof.

41. The method of claim 39 or 40, comprising inserting 2, 3, 4, 5, 6, 7, 8, or more copies of the polynucleotide.

42. The method of any one of claims 39-41 , further comprising introducing one or more single guide RNAs (sgRNA) into the cell that specifically hybridizes to a location within one or more sites comprising exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITAgene, exon 1 of the endogenous TRAC gene, or a combination thereof43. The method of claim 42, wherein 2, 3, 4, or more sgRNAs are introduced into the cell.

44. The method of any one of claims 39-43, further comprising providing a nuclease that introduces a double stranded DNA break to create the insertion site.

45. The method of claim 44, wherein the nuclease is a Cas9 nuclease or a MAD7 nuclease.

46. The method of claim 44 or 45, wherein the method comprises complexing the nuclease with the sgRNA to form a ribonucleoprotein (RNP) complex, wherein the RNP is then introduced into the cell to create the insertion site.

47. The method of claim 46, wherein a ratio of nuclease to sgRNA is 1 :1 to 1 :10.

48. The method of claim 46 or 47, wherein the method includes a first incubation step, wherein the nuclease and sgRNA are incubated for 1 to120 minutes at a temperature of 15 °C to 45 °C to create the RNP complex.

49. The method of claim 48, wherein the method further includes a second incubation step, wherein the RNP is further complexed to the polynucleotide at a concentration of 0.5 pg to 400 pg.

50. The method of claim 49, wherein the second incubation step, wherein the RNP and polynucleotide are incubated for between 1 to 100 minutes at a temperature of 15 °C to 45 °C.

51. The method of any one of claims 46-50, wherein the method further comprises delivering the RNP and polynucleotide complex into the cell52. The method of claim 51 , wherein the RNP and polynucleotide complex is inserted via electroporation.

53. The method of any one of claims 39-52, wherein the polynucleotide is on a plasmid.

54. The method of any one of claims 39-53, wherein the heterologous gene is inserted into the insertion site by homology directed repair.

55. The method of any one of claims 39-54, wherein the mammalian cell is a human cell.

56. The method of any one of claims 39-55, wherein the cell is a primary cell, a pluripotent cell, an embryonic stem cell, or an immune cell.

57. The method of claim 56, wherein the pluripotent cell is a hypoimmune pluripotent cell or an iPSC.

58. The method of any one of claims 39-57, wherein the cell is an iPSC or an ESC derived cell.

59. The method of any one of claim 56-58, wherein the immune cell is a T cell, NK cell, or macrophage.

60. The method of any one of claims 56-59, wherein the immune cell is an innate immune cell.

61. The method of any one of claims 39-60, wherein the cell comprises reduced or eliminated HLA-I, HLA-II, B2M, CIITA, and / or TRAC expression.

62. The method of any one of claims 56-61 , further comprising differentiating the pluripotent cell.

63. The method of claim 62, wherein the pluripotent cell is edited prior to differentiation.

64. The method of claim 62, wherein the pluripotent cell is differentiated prior to editing.

65. The method of any one of claims 62-64, wherein the pluripotent cell is differentiated into a T cell, an NK cell, or a macrophage66. The method of any one of claims 39-65, wherein the SIRPa engager comprises a CD47 ECD.

67. The method of claim 66, wherein the CD47 ECD comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:

168. The method of any one of claims 39-67, wherein the SIRPa engager lacks a functional CD47 ICD.

69. The method of any one of claims 39-68, wherein the SIRPa engager comprises an scFv.

70. The method of claim 69, wherein the scFv comprises heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 20-22 and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 23-25.

