Modified cd47 proteins and their uses

Abstract

The present disclosure provides engineered CD47 proteins and uses thereof. Also disclosed are polynucleotides encoding the engineered CD47 protein, vectors comprising the polynucleotides, cells comprising the engineered proteins and / or the vectors, and compositions comprising the engineered CD47 protein.

Description

MODIFIED CD47 PROTEINS AND THEIR USESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Application No.63 / 534,134, filed August 23, 2023, and United States Provisional Application No. 63 / 534,136, filed August 23, 2023; the contents of which are incorporated herein by reference in their entirety.FIELD OF INVENTION

[0002] The present disclosure generally relates to engineered CD47 proteins and uses thereof. Also disclosed are polynucleotides encoding the engineered CD47 proteins, vectors comprising the polynucleotides, cells comprising the engineered proteins and / or the vectors, and compositions comprising the engineered CD47 proteins.

[0003] International application PCT / US2021 / 065157 and US applications 63 / 282,961, 63 / 270,956, and 63 / 222,954 are incorporated by reference in their entireties. International application PCT / US2023 / 013364 and US application 63 / 311,143 are incorporated by reference in their entireties.SUMMARY

[0004] CD47 is a transmembrane protein that, in humans, is encoded by the CD47 gene (Fig. 1). It is a member of the immunoglobulin (Ig) superfamily. CD47 has a molecular weight of about ~50 kDa. It is glycosylated and ubiquitously expressed by virtually all cells in the human body (Fig. 2). It has a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five membrane-spanning segments, and an alternatively spliced cytoplasmic tail at its C -terminus (Fig. 4). In addition, it has two extracellular regions and two intracellular regions between neighboring membrane-spanning segments. The signal peptide, when it exists on a CD47 isoform, is located at the N-terminus of the IgV-like domain. The human CD47 gene has six naturally-occurring transcripts, five of which each encode a protein isoform of CD47 (Ensembl, Gene: CD47). As such, there are five protein isoforms of human CD47, each with differential expression across various cell and tissue types.

[0005] CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration. CD47 interacts with multiple extracellular ligands, such as TSP-1, integrins, other CD47 proteins, and SIRPa. The CD47 / SIRPa interaction regulates a multitude of intercellular interactions in many body systems, such as the immune system where it regulates lymphocyte homeostasis, dendritic cell (DC) maturation and activation, proper localization of certain DC subsets in secondary lymphoid organs, and cellular transmigration. CD47 on cells, including on donor cells in the context of transplantation or cell therapy applications, can function as a "marker of self" and regulate phagocytosis by binding to SIRPa on the surface of circulating immune cells to deliver an inhibitory "don't kill me" signal. CD47-SIRPa binding results in phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs) on SIRPa, which triggers recruitment of the SHP1 and SHP2 Src homology phosphatases. These phosphatases, in turn, inhibit accumulation of myosin II at the phagocytic synapse, preventing phagocytosis (Fujioka et al., 1996). Phagocytosis of target cells by macrophages is ultimately regulated by a balance of activating signals (e.gjFcyR, CRT, LRP-1) and inhibitory signals (e.g., SIRPa-CD47). Elevated expression of CD47 can help the cell evade immune surveillance, subsequent destruction, and innate immune cell killing. Thus, CD47 can be used as a tolerogenic factor to induce immune tolerance when there is pathological or undesirable activation of an otherwise normal immune response. This can occur, for example, when a patient develops an immune reaction to donor antigens after receiving an allogeneictransplantation or an allogeneic cell therapy, or when the body responds inappropriately to self-antigens implicated in autoimmune diseases. However, there is a need in the art to improve on such uses of CD47.

[0006] In one aspect, the present disclosure provides engineered CD47 proteins comprising one or more amino acid substitutions and / or insertions relative to a wild-type human CD47 protein. In some embodiments, an engineered CD47 protein of the present disclosure exhibits decreased binding with TSP-1 relative to a wild-type human CD47 protein. In some embodiments, an engineered CD47 protein of the present disclosure exhibits decreased CD47 / TSP-l-mediated signaling relative to a wild-type CD47 protein.

[0007] In another aspect, an engineered CD47 protein of the present disclosure exhibits improved binding with SIRPa relative to a wild-type CD47 protein.

[0008] The present disclosure provides, in another aspect, engineered CD47 proteins that have fewer amino acids than the wild-type full-length human CD47 protein. Such engineered proteins afford more efficient cell engineering approaches, including delivery via integrating gene therapy vectors.

[0009] In one aspect, the present disclosure provides genetically engineered cells comprising an engineeredCD47 protein, wherein the engineered CD47 protein exhibits: decreased binding with TSP-1 relative to a wild-type CD47 protein; and / or decreased CD47 / TSP-l-mediated signaling relative to a wild-type CD47 protein.

[0010] In another aspect, the present disclosure provides genetically engineered cells comprising an engineered CD47 protein, wherein the genetically engineered cells: exhibit decreased CD47 / TSP-l-mediated undesirable effects relative to a control cell; and / or exhibit decreased CD47 / TSP-l-mediated exhaustion relative to a control cell; and / or exhibit decreased CD47-mediated undesirable effects relative to a control cell; and / or exhibit increased CD47- mediated desirable effects relative to a control cell; and / or exhibit increased longevity relative to a control cell; and / or exhibit increased persistence relative to a control cell; and / or exhibit increased expansion relative to a control cell, and / or exhibit increased response to its intended therapeutic target, and / or exhibit increased hypoimmunity relative to a control cell; and / or exhibit increased immune evasion relative to a control cell; and / or exhibit increased ability to evade a host immune response relative to a control cell; and / or exhibit increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response relative to a control cell; and / or express the engineered CD47 protein at a first expression level, wherein a control cell expressing a wild-type CD47 protein at the first expression level would experience one or more unwanted effects.

[0011] In another aspect, the present disclosure provides genetically engineered cells comprising an engineered CD47 protein, wherein the engineered CD47 protein exhibits improved binding with SIRPa relative to a wildtype CD47 protein.

[0012] In another aspect, the present disclosure provides genetically engineered cells comprising an engineered CD47 protein, wherein contacting a cell expressing SIRPa with the genetically engineered cell increases CD47 / SIRPa-mediated signaling in the cell expressing SIRPa relative to contacting the cell expressing SIRPa with a control cell.

[0013] In another aspect, the present disclosure provides genetically engineered cells comprising an engineered CD47 protein, wherein contacting a cell expressing SIRPa with the genetically engineered cell increases CD47 / SIRPa-mediated signaling in the cell expressing the engineered CD47 relative to contacting the cell expressing SIRPa with a control cell.

[0014] In another aspect, the present disclosure provides genetically engineered cells comprising an engineered CD47 protein, wherein contacting a cell expressing SIRPa with the genetically engineered cell increasesCD47 / SIRPa-mediated signaling in both the cell expressing SIRPa and the cell expressing CD47 relative to contacting the cell expressing SIRPa with a control cell.

[0015] In another aspect, the present disclosure provides genetically engineered cells comprising an engineered CD47 protein, wherein the genetically engineered cells: exhibit decreased CD47-mediated undesirable effects relative to a control cell; and / or exhibit increased CD47-mediated desirable effects relative to a control cell; and / or exhibit increased longevity relative to a control cell; and / or exhibit increased persistence relative to a control cell; and / or exhibit increased expansion relative to a control cell; and / or exhibit increased response to its intended therapeutic target, and / or exhibit increased hypoimmunity relative to a control cell; and / or exhibit improved immune evasion relative to a control cell; and / or exhibit increased ability to evade a host immune response relative to a control cell; and / or exhibit increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response relative to a control cell; and / or express the engineered CD47 protein at a first expression level, wherein a control cell expressing a wild-type CD47 protein at the first expression level would be killed by one or more immune cells.

[0016] In one aspect, the present disclosure provides genetically engineered cells comprising an engineeredCD47 protein, wherein the engineered CD47 protein exhibits: decreased binding with TSP-1 relative to a wild-type CD47 protein; decreased CD47 / TSP-l-mediated signaling relative to a wild-type CD47 protein; and improved binding with SIRPa relative to a wild-type CD47 protein; and wherein the genetically engineered cells: exhibit decreased CD47 / TSP-1- mediated undesirable effects relative to a control cell; exhibit decreased CD47-mediated undesirable effects relative to a control cell; exhibit decreased CD47 / TSP-l-mediated exhaustion relative to a control cell; exhibit decreased CD47- mediated undesirable effects relative to a control cell; exhibit increased CD47-mediated desirable effects relative to a control cell; exhibit increased longevity relative to a control cell; exhibit increased persistence relative to a control cell; exhibit increased expansion relative to a control cell; exhibit increased response to its intended therapeutic target; exhibit increased hypoimmunity relative to a control cell; exhibit increased immune evasion relative to a control cell; exhibit increased ability to evade a host immune response relative to a control cell; exhibit increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response relative to a control cell; express the engineered CD47 protein at a first expression level, wherein a control cell expressing a wild-type CD47 protein at the first expression level would experience one or more unwanted effects and would be killed by one or more immune cells; and wherein contacting a cell expressing SIRPa with the genetically engineered cell increases CD47 / SIRPa-mediated signaling in both the cell expressing SIRPa and the cell expressing CD47 relative to contacting the cell expressing SIRPa with a control cell.

[0017] In another aspect, the present disclosure provides compositions comprising genetically engineered cells of the present disclosure.

[0018] In another aspect, the present disclosure provides pharmaceutical compositions comprising (i) a genetically engineered cell of the present disclosure, and (ii) a pharmaceutically acceptable excipient.

[0019] In another aspect, the present disclosure provides methods of treating a disease in a subject, the method comprising administering to the subject a genetically engineered cell of the present disclosure, a composition of the present disclosure, or a pharmaceutical composition of the present disclosure.

[0020] In another aspect, the present disclosure provides populations of cells comprising a genetically engineered cell of the present disclosure for use in treating a disease in a subject.

[0021] In another aspect, the present disclosure provides uses of a genetically modified cell of the present disclosure, a population of cells of the present disclosure, a composition of the present disclosure, or a pharmaceutical composition of the present disclosure for use in treating a disease in a subject.

[0022] In another aspect, the present disclosure provides uses of a genetically modified cell of the present disclosure, a population of cells of the present disclosure, a composition of the present disclosure, or a pharmaceutical composition of the present disclosure in the manufacture of a medicament for the treatment of a disease.

[0023] In another aspect, the present disclosure provides methods of making a genetically engineered cell comprising an engineered CD47 protein and a nucleic acid construct encoding the engineered CD47 protein, the method comprising: delivering to a cell a nucleic acid construct comprising a first transgene encoding the engineered CD47 protein, thereby making a genetically engineered cell.

[0024] In another aspect, the present disclosure provides a nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered protein, wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full-length CD47 intracellular domains. In some embodiments, the one or more nucleic acid sequences comprise a nucleic acid sequence at least 80% identical to SEQ ID NO: 17, SEQ ID NO: 16, or SEQ ID NO: 15.

[0025] In another aspect, the present disclosure provides an engineered CD47 protein or a nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered CD47 protein comprising (a) one or more extracellular domains; and (b) one or more membrane tethers. In some embodiments, the one or more extracellular domains comprise: an amino acid substitution at position S82 of SEQ ID NO: 2, an amino acid substitution at position S64 of SEQ ID NO: 3, an amino acid substitution at position S82 of SEQ ID NO: 1, or an amino acid substitution at position S37 of SEQ ID NO: 6; and / or an amino acid substitution at position S83 of SEQ ID NO: 2, an amino acid substitution at position S65 of SEQ ID NO: 3, an amino acid substitution at position S83 of SEQ ID NO: 1, or an amino acid substitution at position S38 of SEQ ID NO: 6; and / or an insertion of one or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 3 SEQ ID NO: 1 or SEQ ID NO: 6; and wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full-length CD47 intracellular domains.

[0026] In another aspect, the present disclosure provides an engineered CD47 protein or a nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered CD47 protein, wherein the engineered CD47 protein comprises an insertion of three amino acids relative to SEQ ID NO: 4 or SEQ ID NO: 5.

[0027] In another aspect, the present disclosure provides an engineered protein encoded by a nucleic acid construct as described herein.

[0028] In another aspect, the present disclosure provides an engineered protein, wherein the engineered protein is or comprises an amino acid sequence at least 80% identical to SEQ ID NO: 5, and wherein the engineered protein does not comprise one or more full-length CD47 intracellular domains.

[0029] In another aspect, the present disclosure provides an engineered protein, wherein the engineered protein is or comprises an amino acid sequence at least 80% identical to SEQ ID NO: 4, and wherein the engineered protein does not comprise one or more full-length CD47 intracellular domains.

[0030] In another aspect, the present disclosure provides an engineered CD47 protein comprising: (a) one or more extracellular domains; and (b) one or more membrane tethers; wherein the one or more extracellular domains comprise: an amino acid substitution at position S82 of SEQ ID NO: 2, an amino acid substitution at position S64 of SEQ ID NO: 3, an amino acid substitution at position S82 of SEQ ID NO: 1, or an amino acid substitution at position S37 of SEQ ID NO: 6; and / or an amino acid substitution at position S83 of SEQ ID NO: 2, an amino acid substitution at position S65 of SEQ ID NO: 3, an amino acid substitution at position S83 of SEQ ID NO: 1, or an amino acid substitution at position S38 of SEQ ID NO: 6; and / or an insertion of one or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 3SEQ ID NO: 1 or SEQ ID NO: 6; and wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full-length CD47 intracellular domains.

[0031] In another aspect, the present disclosure provides a genetically engineered cell comprising an engineered CD47 protein as described herein.

[0032] In another aspect, the present disclosure provides a composition comprising a genetically engineered cell as disclosed herein.

[0033] In another aspect, the present disclosure provides a method comprising administering to a subject a genetically engineered cell, a composition, or a pharmaceutical composition described herein.

[0034] In another aspect, the present disclosure provides a method of treating a disease in a subject, the method comprising administering to the subject a genetically engineered cell, a composition, or a pharmaceutical composition described herein.

[0035] In another aspect, the present disclosure provides a population of cells comprising a genetically engineered cell as described herein for use in treating a disease in a subject.

[0036] In another aspect, the present disclosure provides a composition or pharmaceutical composition for use in treating a disease in a subject.

[0037] In another aspect, the present disclosure provides a use of a genetically engineered cell, a population of cells, a composition, or a pharmaceutical composition as described herein for use in treating a disease in a subject.

[0038] In another aspect, the present disclosure provides a use of a genetically engineered cell, a population of cells, a composition, or a pharmaceutical composition as described herein in the manufacture of a medicament for the treatment of a disease.

[0039] In another aspect, the present disclosure provides a method of making a genetically engineered cell comprising a nucleic acid construct, the method comprising: delivering to a cell a nucleic acid construct as described herein, thereby making a genetically engineered cell.BRIEF DESCRIPTION OF THE DRAWING

[0040] Figure 1 provides a map of the human CD47 gene and illustrates the regions in the human CD47 gene that are protein coding. This map comes from the Ensembl genome database.

[0041] Figure 2A and Figure 2B provide the isoform expression of CD47 ENSG00000196776.14 CD47 molecule (Source: HGNC Symbol;Acc:HGNC: 1682) and illustrate expression of each isoform in various human tissues and human cell types. This data comes from the Genotype-Tissue Expression (GTEx) project database.

[0042] Figure 3 provides the predicted CD47 transmembrane domains and the human CD47 protein topology. In particular, Fig. 3 provides the predicted locations of various domains in the human CD47 protein. This prediction comes from the Universal Protein Resource (UniProt).

[0043] Figure 4 provides the predicted human CD47 tertiary structure from the AlphaFold Protein StructureDatabase.

[0044] Figure 5A, Figure 5B, Figure 5C, and Figure 5D provide a sequence alignment of Isoform 201,Isoform 202, Isoform 203, Isoform 205, and Isoform 206 of the human CD47 protein.

[0045] Figure 6 provides an exemplary graph showing viral titers, as assessed via the Ella automated immunoassay system, of LW comprising exemplary CD47 truncated variants.

[0046] Figure 7 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressingCD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry). Various CD47 variants, including several comprising GPI anchors, were tested.

[0047] Figure 8 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry). Various CD47 variants, including several comprising truncated intracellular domains and alternative transmembrane domains, were tested.

[0048] Figure 9 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressingCD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry). Various CD47 variants, including those comprising truncated intracellular domains and alternative hinge domains, were tested.

[0049] Figure 10 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry). Various CD47 variants, including those comprising truncated intracellular domains and alternative hinge domains, were tested.

[0050] Figure 11 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressingCD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry). Various CD47 variants, including those comprising truncated intracellular domains and alternative hinge domains, were tested.

[0051] Figure 12A, Figure 12B, Figure 12C, Figure 12D, and Figure 12E provide exemplary graphs showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry).

[0052] Figure 13 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry).

[0053] Figure 14 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressingCD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry).

[0054] Figure 15 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressingCD47 after treatment with truncated CD47 LW (as assessed by SIRPa-FC flow cytometry).

[0055] Figure 16 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by SIRPa-FC flow cytometry).

[0056] Figure 17 provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by SIRPa-FC flow cytometry).

[0057] Figure 18A provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by anti-CD47 flow cytometry).

[0058] Figure 18B provides an exemplary graph showing the percentage of CD47KO HEK293T cells expressing CD47 after treatment with truncated CD47 LW (as assessed by SIRPa-FC flow cytometry).

[0059] Figure 19 provides schematics of various exemplary CD47 variants tested, including several variants comprising truncated intracellular domains and alternative transmembrane domains.

[0060] Figure 20 provides graphs showing Gibco iPSC expression of CD47 (left) and isotype control (right) after treatment with control or with truncated CD47 LW, as assessed by flow cytometry.

[0061] Figure 21A and Figure 21B provide exemplary graphs showing Gibco iPSC surface expression ofHLA-I (top row), HLA-II (middle row), and CD47 (bottom row) after treatment with control or with truncated CD47 LW (as assessed by anti-HLA-I, anti-HLA-II, or anti-CD47 flow cytometry, respectively, relative to isotype control). Surface expression of CD47 was also assessed with Quantibrite.

[0062] Figure 22A and Figure 22B provide exemplary graphs showing killing of Gibco iPSC target cells expressing CD47 LW by natural killer (NK) effector cells (top row), in the absence of effector cells (middle row), or after treatment with TritonX (bottom row), as assessed by XCELLIGENCE assay.DETAILED DESCRIPTIONI. Definitions

[0063] All publications, patents, and patent applications referred to herein are 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.

[0064] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings generally understood by a person skilled in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present disclosure, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the Specification as a whole.

[0065] As used herein, the singular terms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise.

[0066] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted.

[0067] The term "about," as used herein when referring to a measurable value such as a sequence length and the like, is meant to encompass variations of 5%, 1 %, 0.5%, or even 0.1 % of the specified amount.