71. The method of any one of claims 39-70, wherein the SIRPa engager comprises a hinge region.

72. The method of claim 71 , wherein the hinge region comprises CD8a hinge region.

73. The method of any one of claims 39-72, wherein the SIRPa engager comprises a heterologous TMD.

74. The method of claim 73, the heterologous TMD is selected from the group consisting of CD47, CD85f, CD349, CD284, CD261, CD 172b, CD277, CD 186, CD156c, CD304, CD254, CD263, CD267, CD337, CD170, CD283, CD133, CD327, CD205, CD232, CD282, CD16b, CD85i, CD85a, CD85c, CD275, CD108, CD358, CD335, CD218b, CD355, CD336, CD160, CD25, CD4, CD8a, CD235a, CD233, CD230, CD90, CD74, CD3d, CD340, CD236, CD61 , CD18, CD54, CD29, CD1a, CD5, CD220, CD2, CD66e, CD51, CD141 , CD115, CD42b, CD221 , CD271 , CD55, CD243, CD98, CD10, CD41, CD14, CD45, CD228, CD16a, CD49e, CD126, CD63, CD48, CD7, CD140b, CD3g, CD117, CD28, CD8b, CD37, CD11b, CD107a, CD331, CD222, CD20, CD79a, CD64, CD32, CD143, CD324, CD42c, CD107b, CD56, CD102, CD49d, CD66a, CD142, CD59, CD62L, CD121a, CD122, CD13, CD155, CD119, CD19, CD116, CD46, CD1e, CD1d, CD227. CD44, CD62P, CD104, CD43, CD140a, CD31, CD152, CD326, CD62E, CD36, CD127, CD49b, CD105, CD35, CD223, CD138, CD325, CD58, CD106, CD53, CD120a, CD224, CD21, CD33, CD22, CD120b, CD11a, CD11c, CD363, CD73, CD88, CD204, CD332, CD9, CD203a, CD334, CD333, CD206, CD49f, CD238, CD252, CD89, CD124, CD181, CD182, CD24, CD95, CD40, CD49c, CD159a, CD159c, CD314, CD27, CD123, CD26, CD82, CD121b, CD34, CD38, CD30, CD1b, CD1c, CD154, CD6, CD52, CD132, CD32, CD66b, CD171, CD191 , CD197, CD185, CD131 , CD50, CD70, CD153, CD144, CD80, CD362, CD68, CD361, CD147, CD309, CD135, CD292, CD103, CD130, CD42d, CD66d, CD66c, CD96, CD110, CD79b, CD200, CD192, CD231 , CD86, CD212, CD118, CD146, CD134, CD158a, CD158bl, CD158b2, CD158e, CD158k, CD158j, CD1581 , CD178, CD295, CD151 , CD97, CD183, CD39, CD239, CD193, CD194, CD195. CD196, CDwl98, CDwl99, CD296, CD298, CD49a, CD322, CD85g, CD184, CD172a, CD156a, CD339, CD156b, CD213al, CD129, CD83, CD125, CD241, CD269, CD202b, CD87, CD164, CD136, CD137, CD249, CD69, CD91, CDw210b, CD167a, CD300c, CD157, CD317, CD148, CD161 , CD215, CD150, CD11d, CD218a, CD210, CD166, CD162, CD213a2, CD242, CD158g, CD158h, CD279, CD111, CD281 , CD226, CD234, CD167b, CD300e, CD276, CD305, CD300g, CD300d, CD109, CD272, CD163, CD302, CD158fl, CD85h, CD85d, CD177, CD158Z, CD158f2, CD85j, CD300f, CD92, CD351, CD112, CD100, CD270, CD101, CD297, CD316, CD352, CD217, CD307b, CD307a, CD307c, CD307d, CD307e, CD114, CD180, CD158d, CD273, CD290, CD244, CD169, CD299, CD318, CD360, CD229, CD248, CD354, CD320, CD93, CD319,CD113, CD163b, CD289, CD288, CD329, CD274, CD353, CD172g, CD315, CD280, CD264, CD300a, CD312, CD84, CD344, CD350, CD246, CD201 , CD338, CD208, CD257, CD328, CD286, CD357, CD294, CD321 , CD265, CD278, ITGA7, ITGA8, ITGA9, ITGA10, ITGA11, CD51 , CD41 , CD29: CD18, CD61 , CD104, and PDGFR TMD.

75. The method of any one of claims 39-74, wherein the SIRPa engager comprises a polypeptide sequence having an amino sequence having at least 85% sequence identity to SEQ ID NO: 5.

76. The method of any one of claims 39-75, wherein the polynucleotide further comprises a heterologous promoter, an enhancer, and / or a polyadenylation sequence.

77. The method of claim 76, wherein the heterologous promoter is operably linked to the heterologous gene.

78. The method of claim 76 or 77, wherein the heterologous promoter is a constitutive promoter, an inducible promoter, a cell-type specific promoter, or a tissue specific promoter79. The method of any one of claims 76-78, wherein the heterologous promoter is selected from the group consisting of EF1a, UBC promoter, EFS promoter, CD11b promoter, sp146 / 147 promoter, CD68 promoter, sp146 / gp9, CD47 promoter, PGK promoter, SV40 promoter, CMV promoter, human b-actin promoter, UAS promoter, AC5 promoter, CaMKIla promoter, GAL1 / GAL10 promoter, GDS promoter, TRE promoter, U6 promoter, H1 promoter, 7SK promoter, DHFR promoter, EGR 1 promoter, TBG promoter, EIF4A1 promoter, FerH promoter, FerL promoter, GAPDH promoter, HSPA5 promoter, HSPA4 promoter, UBB promoter, EBV promoter, MND promoter, LTR of MuMLV promoter, MMTV promoter, RSV promoter, Apolipoprotein E-Human Alpha 1 -Antitrypsin promoter, CAG promoter, CBA promoter, 0K8 promoter, and mU1a promoter.