[0068] Unless the context requires otherwise, the terms "comprise," "comprises," and "comprising," or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

[0069] Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' orientation; and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0070] It is to be understood that this disclosure is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context in which they are used by those skilled in the art.

[0071] As used herein, "affinity" refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., a receptor) and its binding partner (e.g., a ligand). The affinity of a molecule for its partner can generally be represented by the equilibrium dissociation constant (KD) (or its inverse equilibrium association constant, KA). Affinity can be measured by common methods known in the art, including those described herein. See, for example, Pope M.E., Soste M.V., Eyford B.A., Anderson N.L., Pearson T.W., (2009) J. Immunol. Methods. 341(l-2):86-96 and methods described therein.

[0072] As used herein, the terms "percent identity" and "% identity," as applied to nucleic acid or polynucleotide sequences, refer to the percentage of residue matches between at least two nucleic acid or polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.

[0073] Percent identity between nucleic acid or polynucleotide sequences may be determined using a suite of commonly used and freely available sequence comparison algorithms provided by the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (Altschul, S. F. et al. (1990) J. Mol. Biol. 215:403-410), which is available from several sources, including the NCBI, Bethesda, Md., and on the Internet at http: / / www.ncbi.nlm.nih.gov / BLAST / .

[0074] Nucleic acid or polynucleotide sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608; Cassol et al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91-98). The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. The term nucleic acid is used interchangeably with polynucleotide, and (in appropriate contexts) gene, cDNA, and mRNA encoded by a gene.

[0075] As used herein, "percent (%) amino acid sequence identity" with respect to a peptide, polypeptide or protein sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in another peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Percent amino acid sequence identity in the current disclosure is measured using BLAST software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0076] An amino acid substitution refers to the replacement of one amino acid in a polypeptide with another amino acid. Exemplary substitutions are shown in Table 1. Amino acid substitutions may be introduced into a protein of interest and the products screened for a desired activity, for example, retained / improved biological activity.Table 1. Exemplary Amino Acid Substitutions

[0077] Amino acids may be grouped according to common side-chain properties:(1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He;(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;(3) acidic: Asp, Glu;(4) basic: His, Lys, Arg;(5) residues that influence chain orientation: Gly, Pro;(6) aromatic: Trp, Tyr, Phe.

[0078] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. The term, "corresponding to" with reference to nucleotide or amino acid positions of a sequence, such as set forth in the Sequence Listing, refers to nucleotides or amino acid positions identified upon alignment with a target sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, corresponding residues of a similar sequence (e.g., a fragment or species variant) can be determined by alignment to a reference sequence by structural alignment methods. By aligning the sequences, one skilled in the art can identify corresponding residues, for example, using conserved and identical amino acid residues as guides.

[0079] As used herein, an isoform of the human CD47 protein refers to a protein that is translated from the human CD47 gene and is processed by alternative splicing.

[0080] As used herein, a wild-type human CD47 protein refers to a human CD47 protein that is naturally occurring in vivo, such as a wild-type human CD47 protein encoded in / by the human genome. A wild-type human CD47 protein could be any of the isoforms of the naturally occurring human CD47 protein. The amino acid sequences of the five currently known naturally occurring isoforms of CD47 (isoforms 201, 202, 203, 205, 206) are set forth in SEQ ID NOs: 1, 2, 4-5. Isoform CD47-202 (SEQ ID NO:2) is the full-length wild-type human CD47 protein as it is translated containing its signal sequence. The sequence of the mature CD47-202 isoform lacking its signal sequence is set forth in SEQ ID NO:3. A wild-type human CD47 protein may or may not have a signal peptide when it is expressed. For instance, CD47-206 lacks a signal peptide when it is translated. A wild-type human CD47 protein may or may not be glycosylated. A wild-type human CD47 protein could be a proteoglycan.

[0081] As used herein, an engineered CD47 protein refers to a CD47 protein that is not naturally occurring in any species. In other words, an engineered CD47 protein is not a wild-type CD47 protein in any species. In some embodiments, the engineered CD47 protein is an engineered human CD47 protein, meaning it is engineered by using the human wild-type CD47 protein as a starting material and making one or more of the modifications described herein. In some embodiments, the engineered CD47 protein is an engineered humanized CD47 protein, meaning it is engineered by using a non-human (e.g., murine) CD47 protein as a starting material and by humanizing the non-human CD47 sequence in addition to making one or more of the other modifications described herein. In some embodiments, the engineered CD47 protein is an engineered partially-humanized CD47 protein, meaning it is engineered by using a non- human (e.g., murine) CD47 protein as a starting material and by humanizing a portion of the non-human CD47 sequence in addition to making one or more of the other modifications described herein.

[0082] As used herein, an engineered CD47 protein refers to a protein that is not a CD47 protein encoded in / by a native genome, e.g., not a wild-type CD47 protein. Non-limiting examples of engineered CD47 proteins include an engineered CD47 protein having (i) a human CD47 extracellular domain or a portion thereof, at least one humanCD47 transmembrane domain or a portion thereof, and a portion of a human CD47 intracellular domain comprising a deletion of at least one amino acid, wherein the deletion is not a C-terminal deletion of 18 amino acids, (ii) a portion of a human CD47 extracellular domain, at least one human CD47 transmembrane domain or a portion thereof, and a portion of a human CD47 intracellular domain comprising a C-terminal deletion of 18 amino acids, (Hi) a human CD47 extracellular domain or a portion thereof, at least one and fewer than five human CD47 transmembrane domain(s) or portion(s) thereof, and a portion of a human CD47 intracellular domain comprising a C-terminal deletion of 18 amino acids, (iv) a human CD47 extracellular domain or a portion thereof, at least one human CD47 transmembrane domain or a portion thereof, and a signal peptide, wherein the engineered CD47 protein does not comprise an intracellular domain, (v) a human CD47 extracellular domain, and at least one human CD47 transmembrane domain or a portion thereof, wherein the engineered CD47 protein does not comprise an intracellular domain, (vi) a human CD47 extracellular domain or a portion thereof, and at least one and fewer than five human CD47 transmembrane domain(s) or portion(s) thereof, wherein the engineered CD47 protein does not comprise an intracellular domain, (vii) a human CD47 extracellular domain or a portion thereof, and at least one human CD47 transmembrane domain or a portion thereof, no intracellular domain, or a human CD47 intracellular domain comprising a deletion of at least one amino acid, wherein the engineered CD47 protein has an amino acid sequence that has at most 99% identity to SEQ ID NO: 1 and SEQ ID NO:6.

[0083] As used herein, the term "exogenous" in the context of a polynucleotide or polypeptide being expressed is intended to mean that the referenced molecule or the referenced polypeptide is introduced into the cell of interest. The polypeptide can be introduced, for example, by introduction of an encoding nucleic acid into the genetic material of the cells such as by integration into a chromosome or as non-chromosomal genetic material such as a plasmid or expression vector. Therefore, the term as it is used in reference to expression of an encoding nucleic acid refers to introduction of the encoding nucleic acid in an expressible form into the cell. An exogenous polynucleotide can be inserted into at least one allele of the cell using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus based viral vector. In some embodiments, the exogenous polynucleotide is inserted into target locus of at least one allele of the cell.

[0084] An "exogenous" molecule is a molecule, construct, factor and the like that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods. "Normal presence in the cell" is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of neurons is an exogenous molecule with respect to an adult neuron cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally-functioning endogenous molecule.

[0085] An exogenous molecule or factor can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA; can be single- or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplex-forming nucleic acids. See, for example, U.S. Pat. Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin remodeling factors, methylated DNA binding proteins,polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0086] An exogenous molecule or construct can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. In such instances, the exogenous molecule is introduced into the cell at greater concentrations than that of the endogenous molecule in the cell. In some instances, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (Ze., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran- mediated transfer and viral vector-mediated transfer.

[0087] The term "genetic modification" and its grammatical equivalents as used herein can refer to one or more alterations of a nucleic acid, e.g., the nucleic acid within an organism's genome. For example, genetic modification can refer to alterations, additions, and / or deletion of genes or portions of genes or other nucleic acid sequences. A genetically modified cell can also refer to a cell with an added, deleted, and / or altered gene or portion of a gene. A genetically modified cell can also refer to a cell with an added nucleic acid sequence that is not a gene or gene portion. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms, and mechanisms that result in permanent knock-in, knock-down, or knock-out of target genes or portions of genes or nucleic acid sequences. Genetic modifications include, for example, both transient knock-in and mechanisms that result in permanent knock-in of nucleic acids sequences. Genetic modifications also include, for example, reduced or increased transcription, reduced or increased mRNA stability, reduced or increased translation, and reduced or increased protein stability.

[0088] As used herein, a portion of a peptide has fewer amino acids than the reference peptide, and has at least one amino acid from that peptide.

[0089] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells.II. CD47 proteinsOverview of CD47 protein

[0090] This disclosure relates to engineered CD47 proteins and uses thereof.

[0091] CD47, also known as integrin-associated protein (IAP) or MER6, is a transmembrane protein that, in humans, is encoded by the human CD47 gene (SEQ ID NO: 19) (Fig. 1). CD47 is a member of the immunoglobulin (Ig) superfamily and is involved in a range of cellular processes, including apoptosis, proliferation, adhesion, and migration.

[0092] Human CD47 is about ~50 kDa. It is glycosylated and ubiquitously expressed by virtually all cells in the human body (Fig. 2). Historical literature suggests that isoform 202 (i.e., CD47-202, SEQ ID NO: 2) is mainly expressed in the brain, but recent GTEx expression data do not support this conclusion (Fig. 2). As shown in Example 1 herein, isoform CD47-202 (SEQ ID N0:2, SEQ ID NO: 14) and isoform CD47-201 (SEQ ID NO: 1, SEQ ID NO: 13) are expressed at relatively equal levels in Gibco and Rues2 human stem cell lines. Isoforms CD47-206 (SEQ ID N0:6, SEQ ID NO: 18), 205 (SEQ ID NO: 5, SEQ ID NO: 17), 204 (SEQ ID NO: 16) also appear to be highly expressed in these stem cell lines. No evidence of isoform CD47-203 (SEQ ID N0:4, SEQ ID NO: 15) in stem cell lines was detected.

[0093] Human CD47 has a single IgV-like domain at its N-terminus, a highly hydrophobic stretch with five membrane-spanning segments, and an alternatively spliced cytoplasmic tail at its C-terminus (Fig. 4). In addition, it hastwo extracellular regions and two intracellular regions between neighboring membrane-spanning segments. The signal peptide, when it exists on a CD47 isoform, is located at the N-terminus of the IgV-like domain.

[0094] As used herein, a human CD47 extracellular domain refers to the IgV-like domain at the N-terminus of the human CD47 protein. Structurally, the human CD47 extracellular domain is the N-terminal portion of the human CD47 protein that is located outside a cell when the human CD47 protein is anchored in the cell membrane. In some embodiments, the human CD47 extracellular domain has an amino acid sequence corresponding to amino acids 19-141 of SEQ ID N0:2, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 19-141 of SEQ ID NO: 2. In some embodiments, the human CD47 extracellular domain has an amino acid sequence corresponding to amino acids 19-141 of SEQ ID NO:2, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 19-141 of SEQ ID NO: 2.

[0095] As used herein, a human CD47 intracellular domain refers to the cytoplasmic tail at the C-terminus of the human CD47 protein. Structurally, the human CD47 intracellular domain is the C-terminal portion of the human CD47 protein that is located inside a cell when the human CD47 protein is anchored in the cell membrane. The human CD47 intracellular domain is alternatively spliced in vivo. In some embodiments, the human CD47 intracellular domain has an amino acid sequence corresponding to amino acids 290-323 of SEQ ID NO:2, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 290-323 of SEQ ID NO:2. In some embodiments, the human CD47 intracellular domain has an amino acid sequence corresponding to amino acids 290-323 of SEQ ID NO:2, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 290-323 of SEQ ID NO: 2.

[0096] As used herein, a human CD47 transmembrane domain refers to one of the membrane-spanning segments of the human CD47 protein. In some embodiments, the human CD47 transmembrane domain has an amino acid sequence corresponding to amino acids 142-162, 177-197, 208-228, 236-257, or 269-289 of SEQ ID NO:2, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 142-162, 177-197, 208-228, 236-257, or 269-289 of SEQ ID NO:2. In some embodiments, the human CD47 transmembrane domain has an amino acid sequence corresponding to amino acids 142-162, 177-197, 208-228, 236-257, or 269-289 of SEQ ID NO:2, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 142-162, 177-197, 208-228, 236-257, or 269-289 of SEQ ID NO:2.

[0097] As used herein, a signal peptide refers to the short peptide present at the N-terminus of the CD47 protein when the protein is initially translated. Signal peptides are usually cleaved off from a protein by a signal peptidase during or immediately after insertion into a cell membrane. Signal peptides function to prompt a cell to translocate the protein, usually to the plasma membrane. In some embodiments, the signal peptide for a human CD47 protein has an amino acid sequence corresponding to amino acids 1-18 of SEQ ID NO:2, or an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 1-18 of SEQ ID NO:2. In some embodiments, the signal peptide for a human CD47 protein has an amino acid sequence corresponding to amino acids 1-18 of SEQ ID NO: 2, or an amino acid sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with amino acids 1-18 of SEQ ID NO:2.

[0098] The human CD47 gene has six transcripts, five of which encode a protein isoform of CD47 (Ensembl, Gene: CD47). The six transcripts are named CD47-201, CD47-202, CD47-203, CD47-204, CD47-205, and CD47-206 (Ensembl, Gene: CD47, ENSG00000196776). The coding DNA sequence (CDS) of the six transcripts are as set forth inSEQ ID NO: 13-18, respectively. The amino acid sequences of the five protein isoforms are as set forth in SEQ ID NO: 1, 2, 4, 5, 6, respectively (Table 2).

[0099] Transcript CD47-202 (SEQ ID NO: 14) encodes isoform CD47-202 (SEQ ID N0:2), which has 323 amino acids. CD47-202 is the longest transcript of the human CD47 gene. It is designated as the representative transcript in the Ensembl database. In identifying the representative transcript, Ensembl aims to identity the transcript that, on balance, has the highest coverage of conserved exons, highest expression, longest coding sequence and is represented in other key resources, such as NCBI and UniProt. All splice junctions of the CD47-202 transcript are supported by at least one non-suspect mRNA.

[0100] Transcript CD47-201 (SEQ ID NO: 13) encodes isoform CD47-201 (SEQ ID NO: 1), which has 305 amino acids. Isoform CD47-201 has a C-terminal truncation of 18 amino acids from isoform CD47-202. All splice junctions of the CD47-201 transcript are supported by at least one non-suspect mRNA.

[0101] Transcript CD47-203 (SEQ ID NO: 15) encodes isoform CD47-203 (SEQ ID N0:4), which has 86 amino acids. The only support for the transcript model is from a single expressed sequence tag (EST).

[0102] Transcript CD47-204 (SEQ ID NO: 16) does not encode protein. All splice junctions of this transcript are supported by at least one non-suspect mRNA

[0103] Transcript CD47-205 (SEQ ID NO: 17) encodes isoform CD47-205 (SEQ ID N0:5), which has 109 amino acids. Isoform 205 comprises 3 transmembrane domains and a truncated intracellular domain from isoform CD47-202 (SEQ ID NO: 2). The best supporting mRNA for the transcript model is flagged as suspect or the support is from multiple ESTs.

[0104] Transcript CD47-206 (SEQ ID NO: 18) encodes isoform CD47-206 (SEQ ID N0:6), which has 183 amino acids. Isoform 206 comprises a truncated extracellular domain and 5 transmembrane domains from isoform CD47-202 (SEQ ID NO: 2).

[0105] The amino acid sequences of the five isoforms are listed in Table 2. The amino acids corresponding to the various domains in the human CD47 protein are also identified in Table 2 and depicted in Fig. 5. "Intracellular connection" refers to the intracellular region connecting neighboring transmembrane domains, which is positioned inside of a cell (i.e., not outside the cell and not within the cell membrane) but are not positioned at the N-terminus or the C- terminus of the engineered CD47 protein. "Extracellular connection" refers to the extracellular region connecting neighboring transmembrane domains, which is positioned outside of a cell (i.e., not inside the cell and not within the cell membrane). As used herein, the CD47 "intracellular domain" does not include the intracellular connections. Also as used herein, the CD47 "extracellular domain" does not include the extracellular connections.Table 2. Amino Acid SEQ ID NOs for CD47 DomainsEngineered CD47 proteins

[0106] The present disclosure provides engineered CD47 proteins comprising one or more amino acid substitutions and / or insertions relative to a wild-type human CD47 protein. In some embodiments, the one or more amino acid substitutions and / or insertions relative to a wild-type human CD47 protein are described in WO2016179399A1 (incorporated by reference herein). In some embodiments, the one or more amino acid substitutions and / or insertions relative to a wild-type human CD47 protein are described Kaur, Sukhbir, et al. "Heparan sulfate modification of the transmembrane receptor CD47 is necessary for inhibition of T cell receptor signaling by thrombospondin-1." Journal of Biological Chemistry 286.17 (2011): 14991-15002 (incorporated by reference herein). In some embodiments, the one or more amino acid substitutions and / or insertions relative to a wild-type human CD47 protein are described in Ho, Chia Chi M., et al. "Velcro" engineering of high affinity CD47 ectodomain as signal regulatory protein a (SIRPa) antagonists that enhance antibody-dependent cellular phagocytosis." Journal of Biological Chemistry 290.20 (2015): 12650-12663 (incorporated by reference herein).

[0107] In some embodiments, an engineered CD47 protein of the present disclosure exhibits decreased binding with TSP-1 relative to a wild-type human CD47 protein. In some embodiments, an engineered CD47 protein of the present disclosure exhibits decreased CD47 / TSP-l-mediated signaling relative to a wild-type CD47 protein. In some embodiments, decreased binding to TSP-1 is due to a lack of a heparan sulfate proteoglycan modification on the engineered CD47 protein.

[0108] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid substitution at position S82 of SEQ ID NO: 2, an amino acid substitution at position S64 of SEQ ID NO: 3, an amino acid substitution at position S82 of SEQ ID NO: 1, or an amino acid substitution at position S37 of SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid substitution at position S82 of SEQ ID NO: 2. In some embodiments, the term "an engineered CD47 protein comprising an S64A mutation" (or grammatical variants of the term) is used to refer to an engineered CD47 protein of the present disclosure that comprises: an amino acid substitution at position S82 of SEQ ID NO: 2, an amino acid substitution at position S64 ofSEQ ID NO: 3, an amino acid substitution at position S82 of SEQ ID NO: 1, or an amino acid substitution at position S37 of SEQ ID NO: 6.

[0109] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid substitution at position S83 of SEQ ID NO: 2, an amino acid substitution at position S65 of SEQ ID NO: 3, an amino acid substitution at position S83 of SEQ ID NO: 1, or an amino acid substitution at position S38 of SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid substitution at position S83 of SEQ ID NO: 2.

[0110] In some embodiments, an engineered CD47 protein of the present disclosure comprises: amino acid substitutions at positions S82 and S83 of SEQ ID NO: 2, amino acid substitutions at positions S64 and S65 of SEQ ID NO: 3, amino acid substitutions at positions S82 and S83 of SEQ ID NO: 1, or amino acid substitutions at positions S37 and S38 of SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises amino acid substitutions at positions S82 and S83 of SEQ ID NO: 2.

[0111] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid substitution of S82A relative to SEQ ID NO: 2, an amino acid substitution of S64A relative to SEQ ID NO: 3, an amino acid substitution of S82A relative to SEQ ID NO: 1, or an amino acid substitution of S37A relative to SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid substitution of S82A relative to SEQ ID NO: 2.

[0112] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 2, an amino acid substitution of S64V, S64I, S64L, S64F, S64W, S64G, or S64P relative to SEQ ID NO: 3, an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 1, or an amino acid substitution of S37V, S37I, S37L, S37F, S37W, S37G, or S37P relative to SEQ ID NO: 6.

[0113] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid substitution of S83A relative to SEQ ID NO: 2, an amino acid substitution of S65A relative to SEQ ID NO: 3, an amino acid substitution of S83A relative to SEQ ID NO: 1, or an amino acid substitution of S38A relative to SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid substitution of S83A relative to SEQ ID NO: 2.

[0114] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 2, an amino acid substitution of S65V, S65I, S65L, S65F, S65W, S65G, or S65P relative to SEQ ID NO: 3, an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 1, or an amino acid substitution of S38V, S38I, S38L, S38F, S38W, S38G, or S38P relative to SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 2.

[0115] In some embodiments, an engineered CD47 protein of the present disclosure comprises: a first amino acid substitution selected from the group consisting of: an amino acid substitution of S82A relative to SEQ ID NO: 2, an amino acid substitution of S64A relative to SEQ ID NO: 3, an amino acid substitution of S82A relative to SEQ ID NO: 1, and an amino acid substitution of S37A relative to SEQ ID NO: 6; and a second amino acid substitution selected from the group consisting of: an amino acid substitution of S83A relative to SEQ ID NO: 2, an amino acid substitution of S65A relative to SEQ ID NO: 3, an amino acid substitution of S83A relative to SEQ ID NO: 1, and an amino acid substitution ofS38A relative to SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises amino acid substitutions of S82A and S83A relative to SEQ ID NO: 2.

[0116] In some embodiments, an engineered CD47 protein of the present disclosure comprises: a first amino acid substitution selected from the group consisting of: an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 2, an amino acid substitution of S64V, S64I, S64L, S64F, S64W, S64G, or S64P relative to SEQ ID NO: 3, an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 1, and an amino acid substitution of S37V, S37I, S37L, S37F, S37W, S37G, or S37P relative to SEQ ID NO: 6; and a second amino acid substitution selected from the group consisting of: an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 2, an amino acid substitution of S65V, S65I, S65L, S65F, S65W, S65G, or S65P relative to SEQ ID NO: 3, an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 1, and an amino acid substitution of S38V, S38I, S38L, S38F, S38W, S38G, or S38P relative to SEQ ID NO: 6.

[0117] In some embodiments, an engineered CD47 protein of the present disclosure comprises: a first amino acid substitution selected from the group consisting of: an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 2; and a second amino acid substitution selected from the group consisting of: an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 2.

[0118] In some embodiments, an engineered CD47 protein of the present disclosure comprises an elongated N-terminus, which increases the buried surface area between the engineered CD47 protein and SIRPa, resulting in increasing affinity. In some embodiments, an engineered CD47 protein of the present disclosure exhibits improved binding with SIRPa relative to a wild-type CD47 protein.

[0119] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid sequence having at least 80% identity to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and an insertion of one or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises: an amino acid sequence having at least 80% identity to SEQ ID NO: 2; and an insertion of one or more amino acids relative to SEQ ID NO: 2.

[0120] In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of three amino acids relative to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of three amino acids relative to SEQ ID NO: 2.

[0121] In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of the amino acids WQP relative to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of the amino acids WQP relative to SEQ ID NO: 2.

[0122] In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 2 or SEQ ID NO: 1. In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 2.

[0123] In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of the amino acids WQP between A18 and Q19 relative to SEQ ID NO: 2 or SEQ ID NO: 1. In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of the amino acids WQP between A18 and Q19 relative to SEQ ID NO: 2.

[0124] In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid substitution at position Q19 relative to SEQ ID NO: 2 or SEQ ID NO: 1. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid substitution at position Q19 relative to SEQ ID NO: 2.

[0125] In some embodiments, an engineered CD47 protein of the present disclosure comprises: an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 2 and an amino acid substitution of Q19P relative to SEQ ID NO: 2, or an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 1 and an amino acid substitution of Q19P relative to SEQ ID NO: 1. In some embodiments, an engineered CD47 protein of the present disclosure comprises an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 2 and an amino acid substitution of Q19P relative to SEQ ID NO: 2.

[0126] In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2.

[0127] In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence comprising SEQ ID NO: 346. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence comprising SEQ ID NO: 347. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence comprising, from N-terminus to C-terminus: an amino acid sequence comprising SEQ ID NO: 20; an amino acid sequence comprising SEQ ID NO: 348; and an amino acid sequence comprising SEQ ID NO: 3. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence comprising, from N-terminus to C-terminus: an amino acid sequence comprising SEQ ID NO: 20; an amino acid sequence comprising SEQ ID NO: 348; and an amino acid substitution of Q1P relative to SEQ ID NO: SEQ ID NO: 3. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence comprising, from N-terminus to C-terminus: an amino acid sequence comprising SEQ ID NO: 348; and an amino acid sequence comprising SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

[0128] In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence comprising: an amino acid substitution of S82A relative to SEQ ID NO: 2, an amino acid substitution of S64A relative to SEQ ID NO: 3, an amino acid substitution of S82A relative to SEQ ID NO: 1, or an amino acid substitution of S37A relative to SEQ ID NO: 6; an insertion of the amino acids WQP between A18 and Q19 relative to SEQ ID NO: 2 or SEQ ID: 1; and an amino acid substitution of Q19P relative to SEQ ID NO: 2 or SEQ ID NO: 1. In some embodiments, an engineered CD47 protein of the present disclosure comprises an amino acid sequence comprising: an amino acid substitution of S82A relative to SEQ ID NO: 2; an insertion of the amino acids WQP between A18 and Q19 relative to SEQ ID NO: 2; and an amino acid substitution of Q19P relative to SEQ ID NO: 2.

[0129] The present disclosure also provides engineered CD47 proteins that have fewer amino acids than the wild-type full-length human CD47 protein. Such engineered proteins afford more efficient cell engineering approaches, including delivery via integrating gene therapy vectors.

[0130] The wild-type full-length human CD47 protein, as used herein, refers to the isoform CD47-202 as disclosed in the Ensembl database as of the filing date of this patent application. The wild-type full-length human CD47 protein has an amino acid sequence of SEQ ID N0:2, wherein amino acids 1-18 are the signal peptide, amino acids 19- 141 are the extracellular domain, amino acids 142-162, 177-197, 208-228, 236-257, 269-289 are the five transmembrane domains (Fig. 3), and amino acids 290-323 are the intracellular domain. Amino acids 163-176 and 229-235 are the two intracellular connections between the transmembrane domains, and amino acids 198-207 and 257-268 are the two extracellular connections between the transmembrane domains (Fig. 3).

[0131] In some embodiments, the engineered CD47 protein is a C-terminally truncated version of isoform 202 (SEQ ID NO:2). For example, in some embodiments, the C-terminal truncation is consecutive and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, or 161 amino acid(s) long. Preferably, the C- terminal truncation is 158, 123, 92, 64, 31, or 95 amino acids long, resulting in an engineered CD47 protein having an amino acid sequence as set for in SEQ ID NO: 7-12, respectively. In some embodiments, the engineered CD47 protein having a C-terminal truncation of SEQ ID NO: 2 further has an N-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140 consecutive amino acid(s).

[0132] In some embodiments, the engineered CD47 protein is a C-terminally truncated version of isoform 201 (SEQ ID NO: 1). For example, in some embodiments, the C-terminal truncation is consecutive and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, or 143 amino acid(s) long. In some embodiments, the engineered CD47 protein having a C-terminal truncation of SEQ ID NO: 1 further has an N-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 consecutive amino acid(s).

[0133] In some embodiments, the engineered CD47 protein is a C-terminally truncated version of isoform 206(SEQ ID NO: 6). For example, in some embodiments, the C-terminal truncation is consecutive and is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, or 66 amino acid(s) long. In some embodiments, the engineered CD47 protein having a C-terminal truncation of SEQ ID NO:6 further has an N-terminal truncation of 1, 2, 3, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140 consecutive amino acid(s).

[0134] In some embodiments, the engineered CD47 protein comprises a minimal intracellular domain. As used herein, a minimal intracellular domain refers to an intracellular domain that has the minimum number of amino acids required to preserve SIRPa binding of the engineered CD47 protein.

[0135] In some embodiments, the engineered CD47 protein comprises a minimal extracellular domain. As used herein, a minimal extracellular domain refers to an extracellular domain that has the minimum number of amino acids required for the engineered CD47 protein to bind to SIRPa.

[0136] In an aspect, the present disclosure provides an engineered CD47 protein that comprises a humanCD47 extracellular domain or a portion thereof and at least one human CD47 transmembrane domain, wherein when an intracellular domain exists, it is a human CD47 intracellular domain with a deletion of at least one amino acid. In some embodiments, when there are more than one transmembrane domains in the engineered CD47 protein, each of the transmembrane domains are interconnected with intracellular and / or extracellular connection(s).

[0137] In an aspect, the present disclosure provides an engineered CD47 protein that consists essentially of a human CD47 extracellular domain or a portion thereof and at least one human CD47 transmembrane domain. In some embodiments, when there are more than one transmembrane domain in the engineered CD47 protein, each of the transmembrane domains are interconnected with intracellular and / or extracellular connection(s). As used herein, the term "consisting essentially of" includes the specified elements and any additional elements that do not abrogate SIRPa binding of the engineered CD47 protein.

[0138] In an aspect, the present disclosure provides an engineered CD47 protein that consists of a human CD47 extracellular domain or a portion thereof and at least one human CD47 transmembrane domain. In some embodiments, when there are more than one transmembrane domain in the engineered CD47 protein, each of the transmembrane domains are interconnected with intracellular and / or extracellular connection(s).

[0139] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain or a portion thereof, at least one human CD47 transmembrane domain or a portion thereof, and a portion of a human CD47 intracellular domain comprising a deletion of at least one amino acid, wherein the deletion is not a C-terminal deletion of 18 amino acids.

[0140] In some embodiments, the engineered CD47 protein comprises a portion of a human CD47 extracellular domain, at least one human CD47 transmembrane domain or a portion thereof, and a portion of a human CD47 intracellular domain comprising a C-terminal deletion of 18 amino acids.

[0141] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain or a portion thereof, at least one and fewer than five human CD47 transmembrane domain(s) or portion(s) thereof, and a portion of a human CD47 intracellular domain comprising a C-terminal deletion of 18 amino acids.

[0142] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain or a portion thereof, at least one human CD47 transmembrane domain or a portion thereof, and a signal peptide, wherein the engineered CD47 protein does not comprise an intracellular domain.

[0143] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain, and at least one human CD47 transmembrane domain or a portion thereof, wherein the engineered CD47 protein does not comprise an intracellular domain.

[0144] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain or a portion thereof, and at least one and fewer than five human CD47 transmembrane domain(s) or portion(s) thereof, wherein the engineered CD47 protein does not comprise an intracellular domain.

[0145] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain, five human CD47 transmembrane domains and a human CD47 intracellular domain with a C-terminal deletion of 18 amino acids, wherein the amino acid sequence of the engineered CD47 protein is not SEQ ID NO: 1.

[0146] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain and five human CD47 transmembrane domain, wherein the amino acid sequence of the engineered CD47 protein is not SEQ ID N0:6.

[0147] In some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain or a portion thereof, at least one human CD47 transmembrane domain or a portion thereof, and no intracellular domain or a human CD47 intracellular domain comprising a deletion of at least one amino acid, wherein the amino acid sequence of the engineered CD47 protein has at most 99% identity with SEQ ID NO: 1 and SEQ ID N0:6. In other words, in some embodiments, the engineered CD47 protein comprises a human CD47 extracellular domain or a portion thereof, at least one human CD47 transmembrane domain or a portion thereof, and no intracellular domain or a human CD47 intracellular domain comprising a deletion of at least one amino acid, wherein the amino acid sequence of the engineered CD47 protein has 99% identity or less to SEQ ID NO: 1 and has 99% identity or less to SEQ ID NO: 6.

[0148] In some embodiments, the human CD47 extracellular domain in the engineered CD47 protein is a wildtype human CD47 extracellular domain. In some embodiments, the wild-type domain has an amino acid sequence corresponding to amino acids 19-141 of SEQ ID NO: 1, or to amino acids 1-96 of SEQ ID N0:6.

[0149] In some embodiments, the human CD47 extracellular domain in the engineered CD47 protein has an amino acid sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 19-141 of SEQ ID NO: 1, or to amino acids 1-96 of SEQ ID NO:6. In some embodiments, the human CD47 extracellular domain in the engineered CD47 protein has an amino acid sequence with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 19-141 of SEQ ID NO: 1, or to amino acids 1-96 of SEQ ID NO:6.

[0150] In some embodiments, the human CD47 extracellular domain in the engineered CD47 protein is structurally equivalent to a wild-type human CD47 extracellular domain.

[0151] As used herein, "structurally equivalent" refers to two amino acid sequences having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity. Preferably, the sequence variation does not change the engineered CD47 protein's biological activity. In some embodiments, the sequence variation does not prevent the engineered human protein from binding to SIRPa. In some embodiments, the sequence variation does not prevent the engineered human protein from being a tolerogenic factor. In some embodiments, at least a portion of the sequence variation may occur through conservative amino acid substitution(s).