80. The method of any one of claims 76-79, wherein the enhancer is a WPRE or SV40 enhancer.

81. The method of any one of claims 39-80, wherein the polynucleotide comprises a plurality of heterologous genes.

82. The method of claim 81 , wherein the polynucleotide comprises a first heterologous gene encoding the SIRPa engager and a second heterologous gene encoding an Fc receptor.

83. The method of claim 82, wherein the Fc receptor is CD64, CD32, or CD16.

84. The method of claim 83, wherein the CD64 is CD64t that lacks a functional ICD and / or TMD.

85. The method of claim 84, wherein the CD64t comprises an amino acid sequence of SEQ ID NO: 6.

86. The method of any one of claims 81-85, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving linker between the plurality of heterologous genes.

87. The method of claim 86, wherein the self-cleaving linker is a 2A peptide88. The method of claim 87, wherein the 2A peptide is a P2A, E2A, F2A, or T2A peptide89. The method of any one of claims 39-88, wherein the polynucleotide comprises a polynucleotide sequence having at least 85% sequence identity SEQ ID NO: 7.

90. An expression cassette configured to be inserted into an endogenous B2M gene, an endogenous CIITAgene, or an endogenous TRAC gene, wherein the expression cassette comprises a polynucleotide comprising from 5 -3’:(a) a 5’ homology arm homologous to an endogenous sequence upstream of the insertion site;(b) a nucleic acid encoding a SIRPa engager; and(c) a 3’ homology arm homologous to an endogenous sequence downstream of the insertion site.

91. An expression cassette configured to be inserted into an endogenous B2M gene, an endogenous CIITAgene, or an endogenous TRAC gene, wherein the expression cassette comprises a polynucleotide comprising from 5’-3’:(a) a 3’ homology arm homologous to an endogenous sequence downstream of the insertion site;(b) a nucleic acid encoding a SIRPa engager; and(c) a 5’ homology arm homologous to an endogenous sequence upstream of the insertion site.

92. The expression cassette of claim 90 or 91 , wherein the insertion site is within exon 1 of the endogenous B2M gene, exon 2 of the endogenous B2M gene, exon 3 of the endogenous CIITAgene, or exon 1 of the endogenous TRAC gene.

93. The expression cassette of any one of claims 90-92, wherein the 3’ homology arm comprises a polynucleotide sequence having at least 85% sequence identity to any one of SEQ ID NOs: 9, 11 , 13, or 15.

94. The expression cassette of any one of claims 90-93, wherein the 5’ homology arm comprises a polynucleotide sequence having at least an 85% sequence identity to any one of SEQ ID NOs: 8, 10, 12, or 14.

95. The expression cassette of any one of claims 90-94, wherein the SIRPa engager comprises a CD47 ECD.

96. The expression cassette of claim 95, wherein the CD47 ECD comprises the amino acid sequence of SEQ ID NO: 1.

97. The expression cassette of any one of claims 90-96, wherein the SIRPa engager comprises an scFv.

98. The expression cassette of claim 97, wherein the scFv comprises heavy chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 20-22 and light chain CDRs having the amino acid sequences set forth in SEQ ID NOs: 23-25.

99. The expression cassette of any one of claims 90-98, wherein the SIRPa engager lacks a functional CD47 ICD.

100. The expression cassette of any one of claims 90-99, wherein the SIRPa engager comprises a hinge region.

101. The expression cassette of claim 100, wherein the hinge region comprises CD8a hinge region.102 The expression cassette of any one of claims 90-101, wherein the SIRPa engager comprises a heterologous TMD.

103. The expression cassette of claim 102, wherein the heterologous TMD comprises a CD47, CD64, or PDGF TMD.

104. The expression cassette of any one of claims 90-103, wherein the SIRPa engager comprises a polypeptide sequence having the sequence of SEQ ID NO: 5.