[0152] In some embodiments, an engineered protein of the present disclosure comprises one or more membrane tethers. In some embodiments, one or more membrane tethers are or comprise a transmembrane domain. In some embodiments, a transmembrane domain comprises a GPCR transmembrane domain selected from the group consisting of: 5-hydroxytryptamine (serotonin) receptor 1A (HTR1A), 5-hydroxytryptamine (serotonin) receptor IB (HTR1B), 5-hydroxytryptamine (serotonin) receptor ID (HTR1D), 5-hydroxytryptamine (serotonin) receptor IE (HTR1E), 5-hydroxytryptamine (serotonin) receptor IF (HTR1F), 5-hydroxytryptamine (serotonin) receptor 2A (HTR2A), 5- hydroxytryptamine (serotonin) receptor 2B (HTR2B), 5-hydroxytryptamine (serotonin) receptor 2C (HTR2C), 5- hydroxytryptamine (serotonin) receptor 4 (HTR4), 5-hydroxytryptamine (serotonin) receptor 5A (HTR5A), 5- hydroxytryptamine (serotonin) receptor 5B (HTR5BP), 5-hydroxytryptamine (serotonin) receptor 6 (HTR6), 5- hydroxytryptamine (serotonin) receptor 7, adenylate cyclase-coupled (HTR7), cholinergic receptor, muscarinic 1(CHRM1), cholinergic receptor, muscarinic 2 (CHRM2), cholinergic receptor, muscarinic 3 (CHRM3), cholinergic receptor, muscarinic 4 (CHRM4), cholinergic receptor, muscarinic 5 (CHRM5), adenosine Al receptor (AD0RA1), adenosine A2a receptor (AD0RA2A), adenosine A2b receptor (ADORA2B), adenosine A3 receptor (AD0RA3), adhesion G protein- coupled receptor Al (ADGRA1), adhesion G protein-coupled receptor A2 (ADGRA2), adhesion G protein-coupled receptor A3 (ADGRA3), adhesion G protein-coupled receptor Bl (ADGRB1), adhesion G protein-coupled receptor B2 (ADGRB2), adhesion G protein-coupled receptor B3 (ADGRB3), cadherin EGF LAG seven-pass G-type receptor 1 (CELSR1), cadherin EGF LAG seven-pass G-type receptor 2 (CELSR2), cadherin EGF LAG seven-pass G-type receptor 3 (CELSR3), adhesion G protein-coupled receptor DI (ADGRD1), adhesion G protein-coupled receptor D2 (ADGRD2), adhesion G protein-coupled receptor El (ADGRE1), adhesion G protein-coupled receptor E2 (ADGRE2), adhesion G protein-coupled receptor E3 (ADGRE3), adhesion G protein-coupled receptor E4 (ADGRE4P), adhesion G protein-coupled receptor E5 (ADGRE5), adhesion G protein-coupled receptor Fl (ADGRF1), adhesion G protein-coupled receptor F2 (ADGRF2), adhesion G protein-coupled receptor F3 (ADGRF3), adhesion G protein-coupled receptor F4 (ADGRF4), adhesion G protein-coupled receptor F5 (ADGRF5), adhesion G protein-coupled receptor G1 (ADGRG1), adhesion G protein-coupled receptor G2 (ADGRG2), adhesion G protein-coupled receptor G3 (ADGRG3), adhesion G protein-coupled receptor G4 (ADGRG4), adhesion G protein-coupled receptor G5 (ADGRG5), adhesion G protein-coupled receptor G6 (ADGRG6), adhesion G protein-coupled receptor G7 (ADGRG7), adhesion G protein-coupled receptor LI (ADGRL1), adhesion G protein-coupled receptor L2 (ADGRL2), adhesion G protein-coupled receptor L3 (ADGRL3), adhesion G protein-coupled receptor L4 (ADGRL4), adhesion G protein-coupled receptor VI (ADGRV1), adrenoceptor alpha 1A (ADRA1A), adrenoceptor alpha IB (ADRA1B), adrenoceptor alpha ID (ADRA1D), adrenoceptor alpha 2A (ADRA2A), adrenoceptor alpha 2B (ADRA2B), adrenoceptor alpha 2C (ADRA2C), adrenoceptor beta 1 (ADRB1), adrenoceptor beta 2 (ADRB2), adrenoceptor beta 3 (ADRB3), angiotensin II receptor type 1 (AGTR1), angiotensin II receptor type 2 (AGTR2), apelin receptor (APLNR), G protein-coupled bile acid receptor 1 (GPBAR1), neuromedin B receptor (NMBR), gastrin releasing peptide receptor (GRPR), bombesin like receptor 3 (BRS3), bradykinin receptor Bl (BDKRB1), bradykinin receptor B2 (BDKRB2), calcitonin receptor (CALCR), calcitonin receptor like receptor (CALCRL), calcium sensing receptor (CASR), G protein-coupled receptor, class C (GPRC6A), cannabinoid receptor 1 (brain) (CNR1), cannabinoid receptor 2 (CNR2), chemerin chemokine-like receptor 1 (CMKLR1), chemokine (C— C motif) receptor 1 (CCR1), chemokine (C— C motif) receptor 2 (CCR2), chemokine (C— C motif) receptor 3 (CCR3), chemokine (C— C motif) receptor 4 (CCR4), chemokine (C— C motif) receptor 5 (gene / pseudogene) (CCR5), chemokine (C— C motif) receptor 6 (CCR6), chemokine (C— C motif) receptor 7 (CCR7), chemokine (C— C motif) receptor 8 (CCR8), chemokine (C— C motif) receptor 9 (CCR9), chemokine (C— C motif) receptor 10 (CCR10), chemokine (C— X— C motif) receptor 1 (CXCR1), chemokine (C— X— C motif) receptor 2 (CXCR2), chemokine (C— X— C motif) receptor 3 (CXCR3), chemokine (C— X— C motif) receptor 4 (CXCR4), chemokine (C— X— C motif) receptor 5 (CXCR5), chemokine (C— X— C motif) receptor 6 (CXCR6), chemokine (C— X3-C motif) receptor 1 (CX3CR1), chemokine (C motif) receptor 1 (XCR1), atypical chemokine receptor 1 (Duffy blood group) (ACKR1), atypical chemokine receptor 2 (ACKR2), atypical chemokine receptor 3 (ACKR3), atypical chemokine receptor 4 (ACKR4), chemokine (C— C motif) receptor-like 2 (CCRL2), cholecystokinin A receptor (CCKAR), cholecystokinin B receptor (CCKBR), G protein-coupled receptor 1 (GPR1), bombesin like receptor 3 (BRS3), G protein-coupled receptor 3 (GPR3), G protein-coupled receptor 4 (GPR4), G protein-coupled receptor 6 (GPR6), G protein-coupled receptor 12 (GPR12), G protein-coupled receptor 15 (GPR15), G protein-coupled receptor 17 (GPR17), G protein-coupled receptor 18 (GPR18), G protein-coupled receptor 19 (GPR19), G protein-coupled receptor 20 (GPR20), G protein-coupled receptor 21 (GPR21), G protein-coupled receptor 22 (GPR22), G protein-coupled receptor 25 (GPR25), G protein-coupled receptor 26 (GPR26), G protein-coupled receptor 27 (GPR27), G protein-coupled receptor 31 (GPR31), G protein-coupled receptor 32 (GPR32), Gprotein-coupled receptor 33 (gene / pseudogene) (GPR33), G protein-coupled receptor 34 (GPR34), G protein-coupled receptor 35 (GPR35), G protein-coupled receptor 37 (endothelin receptor type B-like) (GPR37), G protein-coupled receptor 37 like 1 (GPR37L1), G protein-coupled receptor 39 (GPR39), G protein-coupled receptor 42 (gene / pseudogene) (GPR42), G protein-coupled receptor 45 (GPR45), G protein-coupled receptor 50 (GPR50), G protein-coupled receptor 52(GPR52), G protein-coupled receptor 55 (GPR55), G protein-coupled receptor 61 (GPR61), G protein-coupled receptor 62(GPR62), G protein-coupled receptor 63 (GPR63), G protein-coupled receptor 65 (GPR65), G protein-coupled receptor 68(GPR68), G protein-coupled receptor 75 (GPR75), G protein-coupled receptor 78 (GPR78), G protein-coupled receptor 79(GPR79), G protein-coupled receptor 82 (GPR82), G protein-coupled receptor 83 (GPR83), G protein-coupled receptor 84(GPR84), G protein-coupled receptor 85 (GPR85), G protein-coupled receptor 87 (GPR87), G protein-coupled receptor 88(GPR88), G protein-coupled receptor 101 (GPR101), G protein-coupled receptor 119 (GPR119), G protein-coupled receptor 132 (GPR132), G protein-coupled receptor 135 (GPR135), G protein-coupled receptor 139 (GPR139), G protein- coupled receptor 141 (GPR141), G protein-coupled receptor 142 (GPR142), G protein-coupled receptor 146 (GPR146), G protein-coupled receptor 148 (GPR148), G protein-coupled receptor 149 (GPR149), G protein-coupled receptor 150 (GPR150), G protein-coupled receptor 151 (GPR151), G protein-coupled receptor 152 (GPR152), G protein-coupled receptor 153 (GPR153), G protein-coupled receptor 160 (GPR160), G protein-coupled receptor 161 (GPR161), G protein- coupled receptor 162 (GPR162), G protein-coupled receptor 171 (GPR171), G protein-coupled receptor 173 (GPR173), G protein-coupled receptor 174 (GPR174), G protein-coupled receptor 176 (GPR176), G protein-coupled receptor 182 (GPR182), G protein-coupled receptor 183 (GPR183), leucine-rich repeat containing G protein-coupled receptor 4 (LGR4), leucine-rich repeat containing G protein-coupled receptor 5 (LGR5), leucine-rich repeat containing G protein- coupled receptor 6 (LGR6), MASI proto-oncogene (MASI), MASI proto-oncogene like (MAS1L), MAS related GPR family member D (MRGPRD), MAS related GPR family member E (MRGPRE), MAS related GPR family member F (MRGPRF), MAS related GPR family member G (MRGPRG), MAS related GPR family member XI (MRGPRX1), MAS related GPR family member X2 (MRGPRX2), MAS related GPR family member X3 (MRGPRX3), MAS related GPR family member X4 (MRGPRX4), opsin 3 (OPN3), opsin 4 (OPN4), opsin 5 (OPN5), purinergic receptor P2Y (P2RY8), purinergic receptor P2Y (P2RY10), trace amine associated receptor 2 (TAAR2), trace amine associated receptor 3 (gene / pseudogene) (TAAR3), trace amine associated receptor 4 (TAAR4P), trace amine associated receptor 5 (TAAR5), trace amine associated receptor 6 (TAAR6), trace amine associated receptor 8 (TAAR8), trace amine associated receptor 9 (gene / pseudogene) (TAAR9), G protein-coupled receptor 156 (GPR156), G protein-coupled receptor 158 (GPR158), G protein-coupled receptor 179 (GPR179), G protein-coupled receptor, class C (GPRC5A), G protein-coupled receptor, class C (GPRC5B), G protein-coupled receptor, class C (GPRC5C), G protein-coupled receptor, class C (GPRC5D), frizzled class receptor 1 (FZD1), frizzled class receptor 2 (FZD2), frizzled class receptor 3 (FZD3), frizzled class receptor 4 (FZD4), frizzled class receptor 5 (FZD5), frizzled class receptor 6 (FZD6), frizzled class receptor 7 (FZD7), frizzled class receptor 8 (FZD8), frizzled class receptor 9 (FZD9), frizzled class receptor 10 (FZD10), smoothened, frizzled class receptor (SMO), complement component 3a receptor 1 (C3AR1), complement component 5a receptor 1 (C5AR1), complement component 5a receptor 2 (C5AR2), corticotropin releasing hormone receptor 1 (CRHR1), corticotropin releasing hormone receptor 2 (CRHR2), dopamine receptor DI (DRD1), dopamine receptor D2 (DRD2), dopamine receptor D3 (DRD3), dopamine receptor D4 (DRD4), dopamine receptor D5 (DRD5), endothelin receptor type A (EDNRA), endothelin receptor type B (EDNRB), formyl peptide receptor 1 (FPR1), formyl peptide receptor 2 (FPR2), formyl peptide receptor 3 (FPR3), free fatty acid receptor 1 (FFAR1), free fatty acid receptor 2 (FFAR2), free fatty acid receptor 3 (FFAR3), free fatty acid receptor 4 (FFAR4), G protein-coupled receptor 42 (gene / pseudogene) (GPR42), gamma-aminobutyric acid (GABA) B receptor, 1 (GABBR1), gamma-aminobutyric acid (GABA) B receptor, 2 (GABBR2), galanin receptor 1 (GALR1), galaninreceptor 2 (GALR2), galanin receptor 3 (GALR3), growth hormone secretagogue receptor (GHSR), growth hormone releasing hormone receptor (GHRHR), gastric inhibitory polypeptide receptor (GIPR), glucagon like peptide 1 receptor (GLP1R), glucagon-like peptide 2 receptor (GLP2R), glucagon receptor (GCGR), secretin receptor (SCTR), follicle stimulating hormone receptor (FSHR), luteinizing hormone / choriogonadotropin receptor (LHCGR), thyroid stimulating hormone receptor (TSHR), gonadotropin releasing hormone receptor (GNRHR), gonadotropin releasing hormone receptor 2 (pseudogene) (GNRHR2), G protein-coupled receptor 18 (GPR18), G protein-coupled receptor 55 (GPR55), G protein-coupled receptor 119 (GPR119), G protein-coupled estrogen receptor 1 (GPER1), histamine receptor Hl (HRH1), histamine receptor H2 (HRH2), histamine receptor H3 (HRH3), histamine receptor H4 (HRH4), hydroxycarboxylic acid receptor 1 (HCAR1), hydroxycarboxylic acid receptor 2 (HCAR2), hydroxycarboxylic acid receptor 3 (HCAR3), KISSI receptor (KISS1R), leukotriene B4 receptor (LTB4R), leukotriene B4 receptor 2 (LTB4R2), cysteinyl leukotriene receptor 1 (CYSLTR1), cysteinyl leukotriene receptor 2 (CYSLTR2), oxoeicosanoid (OXE) receptor 1 (OXER1), formyl peptide receptor 2 (FPR2), lysophosphatidic acid receptor 1 (LPAR1), lysophosphatidic acid receptor 2 (LPAR2), lysophosphatidic acid receptor 3 (LPAR3), lysophosphatidic acid receptor 4 (LPAR4), lysophosphatidic acid receptor 5 (LPAR5), lysophosphatidic acid receptor 6 (LPAR6), sphingosine-l-phosphate receptor 1 (S1PR1), sphingosine-l-phosphate receptor 2 (S1PR2), sphingosine-l-phosphate receptor 3 (S1PR3), sphingosine-l-phosphate receptor 4 (S1PR4), sphingosine-l-phosphate receptor 5 (S1PR5), melanin concentrating hormone receptor 1 (MCHR1), melanin concentrating hormone receptor 2 (MCHR2), melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R), melanocortin 2 receptor (adrenocorticotropic hormone) (MC2R), melanocortin 3 receptor (MC3R), melanocortin 4 receptor (MC4R), melanocortin 5 receptor (MC5R), melatonin receptor 1A (MTNR1A), melatonin receptor IB (MTNRIB), glutamate receptor, metabotropic 1 (GRM1), glutamate receptor, metabotropic 2 (GRM2), glutamate receptor, metabotropic 3 (GRM3), glutamate receptor, metabotropic 4 (GRM4), glutamate receptor, metabotropic 5 (GRM5), glutamate receptor, metabotropic 6 (GRM6), glutamate receptor, metabotropic 7 (GRM7), glutamate receptor, metabotropic 8 (GRM8), motilin receptor (MLNR), neuromedin U receptor 1 (NMUR1), neuromedin U receptor 2 (NMUR2), neuropeptide FF receptor 1 (NPFFR1), neuropeptide FF receptor 2 (NPFFR2), neuropeptide S receptor 1 (NPSR1), neuropeptides B / W receptor 1 (NPBWR1), neuropeptides B / W receptor 2 (NPBWR2), neuropeptide Y receptor Y1 (NPY1R), neuropeptide Y receptor Y2 (NPY2R), neuropeptide Y receptor Y4 (NPY4R), neuropeptide Y receptor Y5 (NPY5R), neuropeptide Y receptor Y6 (pseudogene) (NPY6R), neurotensin receptor 1 (high affinity) (NTSR1), neurotensin receptor 2 (NTSR2), opioid receptor, delta 1 (OPRD1), opioid receptor, kappa 1 (OPRK1), opioid receptor, mu 1 (OPRM1), opiate receptor-like 1 (OPRL1), hypocretin (orexin) receptor 1 (HCRTR1), hypocretin (orexin) receptor 2 (HCRTR2), G protein-coupled receptor 107 (GPR107), G protein-coupled receptor 137 (GPR137), olfactory receptor family 51 subfamily E member 1 (OR51E1), transmembrane protein, adipocyte associated 1 (TPRA1), G protein-coupled receptor 143 (GPR143), G protein-coupled receptor 157 (GPR157), oxoglutarate (alpha-ketoglutarate) receptor 1 (OXGR1), purinergic receptor P2Y (P2RY1), purinergic receptor P2Y (P2RY2), pyrimidinergic receptor P2Y (P2RY4), pyrimidinergic receptor P2Y (P2RY6), purinergic receptor P2Y (P2RY11), purinergic receptor P2Y (P2RY12), purinergic receptor P2Y (P2RY13), purinergic receptor P2Y (P2RY14), parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), platelet-activating factor receptor (PTAFR), prokineticin receptor 1 (PROKR1), prokineticin receptor 2 (PROKR2), prolactin releasing hormone receptor (PRLHR), prostaglandin D2 receptor (DP) (PTGDR), prostaglandin D2 receptor 2 (PTGDR2), prostaglandin E receptor 1 (PTGER1), prostaglandin E receptor 2 (PTGER2), prostaglandin E receptor 3 (PTGER3), prostaglandin E receptor 4 (PTGER4), prostaglandin F receptor (PTGFR), prostaglandin 12 (prostacyclin) receptor (IP) (PTGIR), thromboxane A2 receptor (TBXA2R), coagulation factor II thrombin receptor (F2R), F2R like trypsin receptor 1 (F2RL1), coagulation factor II thrombin receptor like 2 (F2RL2), F2Rlike thrombin / trypsin receptor 3 (F2RL3), pyroglutamylated RFamide peptide receptor (QRFPR), relaxin / insulin-like family peptide receptor 1 (RXFP1), relaxin / insulin-like family peptide receptor 2 (RXFP2), relaxin / insulin-like family peptide receptor 3 (RXFP3), relaxin / insulin-like family peptide receptor 4 (RXFP4), somatostatin receptor 1 (SSTR1), somatostatin receptor 2 (SSTR2), somatostatin receptor 3 (SSTR3), somatostatin receptor 4 (SSTR4), somatostatin receptor 5 (SSTR5), succinate receptor 1 (SUCNR1), tachykinin receptor 1 (TACR1), tachykinin receptor 2 (TACR2), tachykinin receptor 3 (TACR3), taste 1 receptor member 1 (TAS1R1), taste 1 receptor member 2 (TAS1R2), taste 1 receptor member 3 (TAS1R3), taste 2 receptor member 1 (TAS2R1), taste 2 receptor member 3 (TAS2R3), taste 2 receptor member 4 (TAS2R4), taste 2 receptor member 5 (TAS2R5), taste 2 receptor member 7 (TAS2R7), taste 2 receptor member 8 (TAS2R8), taste 2 receptor member 9 (TAS2R9), taste 2 receptor member 10 (TAS2R10), taste 2 receptor member 13 (TAS2R13), taste 2 receptor member 14 (TAS2R14), taste 2 receptor member 16 (TAS2R16), taste 2 receptor member 19 (TAS2R19), taste 2 receptor member 20 (TAS2R20), taste 2 receptor member 30 (TAS2R30), taste 2 receptor member 31 (TAS2R31), taste 2 receptor member 38 (TAS2R38), taste 2 receptor member 39 (TAS2R39), taste 2 receptor member 40 (TAS2R40), taste 2 receptor member 41 (TAS2R41), taste 2 receptor member 42 (TAS2R42), taste 2 receptor member 43 (TAS2R43), taste 2 receptor member 45 (TAS2R45), taste 2 receptor member 46 (TAS2R46), taste 2 receptor member 50 (TAS2R50), taste 2 receptor member 60 (TAS2R60), thyrotropin-releasing hormone receptor (TRHR), trace amine associated receptor 1 (TAAR1), urotensin 2 receptor (UTS2R), arginine vasopressin receptor 1A (AVPR1A), arginine vasopressin receptor IB (AVPR1B), arginine vasopressin receptor 2 (AVPR2), oxytocin receptor (OXTR), adenylate cyclase activating polypeptide 1 (pituitary) receptor type I (ADCYAP1R1), vasoactive intestinal peptide receptor 1 (VIPR1), vasoactive intestinal peptide receptor 2 (VIPR2), and any variant thereof. In some embodiments, a transmembrane domain is or comprises a CD3zeta, CD8a, CD16a, CD28, CD32a, CD32c, CD40, CD47, CD64, ICOS, Dectin-1, DNGR1, EGFR, GPCR, MyD88, PDGFR, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, or VEGFR transmembrane domain.

[0153] Plasma membrane proteins can be attached to the peripheral membrane or can be integral membrane proteins. See, for example, a review in Komath SS, Fujita M, Hart GW, et al. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 4th edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2022. Chapter 12. GPI anchorage refers to the attachment of glycosylphosphatidylinositol, or GPI, to the C-terminus of a protein during posttranslational modification. In some embodiments, a heterologous membrane attachment sequence is a GPI anchor attachment sequence. Proteins that are attached to GPI anchors via their C-terminus are typically found in the outer lipid bilayer. GPI anchors are alternatives to the single transmembrane domain of type-I integral membrane proteins.

[0154] A heterologous GPI anchor attachment sequence can be derived from any known GPI-anchored protein (reviewed in Ferguson MAJ, Kinoshita T, Hart GW. Glycosylphosphatidylinositol Anchors. In: Varki A, Cummings RD, Esko JD, et al., editors. Essentials of Glycobiology. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 2009. Chapter 11). In some embodiments, a heterologous GPI anchor attachment sequence is a GPI anchor attachment sequence from CD14, CD16, CD48, DAF / CD55, CD59, CD80, CD87, or TRAIL-R3. In some embodiments, a heterologous GPI anchor attachment sequence is derived from DAF / CD55. In some embodiments, a heterologous GPI anchor attachment sequence is derived from CD59. In some embodiments, a heterologous GPI anchor attachment sequence is derived from TRAIL-R3. In illustrative embodiments, a heterologous GPI anchor attachment sequence is derived from DAF / CD55, CD59, or TRAIL-R3. In some embodiments, one or both of the activation elements include a heterologous signal sequence to help direct expression of the activation element to the cell membrane. Any signal sequence that is active in the packaging cell line can be used. In someembodiments, a signal sequence is a DAF / CD55 signal sequence. In some embodiments, a signal sequence is a CD59 signal sequence. In some embodiments, a signal sequence is a TRAIL-R3 signal sequence.

[0155] In some embodiments, the engineered CD47 protein comprises one or more wild-type human CD47 transmembrane domains. In some embodiments, the wild-type domain has an amino acid sequence corresponding to amino acids 142-162, 177-197, 208-228, 236-257, or 269-289 of SEQ ID NO:2.

[0156] In some embodiments, the engineered CD47 protein comprises one or more transmembrane domains that are structurally equivalent to a wild-type human CD47 transmembrane domain. In some embodiments, the engineered CD47 protein comprises one or more transmembrane domains having an amino acid sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 142-162, 177-197, 208-228, 236-257, or 269- 289 of SEQ ID NO:2.

[0157] In some embodiments, the human CD47 intracellular domain in the engineered CD47 protein is a wildtype human CD47 intracellular domain. In some embodiments, the wild-type intracellular domain has an amino acid sequence corresponding to amino acids 290-323 of SEQ ID NO:2.

[0158] In some embodiments, the human CD47 intracellular domain in the engineered CD47 protein is structurally equivalent to a wild-type human CD47 intracellular domain. In some embodiments, the human CD47 intracellular domain in the engineered CD47 protein has an amino acid sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to amino acids 290-323 of SEQ ID NO: 2.

[0159] In some embodiments, the engineered CD47 protein comprises an amino acid sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 7.

[0160] In some embodiments, the engineered CD47 protein comprises an amino acid sequence with at least80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:8.

[0161] In some embodiments, the engineered CD47 protein comprises an amino acid sequence with at least80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:9.

[0162] In some embodiments, the engineered CD47 protein comprises an amino acid sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 12.

[0163] In some embodiments, the engineered CD47 protein comprises an amino acid sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 10.

[0164] In some embodiments, the engineered CD47 protein comprises an amino acid sequence with at least80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO: 11.

[0165] In some embodiments, the engineered CD47 protein is a transmembrane protein. A transmembrane protein is an integral membrane protein that spans the entirety of the cell membrane and has both intracellular and extracellular portions. As used herein, "intracellular portion" can include the CD47 intracellular domain and the intracellular connections, when present in the molecule. As used herein, "extracellular portion" can include the CD47 extracellular domain and the extracellular connections, when present in the molecule.

[0166] As used herein, a wild-type human CD47 extracellular domain refers to the extracellular domain of any one of the wild-type human CD47 protein isoforms. A wild-type human CD47 transmembrane domain refers to a transmembrane domain of any one of the wild-type human CD47 protein isoforms. A wild-type human CD47 intracellular domain refers to the intracellular domain of any one of the wild-type human CD47 protein isoforms.

[0167] In some embodiments, the engineered CD47 protein is an engineered human CD47 protein, an engineered humanized CD47 protein, or an engineered partially-humanized CD47 protein.

[0168] As used herein, "humanized" or "humanization" means that the amino acid sequence of the engineered CD47 protein is modified to reduce its immunogenicity in humans. As used herein, "partially-humanized" or "partial humanization" means that a portion of the amino acid sequence of the engineered CD47 protein is modified to reduce the engineered CD47 protein's immunogenicity in humans. For example, in some embodiments, the extracellular domain of the engineered CD47 protein is modified to reduce the engineered CD47 protein's immunogenicity in humans. Humanization is usually achieved by modifying a protein sequence from a non-human source to increase its similarity to its counterpart protein produced naturally in humans. Two major approaches have been used to humanize proteins: rational design and empirical methods. The rational design methods are characterized by protein structural modeling, generating a few variants of the protein and assessing their binding or any other property of interest. In contrast to the rational design methods, empirical methods do not require the structure information of the protein. They depend on the generation of large combinatorial libraries and selection of the desired variants by enrichment technologies such as phage, ribosome or yeast display, or by high throughput screening techniques. These methods rest on selection rather than making assumptions on the impact of mutations on the protein structure. For example, in some embodiments, humanization of the engineered CD47 protein comprises grafting the SIRPa binding region in the engineered CD47 protein onto a human CD47 protein. In other embodiments, humanization of the engineered CD47 protein comprises introducing one or more point mutations in the engineered CD47 protein so that one or more residue(s) in the engineered CD47 protein is substituted with the corresponding residue in a human CD47 protein.

[0169] In some embodiments, an engineered protein of the present disclosure comprises a SIRPa interaction motif comprising a SIRPa antibody. In some embodiments, a SIRPa antibody is selected from the antibodies listed in Table 3 of Int'l App. No. PCT / US2023 / 013364 incorporated herein by reference in its entirety.Glycosylation

[0170] The human CD47 protein is glycosylated. Protein glycosylation involves the covalent attachment of glycans (also called carbohydrates, saccharides, or sugars) to a protein. Based on the amino acid side-chain atoms to which glycans are linked, most protein glycosylations fall within two categories: N-linked glycosylation and O-linked glycosylation. In N-linked glycosylation, glycans are attached to the side-chain nitrogen atoms of asparagine residues in a conserved consensus sequence Asn-Xaa-Ser / Thr (Xaa * Pro), whereas in O-linked glycosylation, glycans are attached to the side-chain oxygen atoms of hydroxyl amino acids, primarily serine and threonine residues.

[0171] The IgV domain of wild type human CD47 protein is N-glycosylated and modified with O-linked glycosaminoglycans. The human CD47 protein can be expressed as a proteoglycan with a molecular weight of >250kDa, having both heparan and chondroitin sulfate glycosaminoglycan (GAG) chains at Ser54and Ser79(Kaur et al., J. Biological Chemistry, 2011). Heparan sulfate (HSGAG) and chondroitin sulfate (CSGAG) are synthesized in the Golgi apparatus, where protein cores made in the rough endoplasmic reticulum are post-translationally modified with O-linked glycosylation by glycosyltransferases forming proteoglycans. In addition, N-linked glycosylation has been identified at four of the five potential modification sites (N16, N32, N55, and N93) in the human CD47 protein (Hatherley et al., Cell, 2008). The numbering of amino acid in this paragraph is based on SEQ ID NO 3 (i.e., mature, full length, wild type CD47).

[0172] In some embodiments, the engineered CD47 protein comprises fewer glycosylation modification sites than a wild-type human CD47 protein. A glycosylation modification site refers to a sequence of consecutive amino acids in a protein that can serve as the attachment site for a glycan. Glycosylation modification sites are also called sequons.In some embodiments, one or more amino acid(s) within the glycosaminoglycan modification site of a wild-type human CD47 protein is deleted or substituted in the engineered CD47 protein. In some embodiments, the engineered CD47 protein has 0, 1, 2, 3, 4, 5, or 6 glycosylation site(s).

[0173] In some embodiments, the engineered CD47 protein comprises fewer glycosylation modifications than a wild-type human CD47 protein. The glycosylation modifications include, but are not limited to, N-glycosylation, O glycosylation, phosphoserine glycosylation, and C-glycosylation. In some embodiments, the engineered CD47 protein has 0, 1, 2, 3, 4, or 5 glycosylation modification(s).

[0174] In some embodiments, the engineered CD47 protein comprises fewer glycosaminoglycan modification sites than a wild-type human CD47 protein. A glycosaminoglycan modification site refers to a sequence of consecutive amino acids in a protein that can serve as the attachment site for a glycosaminoglycan. In some embodiments, one or more amino acid(s) within the glycosaminoglycan modification site of a wild-type human CD47 protein is deleted or substituted in the engineered CD47 protein. In some embodiments, the engineered CD47 protein has 0 or 1 glycosaminoglycan modification site.

[0175] In some embodiments, the engineered CD47 protein comprises fewer glycosaminoglycan chains than a wild-type human CD47 protein. Glycosaminoglycan chains include, but are not limited to, heparan sulfate (HSGAG), chondroitin sulfate (CSGAG), keratan sulfate, and hyaluronic acid. In some embodiments, the engineered CD47 protein has 0 or 1 glycosaminoglycan side chain.

[0176] In some embodiments, the engineered CD47 protein comprises fewer than two heparan and / or chondroitin sulfate glycosaminoglycan modification sites. In some embodiments, one or more amino acid(s) within the glycosaminoglycan modification sites of a wild-type human CD47 protein is deleted or substituted in the engineered CD47 protein. In some embodiments, the engineered CD47 protein comprises one heparan and / or chondroitin sulfate glycosaminoglycan modification site. In some embodiments, the engineered CD47 protein comprises no heparan and / or chondroitin sulfate glycosaminoglycan modification sites.

[0177] In some embodiments, the engineered CD47 protein comprises fewer than two heparan and / or chondroitin sulfate glycosaminoglycan chains. In some embodiments, the engineered CD47 protein has 0 or 1 heparan and / or chondroitin sulfate glycosaminoglycan chains.

[0178] In some embodiments, the engineered CD47 protein comprises fewer N-linked glycosylation sites than a wild-type human CD47 protein. An N-linked glycosylation site is a sequence of consecutive amino acids in a protein that can serve as the attachment site for a saccharide, particularly an N-glycan. In some embodiments, the engineered CD47 protein has 0, 1, 2, 3, or 4 N-linked glycosylation site(s).

[0179] In some embodiments, the engineered CD47 protein comprises fewer N-linked glycosylation modifications than a wild-type human CD47 protein. In some embodiments, the engineered CD47 protein has 0, 1, 2, or 3 N-linked glycosylation modification(s).Biological Activity of Engineered CD47 protein

[0180] In some embodiments the engineered CD47 protein is functionally equivalent to a wild-type humanCD47 protein. As used herein, "functionally equivalent" refers to having at least one type of biological activity of a wildtype human CD47 protein. The types of biological activity of a wild-type human CD47 protein include, but are not limited to, the ability to interact with TSP-1, integrins, another CD47 protein, or SIRPa. Preferably, the engineered CD47 protein can bind to SIRPa.Interaction with TSP-1

[0181] A major secreted ligand for CD47 is thrombospondin-1 (TSP-1). TSP-1 is a homotrimeric glycoprotein, and its C-terminal domain mediates binding to CD47 (Isenberg et al., 2009). At least two additional members of the thrombospondin family bind to CD47, albeit with lower affinity. TSP-1 is the prototypic member of the thrombospondin family of extracellular matrix glycoproteins, which are implicated in regulating cell motility, proliferation, and differentiation. The extracellular IgV domain of CD47 is a receptor for the C-terminal cell-binding domain (CBD) of TSP-1. Mutagenesis studies established that heparan sulfate modification of CD47 is required for high affinity interaction of TSP- 1 with CD47 (Kaur et al., 2011).

[0182] In some embodiments, the engineered CD47 protein lacks one or more thrombospondin-1 binding site(s) compared to a wild-type human CD47 protein. In some embodiments, one or more amino acid(s) within the TSP- 1 site of a wild-type human CD47 protein is deleted or substituted in the engineered CD47 protein. In some embodiments, the engineered CD47 protein lacking one or more TSP-1 binding sites is not glycosylated by one or more heparan sulfate.

[0183] In some embodiments, the engineered CD47 protein binds to TSP-1 with lower affinity compared to a wild-type human CD47 protein. In some embodiments, the engineered CD47 protein binds to TSP-1 with a KD higher than about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM. In some embodiments, the engineered CD47 protein cannot bind to TSP-1.Interaction with integrins

[0184] Early studies of CD47 were based on the use of monoclonal antibodies (mAbs) raised against the CD47 protein purified from placenta. These studies showed a role of CD47 in enhancing the IgG-mediated phagocytosis response in the presence of RGD-containing ligands, such as fibronectin, fibrinogen, vitronectin, or collagen type IV. The same mAbs were also found to block neutrophil transendothelial migration stimulated by interleukin 8 (IL-8) or the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) and to inhibit neutrophil migration across tumor-necrosis-factor-o- (TNFa ) stimulated endothelial cells. These studies revealed that CD47 on both the neutrophils and the endothelial cells was important. Generation of other anti-CD47mAbs, raised against epithelial membrane preparations, showed that CD47 is present at the basolateral membrane of epithelial cell monolayers, that mAbs blocking CD47 on either neutrophils or the epithelial cells delay neutrophil trans-epithelial migration, and that efficient neutrophil chemotaxis correlates with an increased neutrophil cell surface expression of CD47. The basement membrane protein entactin, which contains an RGD sequence, was also found to stimulate neutrophil adhesion and chemotaxis in a CD47- dependent manner in vitro. Generation of CD47-deficient mice further proved the importance of this protein in regulating neutrophil inflammatory responses, by showing an increased sensitivity to bacterial infection due to a delayed neutrophil accumulation in bacterial peritonitis. CD47-deficient neutrophils also show a strongly impaired RGD-stimulated neutrophil adhesion, phagocytosis, and respiratory burst. For ov03 integrin-mediated cellular responses to the extracellular matrix protein vitronectin, CD47 was found to be required for ov03-mediated binding to vitronectin-coated beads, but not ov03- mediated adhesion to vitronectin-coated surfaces. In addition to its original association with ov03 integrins, CD47 has also been shown to interact with and regulate the integrins a2Pi and allbPs on platelets, the a2Pi integrin on smooth muscle cells, the a^Pi integrin on sickle red blood cells and B lymphocytes, the asPi integrin in microglia, and the as integrin in chondrocytes.

[0185] In some embodiments, the engineered CD47 protein lacks one or more integrin binding site(s) compared to a wild-type human CD47 protein. In some embodiments, one or more amino acid(s) within the integrin binding site of a wild-type human CD47 protein is deleted or substituted in the engineered CD47 protein.

[0186] In some embodiments, the engineered CD47 protein binds to integrin with lower affinity compared to a wild-type human CD47 protein. In some embodiments, the engineered CD47 protein binds to integrin with a KD higher than about 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM. In some embodiments, the engineered CD47 protein cannot bind to integrin.

[0187] In some embodiments, the integrin is selected from the group consisting of av03 integrin, allb03 integrin, CI2P1 integrin, cuPi integrin, aepi integrin, and as integrin.Interaction with SIRP proteins

[0188] The signal regulator protein (SIRP) family contains three members, and of these SIRPa and SIRPy are known CD47 receptors. SIRP proteins belong to the Ig family of cell surface glycoproteins, and the first member identified was SIRPa (also known as SHPS-1, CD172a, BIT, MFR, or P84). SIRPa is highly expressed in myeloid cells and neurons, but also in endothelial cells and fibroblasts, and has three extracellular Ig-like domains, one distal IgV-like domain, and two membrane proximal IgC-like domains. In addition, an alternatively spliced form having only one IgV domain has also been reported. In its intracellular tail, SIRPa has two immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which when phosphorylated, can bind the Src homology 2 (SH2) domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2. Additional cytoplasmic binding partners for SIRPa are the adaptor molecules Src kinase- associated protein of 55 kDa homolog / SKAP2 (SKAP55hom / R), Fyn-binding protein / SLP-76-associated phosphoprotein of 130 kDa (FYB / SLAP-130), and the tyrosine kinase PYK2. SIRPa is also a substrate for the kinase activity of the insulin, EGF, and bPDGF receptors. The overexpression of SIRPa in fibroblasts decreases proliferation and other downstream events in response to insulin, EGF, and bPDGF. Since SIRPa is also constitutively associated with the M-CSF receptor c- fms, SIRPa overexpression partially reverses the v-fms phenotype.

[0189] CD47 is a ligand for SIRPa. The glycosylation of CD47 or SIRPa does not seem to be necessary for their interaction, but the level of N-glycosylation of SIRPa has an impact on the interaction, such that over glycosylation reduces the binding of CD47. The long-range disulfide bond between Cys33in the CD47 IgV domain and Cys263in the CD47 transmembrane domain is also important to establish an orientation of the CD47 IgV domain that enhances its binding to SIRPa. The numbering of amino acids is based on SEQ ID NO:3 in this paragraph (i.e., the mature, wild-type, full-length CD47 protein).

[0190] The structure of CD47 and the CD47 / SIRPa complex have been revealed (Hatherley et al., 2008). Two0-strands, corresponding to amino acids 92-100 and 103-113 of SEQ ID NO:3, were identified to be at the core of the CD47 / SIRPa interaction. Amino acids 97 (Glu), 99 (Thr), 100 (Glu), 103 (Arg), 104 (Glu), and 106 (Glu) of SEQ ID NO: 3 were identified as contact residues in the CD47 / SIRPa interaction and play a role in CD47's binding affinity for SIRPa (Hatherley et al., 2008). In preferred embodiments, the two 0-strands and the six contact residues within them are retained in the engineered CD47 proteins disclosed herein to maintain SIRPa binding capability.

[0191] In some embodiments, the engineered CD47 protein comprises at least one SIRPa interaction motif in its extracellular domain. In some embodiments, the amino acids corresponding to amino acids 97, 99, 100, 103, 104, 106 of SEQ ID NO: 3 are retained in the engineered CD47 protein. In some embodiments, the two 0-strands are retained in the engineered CD47 protein.

[0192] In some embodiments, the engineered CD47 protein comprises a disulfide bond between a cysteine within the human CD47 extracellular domain or portion thereof and a cysteine within or between the human CD47 transmembrane domain(s). In some embodiments, the two cysteines are Cys33in the extracellular domain and the Cys263in the transmembrane domain, wherein the numbering is based on SEQ ID NO: 3.

[0193] In some embodiments, the engineered CD47 protein can bind to SIRPa. In some embodiments, the engineered CD47 protein can bind to SIRPa with a binding affinity that is similar to a wild-type human CD47 protein. In some embodiments, the engineered CD47 protein binds to SIRPa with a KDlower than about 0.01 pM, 0.02 pM, 0.03 pM, 0.04 pM, 0.05 pM, 0.06 pM, 0.07 pM, 0.08 pM, 0.09 pM, 0.1 pM, 0.2 pM, 0.3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM. In some embodiments, the engineered CD47 protein can bind to SIRPa with a higher binding affinity than a wild-type human CD47 protein.

[0194] Direct binding studies demonstrate that TSP-1 inhibits SIRPa binding to cells expressing CD47 (Isenberg et al., 2009). This data is consistent with competitive binding of TSP-1 and SIRPa to a single site on CD47, steric inhibition of binding to distinct but proximal sites, or allosteric inhibition. Glycosylation at Ser64with a heparan sulfate chain is necessary for TSP-1 binding but not SIRPa binding (Soto-Pantoja et al., 2015). Therefore, in some embodiments, the engineered CD47 protein that lacks glycosylation at Ser54has enhanced SIRPa binding capacity because TSP-1 binding is hindered.

[0195] The CD47 / SIRPa interaction regulates a multitude of intercellular interactions in many body systems, such as the immune system where it regulates lymphocyte homeostasis, dendritic cell (DC) maturation and activation, proper localization of certain DC subsets in secondary lymphoid organs, and cellular transmigration. The CD47 / SIRPa interaction also regulates cells of the nervous system. An interaction between these two proteins also plays an important role in bone remodeling. Cellular responses regulated by the CD47 / SIRPa interaction are often dependent on a bidirectional signaling through both receptors.

[0196] CD47 on host cells can function as a "marker of self" and regulate phagocytosis by binding to SIRPa on the surface of circulating immune cells to deliver an inhibitory "don't kill me" signal. As disclosed above, SIRPa encodes an Ig-superfamily receptor expressed on the surface of macrophages and dendritic cells, whose cytoplasmic region contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) that can trigger a cascade to inhibit phagocytosis. CD47-SIRPa binding results in phosphorylation of ITIMs on SIRPa, which triggers recruitment of the SHP1 and SHP2 Src homology phosphatases. These phosphatases, in turn, inhibit accumulation of myosin II at the phagocytic synapse, preventing phagocytosis (Fujioka et al., 1996). Phagocytosis of target cells by macrophages is ultimately regulated by a balance of activating signals (FcyR, CRT, LRP-1) and inhibitory signals (SIRPa-CD47). Elevated expression of CD47 can help the cell evade immune surveillance and subsequent destruction. Elevated expression of CD47 can help the cell evade innate immune cell killing.