105. The expression cassette of any one of claims 90-104, wherein the expression cassette further comprises a heterologous promoter, an enhancer, and / or a polyadenylation sequence106. The expression cassette of claim 105, wherein the heterologous promoter is operably linked to the heterologous gene.

107. The expression cassette of claim 105 or 106, wherein the heterologous promoter is a constitutive promoter, an inducible promoter, a cell-type specific promoter, or a tissue-specific promoter.

108. The expression cassette of any one of claims 105-107, wherein the heterologous promoter is selected from the group consisting of EF1a, UBC promoter, EFS promoter, CD11 b promoter, sp146 / 147 promoter, CD68 promoter, sp146 / gp9, CD47 promoter, PGK promoter, SV40 promoter, CMV promoter, human b-actin promoter, UAS promoter, AC5 promoter, CaMKIla promoter, GAL1 / GAL10 promoter, GDS promoter, TRE promoter, U6 promoter, H1 promoter, 7SK promoter, DHFR promoter, EGR 1 promoter, TBG promoter, EIF4A1 promoter, FerH promoter, FerL promoter, GAPDH promoter, HSPA5 promoter, HSPA4 promoter, UBB promoter, EBV promoter, MND promoter, LTR of MuMLV promoter, MMTV promoter, RSV promoter, Apolipoprotein E-Human Alpha 1- Antitrypsin promoter, CAG promoter, CBA promoter, CK8 promoter, and mU1a promoter109. The expression cassette of any one of claims 105-108, wherein the enhancer is a WPRE or SV40 enhancer.

110. The expression cassette of any one of claims 90-109, wherein the expression cassette comprises a plurality of heterologous genes.111 The expression cassette of claim 110, wherein the expression cassette comprises a first heterologous gene encoding the SIRPa engager and a second heterologous gene encoding an Fc receptor.

112. The expression cassette of claim 111, wherein the Fc receptor is CD64, CD32, or CD16.113 The expression cassette of claim 112, wherein the CD64 is CD64t that lacks a functional ICD and / or TMD114. The expression cassette of claim 113, wherein the CD64t comprises an amino acid sequence of SEQ ID NO: 6.

115. The expression cassette of any one of claims 110-114, wherein the expression cassette comprises a polynucleotide encoding a self-cleaving linker between the plurality of heterologous genes.

116. The expression cassette of claim 115, wherein the self-cleaving linker is a 2A peptide117. The expression cassette of claim 116, wherein the 2A peptide is a P2A, E2A, F2A, or T2A peptide.

118. The expression cassette of any one of claims 90-117, wherein the expression cassette comprises a polynucleotide sequence having at least an 85% sequence identity to SEQ ID NO: 7.

119. A pharmaceutical composition comprising the cell of any one of claims 1-38 and a pharmaceutically acceptable carrier.

120. A method of treating a disease in a subject, comprising transplanting the cell of any one of claims 1-38 or the pharmaceutical composition of claim 119 into the subject.

121. The method of claim 120, wherein said disease is Type 1 diabetes, a cardiac disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease.

122. The method of claim 121 , wherein the cancer is a blood cancer.

123. The method of claim 122, wherein the blood cancer is leukemia, lymphoma, or myeloma.

124. The cell of any one of claims 1-38 for transplanting into a subject for treating a disease.

125. The cell of claim 124, wherein the disease is Type 1 diabetes, a cardiac disease, a neurological disease, an endocrine disease, cancer, blindness, or a vascular disease.

126. The cell of claim 125, wherein the cancer is a blood cancer.

127. The cell of claim 126, wherein the blood cancer is leukemia, lymphoma, or myeloma.128 A kit, comprising the pharmaceutical composition of claim 119 and instructions for its use129. A polypeptide comprising a SIRPa engager and a CD64.

130. The polypeptide of claim 129, wherein the polypeptide comprises a self-cleaving peptide between the SIRPa engager and the CD64.

131. The polypeptide of claim 130, wherein the self-cleaving peptide is a 2A peptide.

132. The polypeptide of claim 129 or 130, wherein the polypeptide comprises the structure form the amino terminus to the carboxy terminus SIRPa engager-2A-CD64133. The polypeptide of claim 129 or 130, wherein the polypeptide comprises the structure from the amino terminus to the carboxy terminus CD64-2A- SIRPa engager.

134. The polypeptide of any one of claims 129-133, wherein the CD64 is CD64t.

135. A polynucleotide encoding the polypeptide of any one of claims 129-134.

Images