[0197] In some embodiments, the engineered CD47 protein is a tolerogenic factor. As used herein, tolerogenic factor is an agent that induces immune tolerance when there is pathological or undesirable activation of the normal immune response. This can occur, for example, when a patient develops an immune reaction to donor antigens after receiving an allogeneic transplantation or an allogeneic cell therapy, or when the body responds inappropriately to self-antigens implicated in autoimmune diseases. In some embodiments, "tolerogenic factor" includes hypoimmunity factors, complement inhibitors, and other factors that modulate or affect the ability of a cell to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment. In some embodiments, the tolerogenic factor is genetically modified to achieve additional functions.

[0198] In some embodiments, the engineered CD47 protein can inhibit phagocytosis, release of cytotoxic agents, and / or other mechanisms of cell-mediated killing.Interactions between CD47 molecules

[0199] It has also been shown that CD47 mediates cell adhesion interactions in the absence of any known CD47 ligands. This cell-cell adhesion, which requires CD47 but not any of its known ligands, suggests that homotypic binding can also occur between the IgV domains of CD47 on opposing cells (Rebres et al., 2005). This interaction may require an unidentified trypsin-sensitive protein (X) to mediate cell-cell adhesion, but the potential should be considered that this cell-cell interaction and homotypic binding of proteolytically shed CD47 IgV domain (Maile et al., 2010) or CD47 in exosomes (Kaur et al., 2014) to cell surface CD47 could elicit CD47 signal transduction. However, direct evidence for CD47-CD47 binding and signaling resulting from homotypic CD47 binding is lacking. In some embodiments, the engineered CD47 protein cannot interact with another CD47 molecule.III. Polynucleotides

[0200] In another aspect, the present disclosure provides a polynucleotide encoding the engineered CD47 protein disclosed herein.

[0201] A polynucleotide encoding the engineered CD47 protein disclosed herein can be obtained by methods known in the art. For example, the polynucleotide can be obtained from cloned DNA (e.g., from a DNA library), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA or fragments thereof, purified from the desired cell. When the polynucleotides are produced by recombinant means, any method known to those skilled in the art for identification of nucleic acids that encode desired genes can be used. Any method available in the art can be used to obtain a full length (i.e. encompassing the entire coding region) cDNA or genomic DNA encoding a desired human CD47 protein, such as from a cell or tissue source. Modified or variant polynucleotides, including truncated forms of CD47 such as provided herein, can be engineered from a wildtype polynucleotide using standard recombinant DNA methods. Polynucleotides can be cloned or isolated using any available methods known in the art for cloning and isolating nucleic acid molecules. Such methods include PCR amplification of nucleic acids and screening of libraries, including nucleic acid hybridization screening, antibody-based screening, and activity-based screening.

[0202] Methods for amplification of polynucleotides can be used to isolate polynucleotides encoding a desired protein, including for example, polymerase chain reaction (PCR) methods. PCR can be carried out using any known methods or procedures in the art. Exemplary methods include use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp). A nucleic acid containing gene of interest can be used as a source material from which a desired polypeptide-encoding nucleic acid molecule can be amplified. For example, DNA and mRNA preparations, cell extracts, tissue extracts from an appropriate source (e.g. testis, prostate, breast), fluid samples (e.g. blood, serum, saliva), samples from healthy and / or diseased subjects can be used in amplification methods. The source can be from any eukaryotic species including, but not limited to, vertebrate, mammalian, human, porcine, bovine, feline, avian, equine, canine, and other primate sources. Nucleic acid libraries also can be used as a source material. Primers can be designed to amplify a desired polynucleotide. For example, primers can be designed based on expressed sequences from which a desired polynucleotide is generated. Primers can be designed based on back-translation of a polypeptide amino acid sequence. If desired, degenerate primers can be used for amplification. Oligonucleotide primers that hybridize to sequences at the 3' and 5' termini of the desired sequence can be uses as primers to amplify by PCR from a nucleic acidsample. Primers can be used to amplify the entire full-length polynucleotide, or a truncated sequence thereof. Nucleic acid molecules generated by amplification can be sequenced and confirmed to encode a desired polypeptide.IV. Vectors

[0203] In another aspect, the present disclosure provides a vector comprising a polynucleotide that encodes the engineered CD47 protein disclosed herein.

[0204] Any methods known to those skilled in the art for the insertion of DNA fragments into a vector can be used to construct expression vectors comprising a polynucleotide disclosed herein. These methods can include in vitro recombinant DNA and synthetic techniques and in vivo (genetic) recombination. The polynucleotide disclosed herein can be operably linked to control sequences in the expression vector(s) to ensure the expression of the engineered CD47 protein. Such control sequences may include, but are not limited to, leader or signal sequences, promoters (e.g., naturally associated or heterologous promoters), ribosomal binding sites, enhancer or activator elements, translational start and termination sequences, and transcription start and termination sequences, and are chosen to be compatible with the host cell chosen to express the engineered CD47 protein. Constitutive or inducible promoters as known in the art are also contemplated. The promoters may be either naturally occurring promoters, hybrid promoters that combine elements of more than one promoter, or synthetic promoters. An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome such as in a gene locus. In some embodiment, the expression vector includes a selectable marker gene to allow the selection of transformed host cells. Some embodiments, include an expression vector comprising a nucleotide sequence encoding a variant polypeptide operably linked to at least one regulatory control sequence. Regulatory control sequence for use herein include promoters, enhancers, and other expression control elements. In some embodiments, an expression vector is designed for the choice of the host cell to be transformed, the particular variant polypeptide desired to be expressed, the vector's copy number, the ability to control that copy number, and / or the expression of any other protein encoded by the vector, such as antibiotic markers.

[0205] Examples of suitable mammalian promoters include, for example, promoters from the following genes: elongation factor 1 alpha (EFla) promoter, CAG promoter, ubiquitin / S27a promoter of the hamster (WO 97 / 15664), Simian vacuolating virus 40 (SV40) early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, the long terminal repeat region of Rous Sarcoma Virus (RSV), mouse mammary tumor virus promoter (MMTV), Moloney murine leukemia virus Long Terminal repeat region, and the early promoter of human Cytomegalovirus (CMV).Examples of other heterologous mammalian promoters are the actin, immunoglobulin or heat shock promoter(s). In additional embodiments, promoters for use in mammalian host cells can be obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40). In further embodiments, heterologous mammalian promoters are used. Examples include the actin promoter, an immunoglobulin promoter, and heat-shock promoters. The early and late promoters of SV40 are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature 273: 113-120 (1978)). The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll restriction enzyme fragment (Greenaway et al., Gene 18: 355-360 (1982)). The foregoing references are incorporated by reference in their entirety.

[0206] In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide.

[0207] The vector can include, but is not limited to, viral vectors and plasmid DNA. Viral vectors can include, but are not limited to, adenoviral vectors, lentiviral vectors, retroviral vectors, and adeno-associated viral vectors. Commonly, expression vectors contain selection markers such as ampicillin-resistance, hygromycin-resistance, tetracycline resistance, kanamycin resistance, or neomycin resistance to permit detection of those cells transformed with the desired DNA sequences. Suitable vectors, promoter, and enhancer elements are known in the art; many are commercially available for generating subject recombinant constructs. In some embodiments, the vector is a polycistronic vector. In some embodiments, the vector is a bicistronic vector or a tricistronic vector. Bicistronic or multicistronic expression vectors may include (1) multiple promoters fused to each of the open reading frames; (2) insertion of splicing signals between genes; (3) fusion of genes whose expressions are driven by a single promoter; and (4) insertion of proteolytic cleavage sites between genes (self-cleavage peptide) or insertion of internal ribosomal entry sites (IRESs) between genes.

[0208] A polycistronic vector is used to co-express multiple genes in the same cell. Two strategies are most commonly used to construct a multicistronic vector. First, an Internal Ribosome Entry Site (IRES) element is typically used for bi-cistronic vectors. The IRES element, acting as another ribosome recruitment site, allows initiation of translation from an internal region of the mRNA. Thus, two proteins are translated from one mRNA. IRES elements are quite large (usually 500-600 bp) (Pelletier et al., 1988; Jang et al., 1988). The engineered CD47 proteins disclosed herein have a smaller size compared to the wild-type full-length human CD47, and thus could be used with IRES element in a multicistronic vectors having limited packaging capacity.

[0209] The second strategy relies on "self-cleaving" 2A peptides. These peptides, first discovered in picornaviruses, are short (about 20 amino acids) and produce equimolar levels of multiple genes from the same mRNA. The term "self-cleaving" is not entirely accurate, as these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (Kim et al., 2011). The "cleavage" occurs between the glycine and proline residues found on the C-terminus. Thus, the upstream cistron will have a few additional residues added to the end, while the downstream cistron will start with the proline.

[0210] In some embodiments, the polycistronic vectors used in the context of this disclosure are the polycistronic vectors described in US applications 63 / 270,956 and 63 / 222,954.

[0211] In some embodiments, the vector herein is a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule, including into the cell or into the genome of a cell. The transferred nucleic acid is generally linked to, e.g., inserted into, the vector nucleic acid molecule. A vector may include sequences that direct autonomous replication in a cell or may include sequences sufficient to allow integration into host cell DNA. Useful vectors include, for example, plasmids {e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, e.g., replication defective retroviruses and lentiviruses. Non- viral vectors may require a delivery vehicle to facilitate entry of the nucleic acid molecule into a cell.

[0212] A viral vector can comprise a nucleic acid molecule that includes virus-derived nucleic acid elements that typically facilitate transfer of the nucleic acid molecule or integration into the genome of a cell or to a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components and sometimes also host cell components in addition to nucleic acid(s). A viral vector can comprise, e.g., a virus or viral particle capable oftransferring a nucleic acid into a cell, or to the transferred nucleic acid (e.g., as naked DNA). Viral vectors and transfer plasmids can comprise structural and / or functional genetic elements that are primarily derived from a virus. A retroviral vector can comprise a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, that are primarily derived from a retrovirus.

[0213] In some vectors described herein, at least part of one or more protein coding regions that contribute to or are essential for replication may be absent compared to the corresponding wild-type virus. This makes the viral vector replication-defective. In some embodiments, the vector is capable of transducing a target non-dividing host cell and / or integrating its genome into a host genome.

[0214] In some embodiments, the retroviral nucleic acid comprises one or more of (e.g., all of): a 5' promoter{e.g., to control expression of the entire packaged RNA), a 5' LTR {e.g., that includes R (polyadenylation tail signal) and / or U5 which includes a primer activation signal), a primer binding site, a psi packaging signal, a RRE element for nuclear export, a promoter directly upstream of the transgene to control transgene expression, a transgene (or other exogenous agent element), a polypurine tract, and a 3' LTR {e.g., that includes a mutated U3, a R, and U5). In some embodiments, the retroviral nucleic acid further comprises one or more of a cPPT, a WPRE, and / or an insulator element (e.g., as described in Browning et al., "Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy," Biomedicines, 4(1):4 (2016)).

[0215] A retrovirus typically replicates by reverse transcription of its genomic RNA into a linear doublestranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. The structure of a wildtype retrovirus genome often comprises a 5' long terminal repeat (LTR) and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome and gag, pol and env genes encoding the packaging components which promote the assembly of viral particles. More complex retroviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell. In the provirus, the viral genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are involved in proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.

[0216] The LTRs themselves are typically similar {e.g., identical) sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA and U5 is derived from the sequence unique to the 5' end of the RNA. The sizes of the three elements can vary considerably among different retroviruses.

[0217] For the viral genome, the site of transcription initiation is typically at the boundary between U3 and R in one LTR and the site of poly (A) addition (termination) is at the boundary between R and U5 in the other LTR. U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins. Some retroviruses comprise any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tot, rev, tax and rex.

[0218] With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (IN), which mediate replication of the genome. The env gene encodes the surface (SU) glycoprotein andthe transmembrane (TM) protein of the virion, which form a complex that interacts specifically with cellular receptor proteins. This interaction promotes infection, e.g., by fusion of the viral membrane with the cell membrane.

[0219] In a replication-defective retroviral vector genome gag, pol and env may be absent or not functional. The R regions at both ends of the RNA are typically repeated sequences. U5 and U3 represent unique sequences at the 5' and 3' ends of the RNA genome respectively. Retroviruses may also contain additional genes which code for proteins other than gag, pol and env. Examples of additional genes include (in HIV), one or more of vif, vpr, vpx, vpu, tat, rev and nef. EIAV has (amongst others) the additional gene S2.

[0220] Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to:Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus(MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV) and human immunodeficiency virus (HIV).

[0221] In some embodiments the retrovirus is a Gammretrovirus. In some embodiments the retrovirus is anEpsilonretrovirus. In some embodiments the retrovirus is an Alpharetrovirus. In some embodiments the retrovirus is a Betaretrovirus. In some embodiments the retrovirus is a Deltaretrovirus. In some embodiments the retrovirus is a Spumaretrovirus. In some embodiments the retrovirus is an endogenous retrovirus. In some embodiments the retrovirus is a lentivirus.

[0222] In some embodiments, a retroviral or lentivirus vector further comprises one or more insulator elements, e.g., an insulator element described in Browning et al., "Insulators to Improve the Safety of Retroviral Vectors for HIV Gene Therapy," Biomedicines, 4(1):4 (2016). In various embodiments, the vectors comprise a promoter operably linked to a polynucleotide encoding an exogenous agent. The vectors may have one or more LTRs, wherein either LTR comprises one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vectors may further comprise one of more accessory elements to increase transduction efficiency {e.g., a cPPT / FLAP), viral packaging {e.g., a Psi (Y) packaging signal, RRE), and / or other elements that increase exogenous gene expression {e.g., poly (A) sequences), and may optionally comprise a WPRE or HPRE. In some embodiments, a lentiviral nucleic acid comprises one or more of, e.g., all of, e.g, from 5' to 3', a promoter {e.g, CMV), an R sequence {e.g., comprising TAR), a U5 sequence {e.g, for integration), a PBS sequence {e.g., for reverse transcription), a DIS sequence {e.g., for genome dimerization), a psi packaging signal, a partial gag sequence, an RRE sequence {e.g., for nuclear export), a cPPT sequence {e.g., for nuclear import), a promoter to drive expression of the exogenous agent, a gene encoding the exogenous agent, a WPRE sequence {e.g., for efficient transgene expression), a PPT sequence {e.g., for reverse transcription), an R sequence {e.g., for polyadenylation and termination), and a U5 signal {e.g., for integration).

[0223] Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In some embodiments, HIV based vector backbones (i.e., HIV cis-acting sequence elements) are used. A lentivirus vector can comprise a viral vector or plasmid containing structural and functional genetic elements, or portions thereof, including LTRs that are primarily derived from a lentivirus.

[0224] In embodiments, a lentivirus vector {e.g., lentiviral expression vector) may comprise a lentiviral transfer plasmid {e.g., as naked DNA) or an infectious lentiviral particle. With respect to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is to be understood that the sequences of these elements can be present in RNA form in lentiviral particles and can be present in DNA form in DNA plasmids.

[0225] In embodiments, a lentivirus vector is a vector with sufficient retroviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle capable of infecting a target cell. Infection of the target cell can comprise reverse transcription and integration into the target cell genome. The recombinant lentivirus vector (RLV) typically carries non-viral coding sequences which are to be delivered by the vector to the target cell. In embodiments, an RLV is incapable of independent replication to produce infectious retroviral particles within the target cell. Usually the RLV lacks a functional gag-pol and / or env gene and / or other genes involved in replication. The vector may be configured as a split-intron vector, e.g., as described in PCT patent application WO 99 / 15683, which is herein incorporated by reference in its entirety.

[0226] In some embodiments, the lentivirus vector comprises a minimal viral genome, e.g., the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell, e.g., as described in WO 98 / 17815, which is herein incorporated by reference in its entirety.

[0227] A minimal lentiviral genome may comprise, e.g., (5')R-U5-one or more first nucleotide sequences-U3-R(3'). However, the plasmid vector used to produce the lentiviral genome within a source cell can also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a source cell. These regulatory sequences may comprise the natural sequences associated with the transcribed retroviral sequence, e.g., the 5' U3 region, or they may comprise a heterologous promoter such as another viral promoter, for example the CMV promoter. Some lentiviral genomes comprise additional sequences to promote efficient virus production. For example, in the case of HIV, rev and RRE sequences may be included.V. Cells

[0228] In another aspect, the present disclosure provides a cell comprising a polynucleotide encoding an engineered CD47 protein as disclosed herein, and / or a vector comprising a polynucleotide that encodes an engineered CD47 protein as disclosed herein.

[0229] In another aspect, the present disclosure provides a cell comprising an engineered CD47 protein as disclosed herein. In some embodiments, the engineered CD47 protein is introduced into a cell in the form of a nucleic acid molecule encoding the engineered CD47 protein. The process of introducing the nucleic acid molecule into the cell can be achieved by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using a viral vector. In some embodiments, the nucleic acid molecule comprises DNA. In some embodiments, the nucleic acid molecule comprises a modified DNA. In some embodiments, the nucleic acid molecule comprises mRNA. In some embodiments, the nucleic acid molecule comprises a modified mRNA.

[0230] In some embodiments, the engineered CD47 protein is delivered using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide.

[0231] In some embodiments, the engineered CD47 protein is delivered using one or more gene editing systems. In some embodiments, the gene editing system is CRISPR / Cas. In some embodiments, the gene editing system is one or more of the CRISPR / Cas systems described herein. In some embodiments, the engineered CD47 protein is delivered using Transcription Activator-Like Effector Nucleases (TALEN) methodologies. In some embodiments, the gene editing system is one or more of the TALEN methodologies described herein.

[0232] In some embodiments, the engineered CD47 protein is delivered using zinc finger nuclease (ZFN). In some embodiments, the gene editing system is one or more of the ZFN methodologies described herein.

[0233] In some embodiments, the engineered CD47 protein is delivered using a meganuclease. In some embodiments, the gene editing system is one or more of the meganuclease methodologies described herein.

[0234] In some embodiments, the cell is a stem cell.

[0235] In some embodiments, the cell is a pluripotent stem cell. Pluripotent stem cells are cells that have the capacity to self-renew by dividing and to develop into the three primary germ cell layers of the early embryo and therefore into all cells of the adult body, but not extra-embryonic tissues such as the placenta. Embryonic stem cells and induced pluripotent stem cells are pluripotent stem cells.

[0236] In some embodiments, the cell is an embryonic stem cell (ESC). Embryonic stem cells are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo.

[0237] In some embodiments, the cell is an induced pluripotent stem cell (iPSC). iPSCs are derived from adult somatic cells that have been genetically reprogrammed back into an embryonic-like pluripotent state that enables the development of an unlimited source of any type of cell needed for therapeutic purposes.

[0238] "Pluripotent stem cells" as used herein have the potential to differentiate into any of the three germ layers: endoderm (e.g., the stomach lining, gastrointestinal tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.) or ectoderm (e.p., epidermal tissues and nervous system tissues). The term "pluripotent stem cells," as used herein, also encompasses induced pluripotent stem cells (iPSCs or iPS cells), or a type of pluripotent stem cell derived from a non-pluripotent cell. In some embodiments, a pluripotent stem cell is produced or generated from a cell that is not a pluripotent cell. In other words, pluripotent stem cells can be direct or indirect progeny of a non- pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the induction of iPS cells are known in the art and are further described below. (See, e.g, Zhou etal., Stem Cells 27 (11): 2667-74 (2009); Huangfu eta / ., Nature Biotechnol. 26 (7): 795 (2008); Woltjen et al., Nature 458 (7239): 766-770 (2009); and Zhou etaL, Cell Stem Cell 8:381-384 (2009); each of which is incorporated by reference herein in their entirety.) As used herein, "hiPSCs" are human induced pluripotent stem cells. In some embodiments, "pluripotent stem cells," as used herein, also encompasses mesenchymal stem cells (MSCs), and / or embryonic stem cells (ESCs).

[0239] In some embodiments, the cell is a pancreatic islet cell. In some embodiments, the cell is a primary pancreatic islet cell.

[0240] In some embodiments, the cell is differentiated from a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an iPSC or an ESC.

[0241] In some embodiments, the cell is a T cell. In some embodiments, the cell is a primary T cell. In some embodiments, the cell is a T cell comprising a chimeric antigen receptor (CAR), such as comprising a polynucleotide encoding a CAR and / or comprising the CAR, or otherwise expressing the CAR from the polynucleotide. In some embodiments, the cell is a CAR-T cell. In some embodiments, the T cell is differentiated from a pluripotent stem cell. In some embodiments, the pluripotent stem cell is an iPSC or an ESC.

[0242] A T cell is a type of lymphocyte. T cells are one of the white blood cells of the immune system and play a central role in the adaptive immune response. CAR-T cells are T cells that have been genetically engineered to produce an artificial T cell receptor. Chimeric antigen receptors (CARs, also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors) are receptor proteins that have been engineered to give T cells theability to target a specific protein. The receptors are chimeric because they combine both antigen-binding and T cell activating functions into a single receptor. CAR-T cells can be both CD4+ and CD8+, with a 1-to-l ratio of both cell types providing synergistic antitumor effects. CAR-T cells can be derived from T cells in a patient's own blood (autologous) or derived from the T cells of another healthy donor (allogeneic). T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLL™ separation, antibody-conjugated bead-based methods such as MACS™ separation (Miltenyi).

[0243] In some embodiments, the cell is selected from, but not limited to, cardiac cells, cardiac progenitor cells, neural cells, glial progenitor cells, endothelial cells, T cells, B cells, pancreatic islet cells, retinal pigmented epithelium cells, hepatocytes, thyroid cells, skin cells, blood cells, plasma cells, platelets, renal cells, epithelial cells, CAR- T cells, NK cells, and CAR-NK cells.

[0244] In some embodiments, the cell is a primary cell. Primary cells are isolated directly from human or animal tissue using enzymatic or mechanical methods. Once isolated, they are placed in an artificial environment in plastic or glass containers supported with specialized medium containing essential nutrients and growth factors to support proliferation. Primary cells could be of two types: adherent or suspension. Adherent cells require attachment for growth and are said to be anchorage-dependent cells. Adherent cells are usually derived from tissues of organs. Suspension cells do not require attachment for growth and are said to be anchorage-independent cells. Most suspension cells are isolated from the blood system, but some tissue-derived cells can also be used in suspension, such as hepatocytes or intestinal cells. Although primary cells usually have a limited lifespan, they offer a number of advantages compared to cell lines. Primary cell culture enables researchers to study donors and not just cells. Several factors such as age, medical history, race, and sex can be considered when building an experimental model. With a growing trend towards personalized medicine, such donor variability and tissue complexity can be achieved with use of primary cells, but are difficult to replicate with cell lines that are more systematic and uniform in nature and do not capture the true diversity of a living tissue.

[0245] In some embodiments, the cell is a differentiated cell. Differentiated cells are cells that have undergone differentiation. They are mature cells that perform a specialized function. Some examples of differentiated cells are epithelial cells, skin fibroblasts, endothelial cells lining the blood vessels, smooth muscle cells, liver cells, nerve cells, human cardiac muscle cells, etc. Generally, these cells have a unique morphology, metabolic activity, membrane potential, and responsiveness to signals facilitating their function in a body tissue or organ.Hypolmmunogenlc cells

[0246] In some embodiments, the cells described herein are hypoimmunogenic cells. As used herein to characterize a cell, the term "hypoimmunogenic" generally means that such cell is less prone to innate or adaptive immune rejection by a subject into which such cells are transplanted, e.g., the cell is less prone to allorejection by a subject into which such cells are transplanted. For example, in some embodiments, relative to a cell of the same cell type that does not comprise the modifications, such a hypoimmunogenic cell may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, 100%, or any amount in between, less prone to innate or adaptive immune rejection by a subject into which such cells are transplanted. In some embodiments, genome editing technologies are used to modulate the expression of MHC I and MHC II genes, and thus, contribute to generation of ahypoimmunogenic cell. In some embodiments, a hypoimmunogenic cell evades immune rejection in an MHC-mismatched allogeneic recipient. In some embodiments, differentiated cells produced from the hypoimmunogenic stem cells outlined herein evade immune rejection when administered (e.g., transplanted or grafted) to an MHC-mismatched allogeneic recipient. In some embodiments, a hypoimmunogenic cell is protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection. Detailed descriptions of hypoimmunogenic cells, methods of producing such cells, and methods of using such cells are found in W02016183041 filed May 9, 2015; WO2018132783 filed January 14, 2018; WO2018176390 filed March 20, 2018; W02020018615 filed July 17, 2019; W02020018620 filed July 17, 2019; PCT / US2020 / 44635 filed July 31, 2020; US62 / 881,840 filed August 1, 2019; US62 / 891,180 filed August 23, 2019; 11563 / 016,190, filed April 27, 2020; and 11563 / 052,360 filed July 15, 2020, WO2023158836, filed February 2023, the disclosures including the examples, sequence listings, and figures of which are incorporated herein by reference in their entirety.

[0247] Hypoimmunogenicity of a cell can be determined by evaluating the immunogenicity of the cell such as the cell's ability to elicit adaptive and innate immune responses or to avoid eliciting such adaptive and innate immune responses. Such immune response can be measured using assays recognized by those skilled in the art. In some embodiments, an immune response assay measures the effect of a hypoimmunogenic cell on T cell proliferation, T cell activation, T cell killing, donor specific antibody generation, NK cell proliferation, NK cell activation, and macrophage activity. In some cases, hypoimmunogenic cells and derivatives thereof undergo decreased killing by T cells and / or NK cells upon administration to a subject. In some instances, the cells and derivatives thereof show decreased macrophage engulfment compared to an unmodified or wild-type cell. In some embodiments, a hypoimmunogenic cell elicits a reduced or diminished immune response in a recipient subject compared to a corresponding unmodified wild-type cell. In some embodiments, a hypoimmunogenic cell is nonimmunogenic or fails to elicit an immune response in a recipient subject.

[0248] In some embodiments, the expression of major histocompatibility (MHC) class I and MHC class II proteins are disrupted in the cell. MHC class I and class II proteins play a role in the adaptive branch of the immune system. Both classes of proteins share the task of presenting peptides on the cell surface for recognition by T cells. Immunogenic peptide-MHC class I (pMHCI) complexes are presented on nucleated cells and are recognized by cytotoxic CD8+ T cells. The presentation of peptide-MHC class II (pMHCII) by antigen-presenting cells (e.g., dendritic cells (DCs), macrophages, or B cells), on the other hand, can activate CD4+ T cells, leading to the coordination and regulation of effector cells. In all cases, it is a clonotypic T cell receptor that interacts with a given pMHC complex, potentially leading to sustained cell-cell contact formation and T cell activation.

[0249] In some embodiments the cells described herein express reduced levels of MHC class I proteins relative to a wild-type or control cell. In some embodiments, the cells express reduced levels of MHC class II proteins relative to a wild-type or control cell. In some embodiments, the cells express reduced levels of both MHC class I and class II proteins relative to a wild-type or control cell.

[0250] In some embodiments, the cells do not express any MHC class I proteins. In some embodiments, the cells do not express any MHC class II proteins. In some embodiments, the cells do not express any MHC class I and do not express any MHC class II proteins.

[0251] In some embodiments, the MHC proteins discussed herein are HLA proteins.

[0252] "HLA" or "human leukocyte antigen" complex is a gene complex encoding the MHC proteins in humans. The cell-surface proteins that make up the HLA complex are responsible for the regulation of the immune response to antigens. In humans, there are two MHCs, class I and class II, or "HLA-I" and "HLA-II." HLA-I includes threeproteins, HLA-A, HLA-B, and HLA-C, which present peptides from the inside of the cell, and antigens presented by the HLA-I complex attract killer T-cells (also known as CD8+ T-cells or cytotoxic T cells). The HLA-I proteins are associated with p-2 microglobulin (B2M). HLA-II includes five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which present antigens from outside the cell to T lymphocytes. The HLA-II proteins are associated with Class II transactivator (CIITA). This stimulates CD4+ cells (also known as T-helper cells). It should be understood that the use of either "MHC" or "HLA" is not meant to be limiting, though the term "HLA" typically indicates that the genes are from humans. Thus, as it relates to mammalian cells, these terms may be used interchangeably herein.

[0253] In some embodiments, the expression of MHC class II proteins is reduced by knocking out or by reducing expression of CIITA. In some embodiments, the expression of MHC II genes is modulated (<?.£?., reduced or eliminated) by targeting and modulating (e.g., reducing or eliminating) Class II transactivator (CIITA) expression. In some embodiments, the modulation occurs using a CRISPR / Cas system. CIITA is a member of the LR or nucleotide binding domain (NBD) leucine-rich repeat (LRR) family of proteins and regulates the transcription of MHC II by associating with the MHC enhanceosome. In some embodiments, the polynucleotide sequence being targeted for modulation is a variant of CIITA, a homolog of CIITA, or an ortholog of CIITA.

[0254] In some embodiments, reduced or eliminated expression of CIITA reduces or eliminates expression of one or more of the following MHC class II molecules: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0255] In some embodiments, the cells described herein comprise gene modifications at the gene locus encoding the CIITA protein. In other words, the cells comprise a genetic modification at the CIITA locus. In some instances, the nucleotide sequence encoding the CIITA protein is set forth in RefSeq. No. NM_000246.4 and NCBI Genbank No. U18259. In some instances, the CIITA gene locus is described in NCBI Gene ID No. 4261. In certain cases, the amino acid sequence of CIITA is depicted as NCBI GenBank No. AAA88861.1. Additional descriptions of the CIITA protein and gene locus can be found in Uniprot No. P33076, HGNC Ref. No. 7067, and OMIM Ref. No. 600005.

[0256] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the CIITA gene. In some embodiments, the genetic modification targeting the CIITA gene is generated by a rare-cutting endonuclease comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOS:5184-36352 of Table 12 of W02016183041, which is herein incorporated by reference. In some embodiments, the cell has a reduced ability to induce an innate and / or an adaptive immune response in a recipient subject. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CIITA gene.

[0257] Assays to test whether the CIITA gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the CIITA gene can be confirmed by PCR and the reduction of HLA-II expression can be confirmed by FACS analysis. In another embodiment, CIITA protein expression is detected using a Western blot of cells lysates probed with antibodies to the CIITA protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the inactivating genetic modification. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell by viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide. In some embodiments, theexogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus based viral vector.

[0258] In some embodiments, the expression of MHC class I proteins is reduced by knocking out or by reducing expression of B2M. In some embodiments, the expression of MHC-I genes is modulated (e.g., reduced or eliminated) by targeting and modulating {e.g., reducing or eliminating) expression of the accessory chain B2M. In some embodiments, the modulation occurs using a CRISPR / Cas system. By modulating (e.p., reducing or deleting) expression of B2M, surface trafficking of MHC-I molecules is blocked and the cell rendered hypoimmunogenic. In some embodiments, the cell has a reduced ability to induce an innate and / or an adaptive immune response in a recipient subject.

[0259] In some embodiments, the polynucleotide sequence being targeted for modulation is a variant of B2M, a homolog of B2M, or an ortholog of B2M.

[0260] In some embodiments, decreased or eliminated expression of B2M reduces or eliminates expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0261] In some embodiments, the cells described herein comprise gene modifications at the gene locus encoding the B2M protein. In other words, the cells comprise a genetic modification at the B2M locus. In some instances, the nucleotide sequence encoding the B2M protein is set forth in RefSeq. No. NM_004048.4 and Genbank No. AB021288.1. In some instances, the B2M gene locus is described in NCBI Gene ID No. 567. In certain cases, the amino acid sequence of B2M is set forth in NCBI GenBank No. BAA35182.1. Additional descriptions of the B2M protein and gene locus can be found in Uniprot No. P61769, HGNC Ref. No. 914, and OMIM Ref. No. 109700.

[0262] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the B2M gene. In some embodiments, the genetic modification targeting the B2M gene is generated by a rare- cutting endonuclease comprising a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOS:81240-85644 of Table 15 of W02016183041, which is herein incorporated by reference. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the B2M gene. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus based viral vector.

[0263] Assays to test whether the B2M gene has been inactivated are known and described herein. In some embodiments, the resulting genetic modification of the B2M gene can be confirmed by PCR and the reduction of HLA-I expression can be confirmed by FACS analysis. In another embodiment, B2M protein expression is detected using a Western blot of cells lysates probed with antibodies to the B2M protein. In another embodiment, reverse transcriptase polymerase chain reactions (RT-PCR) are used to confirm the presence of the inactivating genetic modification.

[0264] In some embodiments of the T cells, iPSCs, or ESCs described herein, T cell receptor alpha chain (TRAC) and / or T cell receptor beta chain (TRBC) genes are knocked out, or their expression is reduced in the cells.

[0265] The T-cell receptor (TCR) is a protein complex found on the surface of T cells that is responsible for recognizing fragments of antigen as peptides bound to MHC molecules. The TCR is a disulfide-linked membrane- anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (p) chains (encoded by TRAC and TRBC genes, respectively) expressed as part of a complex with the invariant CD3 chain molecules. T cells expressing this receptor are referred to as a:p (or aP) T cells, though a minority of T cells express an alternate receptor, formed by variable gamma (y) and delta (6) chains, referred as y6 T cells. Each chain is composed of two extracellular domains: a variable region and a constant region, both of these immunoglobulin superfamily (IgSF) domains forming antiparallel p-sheets. The constant region is proximal to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / MHC complex. It has been reported that knockout of endogenous TRAC and / or TRBC genes could increase expression and function of T cells expressing transgenic T cell receptor. In some embodiments the cells described herein express reduced levels of MHC class I proteins and / or MHC class II proteins relative to a wild-type or control cell.

[0266] In some embodiments, the cells comprise increased expression of wild-type and / or engineered CD47 protein relative to a wild-type cell or a control cell of the same cell type. In some embodiments, the wild-type cell or the control cell is a starting material. As used herein, a starting material refers to a raw material upon which one or more of the modifications described herein are made in order to produce the engineered CD47 protein as described herein, the polynucleotide encoding the engineered CD47 protein as described herein, the vector as described herein, the cell comprising the engineered CD47 protein as described herein, or the composition comprising the engineered CD47 protein or the cell as described herein.

[0267] As used herein, a "wild-type cell" or a "wt cell" means any cell found in nature. In some embodiments, wild-type cells include primary cells and T cells found in nature. As used herein, a "control cell" is a cell whose CD47 gene is unaltered, but in which other modifications may be made. In some embodiments, the control cell is an engineered cell that may contain nucleic acid changes resulting in reduced expression of MHC I protein and / or MHC II protein and / or T-cell receptors. In some embodiments, the control cell is an engineered cell that has B2M knocked out, or comprises reduced expression of B2M. In some embodiments, the control cell is an engineered cell that has CIITA knocked out, or comprises reduced expression of CIITA. In some embodiments, the control cell is an engineered cell that has TRAC and / or TRBC knocked out, or comprises reduced expression of TRAC and / or TRBC.

[0268] In some embodiments, the control cell is an iPSC, an ESC, or a progeny that contains nucleic acid changes resulting in pluripotency. In some embodiments, the control cell is an iPSC, an ESC, or a progeny that has B2M knocked out, or comprises reduced expression of B2M. In some embodiments, the control cell is an iPSC, an ESC, or a progeny that has CIITA knocked out, or comprises reduced expression of CIITA. In some embodiments, the control cell is an iPSC, an ESC, or a progeny that has TRAC and / or TRBC knocked out, or comprises reduced expression of TRAC and / or TRBC.

[0269] In some embodiments, the control cell is a primary T cell or a progeny that contains nucleic acid changes resulting in reduced expression of MHC I protein and / or MHC II protein and / or T-cell receptors. In some embodiments, the control cell is a primary T cell or a progeny that has B2M knocked out, or comprises reduced expression of B2M. In some embodiments, the control cell is a primary T cell or a progeny that has CIITA knocked out, or comprises reduced expression of CIITA. In some embodiments, the control cell is a primary T cell or a progeny that has TRAC and / or TRBC knocked out, or comprises reduced expression of TRAC and / or TRBC.

[0270] In some embodiments, the starting material is a primary cell collected from a donor. In some embodiments, the starting material is a primary blood cell collected from a donor, e.g., via a leukopak. For example, insome embodiments, the starting material are unmodified T cells obtained from a donor. In some embodiments, the starting material is an iPSC cell line. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes other than human CD47 gene.

[0271] In some embodiments, the engineered and hypoimmunogenic cells described are derived from an iPSC or a progeny thereof. As used herein, the term "derived from an iPSC or a progeny thereof" encompasses the initial IPSC that is generated and any subsequent progeny thereof. As used herein, the term "progeny" encompasses, e.g., a first- generation progeny, i.e., the progeny is directly derived from, obtained from, obtainable from or derivable from the initial iPSC by, e.g., traditional propagation methods. The term "progeny" also encompasses further generations such as second, third, fourth, fifth, sixth, seventh, or more generations, i.e., generations of cells which are derived from, obtained from, obtainable from or derivable from the former generation by, e.g., traditional propagation methods. The term "progeny" also encompasses modified cells that result from the modification or alteration of the initial iPSC or a progeny thereof.

[0272] In some embodiments, knocking down (e.g., decreasing, eliminating, or inhibiting) gene expression can be achieved by RNA silencing or RNA interference (RNAi). Useful RNAi methods include those that utilize synthetic RNAi molecules, short interfering RNAs (siRNAs), PlWI-interacting NRAs (piRNAs), short hairpin RNAs (shRNAs), microRNAs (miRNAs), and other transient knock down methods recognized by those skilled in the art. Reagents for RNAi including sequence specific shRNAs, siRNA, miRNAs and the like are commercially available.

[0273] The term "engineered cell" as used herein refers to a cell that has been altered in at least some way by human intervention, including, for example, by genetic alterations or modifications such that the engineered cell differs from a wild-type cell.

[0274] The terms "decrease," "reduced," "reduction," and "decreased" are all used herein generally to mean a lowering by a statistically significant amount. However, for avoidance of doubt, "decrease," "reduced," "reduction," "decreased" means a lowering by at least 10% as compared to a reference level, for example a lowering by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% lowering (i.e. absent level as compared to a reference sample), or any lowering between 10-100% as compared to a reference level. In some embodiments, the cells are engineered to have reduced expression of one or more targets relative to an unaltered or unmodified wild-type cell.

[0275] In some embodiments, the provided modified cells are modified such that they are able to evade immune recognition and responses when administered to a patient (e.g., recipient subject). The cells can evade killing by immune cells in vitro and in vivo. In some embodiments, the cells evade killing by macrophages and NK cells. In some embodiments, the cells are ignored by immune cells or a subject's immune system. In other words, the cells administered in accordance with the methods described herein are not detectable by immune cells of the immune system. In some embodiments, the cells are cloaked and therefore avoid immune rejection.

[0276] Methods of determining whether a modified cell provided herein evades immune recognition include, but are not limited to, IFN-y Elispot assays, microglia killing assays, cell engraftment animal models, cytokine release assays, ELISAs, killing assays using bioluminescence imaging or chromium release assay or Xcelligence analysis, mixed- lymphocyte reactions, immunofluorescence analysis, etc.

[0277] In some embodiments, the immunogenicity of the cells is evaluated in a complement-dependent cytotoxicity (CDC) assay. CDC can be assayed in vitro by incubating cells with IgG or IgM antibodies targeting an HLA- independent antigen expressed on the cell surface in the presence of serum containing complement and analyzing cellkilling. In some embodiments, CDC can be assayed by incubating cells with ABO blood type incompatible serum, wherein the cells comprise A antigens or B antigens, and the serum comprises antibodies against the A antigens and / or B antigens of the cells.

[0278] In some embodiments, once the modified cells have been modified or generated as described herein, they may be assayed for their hypoimmunogenicity. Any of a variety of assays can be used to assess if the cells are hypoimmunogenic or can evade the immune system. Exemplary assays include any as is described in W02016183041 and WO2018132783.

[0279] In some embodiments, the modified cells described herein survive in a host without stimulating the host immune response for one week or more (e.g., one week, two weeks, one month, two months, three months, 6 months, one year, two years, three years, four years, five years or more, e.g., for the life of the cell and / or its progeny). The cells maintain expression of the transgenes and / or are deleted or reduced in expression of target genes for as long as they survive in the host. In some aspects, if the transgenes are no longer expressed and / or if target genes are expressed the modified cells may be removed by the host's immune system. In some embodiments, the persistence or survival of the modified cells may be monitored after their administration to a recipient by further expressing a transgene encoding a protein that allows the cells to be detected in vivo (e.g., a fluorescent protein, such as GFP, a truncated receptor or other surrogate marker or other detectable marker).

[0280] The hypoimmunogenic cells are administered in a manner that permits them to engraft to the intended tissue site and reconstitute or regenerate the functionally deficient area. In some embodiments, the hypoimmunogenic cells are assayed for engraftment (e.g., successful engraftment). In some embodiments, the engraftment of the hypoimmunogenic cells is evaluated after a pre-selected amount of time. In some embodiments, the engrafted cells are monitored for cell survival. For example, the cell survival may be monitored via bioluminescence imaging (BLI), wherein the cells are transduced with a luciferase expression construct for monitoring cell survival. In some embodiments, the engrafted cells are visualized by immunostaining and imaging methods known in the art. In some embodiments, the engrafted cells express known biomarkers that may be detected to determine successful engraftment. For example, flow cytometry may be used to determine the surface expression of particular biomarkers. In some embodiments, the hypoimmunogenic cells are engrafted to the intended tissue site as expected (e.g., successful engraftment of the hypoimmunogenic cells). In some embodiments, the hypoimmunogenic cells are engrafted to the intended tissue site as needed, such as at a site of cellular deficiency. In some embodiments, the hypoimmunogenic cells are engrafted to the intended tissue site in the same manner as a cell of the same type not comprising the modifications.

[0281] In some embodiments, administering the populations of modified cells (e.g., modified beta islet cells comprising modifications including reduced CD142 expression) or combinations (e.g., administering a population of modified cells in combination with an anti-coagulant agent) improves survival and engraftment by allowing cells to avoid or reduce IBMIR that occurs as a result of exposure of the cells to blood during transplant. In some embodiments, the reduction in IBMIR reduces the amount of cell loss (e.g., loss of transplanted islets) that occurs during transplant.

[0282] In some embodiments, the hypoimmunogenic cells are assayed for function. In some embodiments, the hypoimmunogenic cells are assayed for function prior to their engraftment to the intended tissue site. In some embodiments, the hypoimmunogenic cells are assayed for function following engraftment to the intended tissue site. In some embodiments, the function of the hypoimmunogenic cells is evaluated after a pre-selected amount. In some embodiments, the function of the engrafted cells is evaluated by the ability of the cells to produce a detectable phenotype. For example, engrafted beta islet cells function may be evaluated based on the restoration of lost glucose control due to diabetes. In some embodiments, the function of the hypoimmunogenic cells is as expected (e.g.,successful function of the hypoimmunogenic cells while avoiding antibody-mediated rejection). In some embodiments, the function of the hypoimmunogenic cells is as needed, such as sufficient function at a site of cellular deficiency while avoiding antibody-mediated rejection. In some embodiments, the modified cells function in the same manner as a nonmodified cell of the same type.

[0283] In some embodiments, genetically engineered cells of the present disclosure comprise an engineeredCD47 protein of the present disclosure, wherein the engineered CD47 protein exhibits: decreased binding with TSP-1 relative to a wild-type CD47 protein; and / or decreased CD47 / TSP-l-mediated signaling relative to a wild-type CD47 protein. In some embodiments, genetically engineered cells of the present disclosure comprise an engineered CD47 protein of the present disclosure and: exhibit decreased CD47 / TSP-l-mediated undesirable effects relative to a control cell; and / or exhibit decreased CD47 / TSP-l-mediated exhaustion relative to a control cell; and / or exhibit decreased CD47- mediated undesirable effects relative to a control cell; and / or exhibit increased CD47-mediated desirable effects relative to a control cell; and / or exhibit increased longevity relative to a control cell; and / or exhibit increased persistence relative to a control cell; and / or exhibit increased expansion relative to a control cell, and / or exhibit increased response to its intended therapeutic target, and / or exhibit increased hypoimmunity relative to a control cell; and / or exhibit increased immune evasion relative to a control cell; and / or exhibit increased ability to evade a host immune response relative to a control cell; and / or exhibit increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response relative to a control cell; and / or express the engineered CD47 protein at a first expression level, wherein a control cell expressing a wild-type CD47 protein at the first expression level would experience one or more unwanted effects. In some embodiments, a control cell comprises a wild-type cell, an unmodified cell, a cell that has not been engineered to express CD47 or a cell that has been engineered to express wild-type CD47. In some embodiments, a first expression level comprises 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.25x, 3.5x, 3.75x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, lOx, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx the level of wild-type CD47 protein in an unmodified cell. In some embodiments, CD47 / TSP-l-mediated signaling and / or CD47 / TSP-l-mediated exhaustion comprises autocrine signaling. In some embodiments, CD47 / TSP-l-mediated signaling and / or CD47 / TSP-l-mediated exhaustion comprises paracrine signaling. In some embodiments, CD47 / TSP-l-mediated signaling and / or CD47 / TSP-1- mediated exhaustion comprises both autocrine signaling and paracrine signaling. In some embodiments, increased longevity, increased persistence, increased expansion, increased response to its intended therapeutic target, increased immune evasion, increased ability to evade a host immune response, and / or increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response comprises reduced CD47 / TSP-l-mediated exhaustion via autocrine signaling. In some embodiments, increased longevity, increased persistence, increased expansion, increased response to its intended therapeutic target, increased immune evasion, increased ability to evade a host immune response, and / or increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response comprises reduced CD47 / TSP-1- mediated exhaustion via paracrine signaling. In some embodiments, increased longevity, increased persistence, increased expansion, increased response to its intended therapeutic target, increased immune evasion, increased ability to evade a host immune response, and / or increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response comprises reduced CD47 / TSP-l-mediated exhaustion via both autocrine signaling and paracrine signaling. In some embodiments, exhaustion comprises increased expression of PD1, LAG3, TIM3, CD39, CD101, or a combination thereof. In some embodiments, exhaustion comprises decreased or eliminated cytokine expression.

[0284] In some embodiments, genetically engineered cells of the present disclosure comprise an engineered CD47 protein of the present disclosure, wherein the engineered CD47 protein exhibits improved binding with SIRPa relative to a wild-type CD47 protein. In some embodiments, genetically engineered cells of the present disclosurecomprise an engineered CD47 protein of the present disclosure, wherein contacting a cell expressing SIRPa with a genetically engineered cell increases CD47 / SIRPa-mediated signaling in the cell expressing SIRPa relative to contacting the cell expressing SIRPa with a control cell. In some embodiments, genetically engineered cells of the present disclosure comprise an engineered CD47 protein of the present disclosure, wherein contacting a cell expressing SIRPa with a genetically engineered cell increases CD47 / SIRPa-mediated signaling in the cell expressing the engineered CD47 relative to contacting the cell expressing SIRPa with a control cell. In some embodiments, genetically engineered cells of the present disclosure comprise an engineered CD47 protein of the present disclosure, wherein contacting a cell expressing SIRPa with a genetically engineered cell increases CD47 / SIRPa-mediated signaling in both the cell expressing SIRPa and the cell expressing CD47 relative to contacting the cell expressing SIRPa with a control cell. In some embodiments, genetically engineered cells of the present disclosure comprise an engineered CD47 protein of the present disclosure, wherein the genetically engineered cells: exhibit decreased CD47-mediated undesirable effects relative to a control cell; and / or exhibit increased CD47-mediated desirable effects relative to a control cell; and / or exhibit increased longevity relative to a control cell; and / or exhibit increased persistence relative to a control cell; and / or exhibit increased expansion relative to a control cell; and / or exhibit increased response to its intended therapeutic target, and / or exhibit increased hypoimmunity relative to a control cell; and / or exhibit improved immune evasion relative to a control cell; and / or exhibit increased ability to evade a host immune response relative to a control cell; and / or exhibit increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response relative to a control cell; and / or express the engineered CD47 protein at a first expression level, wherein a control cell expressing a wild-type CD47 protein at the first expression level would be killed by one or more immune cells. In some embodiments, a control cell comprises a wildtype cell, an unmodified cell, a cell that has not been engineered to express CD47 or a cell that has been engineered to express wild-type CD47. In some embodiments, a first expression level comprises 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.25x, 3.5x, 3.75x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, lOx, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx the level of wild-type CD47 protein in an unmodified cell.

[0285] In some embodiments, genetically engineered cells of the present disclosure comprise an engineered CD47 protein of the present disclosure, wherein the engineered CD47 protein exhibits: decreased binding with TSP-1 relative to a wild-type CD47 protein; decreased CD47 / TSP-l-mediated signaling relative to a wild-type CD47 protein; and improved binding with SIRPa relative to a wild-type CD47 protein; and wherein the genetically engineered cells: exhibit decreased CD47 / TSP-l-mediated undesirable effects relative to a control cell; exhibit decreased CD47-mediated undesirable effects relative to a control cell; exhibit decreased CD47 / TSP-l-mediated exhaustion relative to a control cell; exhibit decreased CD47-mediated undesirable effects relative to a control cell; exhibit increased CD47-mediated desirable effects relative to a control cell; exhibit increased longevity relative to a control cell; exhibits increased persistence relative to a control cell; exhibit increased expansion relative to a control cell; exhibit increased response to its intended therapeutic target; exhibit increased hypoimmunity relative to a control cell; exhibit increased immune evasion relative to a control cell; exhibit increased ability to evade a host immune response relative to a control cell; exhibit increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response relative to a control cell; express the engineered CD47 protein at a first expression level, wherein a control cell expressing a wild-type CD47 protein at the first expression level would experience one or more unwanted effects and would be killed by one or more immune cells; and wherein contacting a cell expressing SIRPa with the genetically engineered cell increases CD47 / SIRPa-mediated signaling in both the cell expressing SIRPa and the cell expressing CD47 relative to contacting the cell expressing SIRPa with a control cell. In some embodiments, a control cell comprises a wild-type cell, an unmodified cell, a cell that has not been engineered to express CD47 or a cell that has been engineered to express wild-type CD47.In some embodiments, a first expression level comprises 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.25x, 3.5x, 3.75x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x, 9.5x, lOx, 15x, 20x, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx the level of wild-type CD47 protein in an unmodified cell. In some embodiments, CD47 / TSP-l-mediated signaling and / or CD47 / TSP-l-mediated exhaustion comprises autocrine signaling. In some embodiments, CD47 / TSP-l-mediated signaling and / or CD47 / TSP-l-mediated exhaustion comprises paracrine signaling. In some embodiments, CD47 / TSP-1- mediated signaling and / or CD47 / TSP-l-mediated exhaustion comprises both autocrine signaling and paracrine signaling. In some embodiments, increased longevity, increased persistence, increased expansion, increased response to its intended therapeutic target, increased immune evasion, increased ability to evade a host immune response, and / or increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response comprises reduced CD47 / TSP-1- mediated exhaustion via autocrine signaling. In some embodiments, increased longevity, increased persistence, increased expansion, increased response to its intended therapeutic target, increased immune evasion, increased ability to evade a host immune response, and / or increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response comprises reduced CD47 / TSP-l-mediated exhaustion via paracrine signaling. In some embodiments, increased longevity, increased persistence, increased expansion, increased response to its intended therapeutic target, increased immune evasion, increased ability to evade a host immune response, and / or increased ability to evade a natural killer (NK) cell, monocyte, or macrophage response comprises reduced CD47 / TSP-l-mediated exhaustion via both autocrine signaling and paracrine signaling. In some embodiments, exhaustion comprises increased expression of PD1, LAG3, TIM3, CD39, CD101, or a combination thereof. In some embodiments, exhaustion comprises decreased or eliminated cytokine expression.Instant blood-mediated inflammatory reaction

[0286] In some embodiments, the modified cells provided herein evade an instant blood-mediated inflammatory reaction. A major contributor to the poor outcome of clinical islet transplantation is the occurrence of the destructive instant blood mediated inflammatory reaction (IBMIR), which leads to loss of transplanted tissue when the islets encounter the blood in the portal vein (Bennet et al., (1995) Diabetes 48: 1907-1914; Moberg et al., (2002) Lancet 360:2039-2045). This reaction is triggered by tissue factor (TF) expression by the endocrine cells of the islets, combined with an array of other proinflammatory events, such as the expression of MCP-1 (Piemonti et al., (2002) Diabetes 51:55- 65), IL-8, and MIF (Waeber et al., (1997) Proc Natl Acad Sci USA 94:4782-4787; Johansson et al., (2006) Am J Transplantation 6(2):305).

[0287] Instant blood-mediated inflammatory reaction (IBMIR) is a nonspecific inflammatory and thrombotic reaction that can occur when cells expressing CD142 come into contact with blood. IBMIR is initiated rapidly by exposure to human blood in the portal vein. It is characterized by activation of complement, platelets, and the coagulation pathway, which in turn leads to the recruitment of neutrophils. IBMIR causes significant loss of transplanted islets. In some embodiments, provided herein are compositions (e.g., modified cells comprising reduced expression of CD142 in combination with one or more of the other modifications described herein), combinations (e.g., a combination comprising any of the populations of modified cells described herein and an anti-coagulant agent that reduces coagulation), and methods (e.g., methods of treating a patient comprising administering any of the populations of modified cells described herein and anti-coagulant agent that reduces coagulation) that reduce an IBMIR associated with transplantation of the cells or exposure of the cells to blood.

[0288] In some embodiments, IBMIR can be assayed in vitro, for example, in an in vitro tubing loop model of IBMIR, which has been previously described in U.S. Pat. No. 7,045,502, which is herein incorporated by reference in its entirety.

[0289] In some embodiments, IBMIR can be assayed in vivo (e.g., in a mammal or in a human patient) by drawing blood samples during the peritransplant period and evaluating plasma levels of thrombin-anti-thrombin III complex (TAT), C-peptide, factor XIa-antithrombin (FXIa-AT), factor Xlla-antithrombin (FXIIa-AT), thrombinantithrombin (TAT) plasmin-alpha 2 antiplasmin (PAP), and / or complement C3a. sin some embodiments, IBMIR is associated with increased levels of TAT, C-peptide, FXIa-AT, FXIIa-AT, PAP, and / or complement C3a during infusion of transplanted cells and / or in a period of time following transplant (e.g., up to 3, 5, 10, or more than 10 hours after transplant). In some embodiments, IBMIR can be assayed by monitoring counts of free circulating platelets, wherein a decrease in the counts of platelets during or following transplantation is associated with IBMIR (e.g., with platelet consumption due to IBMIR).Complement-dependent cytotoxicity

[0290] In some embodiments, the modified cells (e.g., beta islets) provided herein evade complement dependent cytotoxicity (CDC). In some embodiments, the CDC is secondary to a thrombotic reaction of IBMIR. In some embodiments, the CDC occurs independently of IBMIR.

[0291] In some embodiments, susceptibility of cells to CDC can be analyzed in vitro according to standard protocols understood by one of ordinary skill in the art. In some embodiments, CDC can be analyzed in vitro by mixing serum comprising the components of the complement system (e.g., human serum), with target cells bound by an antibody (e.g., an IgG or IgM antibody), and then to determine cell death. In some embodiments, susceptibility of cells to CDC can be analyzed in vitro by incubating cells in the presence of ABO-incompatible or Rh factor incompatible serum, comprising the components of the complement system and antibodies against ABO type A, ABO type B, and / or Rh factor antigens of the cells.

[0292] A common CDC assay determines cell death via pre-loading the target cells with a radioactive compound. As cells die, the radioactive compound is released from them. Hence, the efficacy of the antibody to mediate cell death is determined by the radioactivity level. Unlike radioactive CDC assays, non-radioactive CDC assays often determine the release of abundant cell components, such as GAPDH, with fluorescent or luminescent determination. In some embodiments, cell killing by CDC can be analyzed using a label-free platform such as xCELLigence™ (Agilent).VI. Compositions

[0293] In another aspect, the present disclosure provides a composition comprising the engineered CD47 protein disclosed herein.

[0294] In another aspect, the present disclosure provides a composition comprising the cell that comprises a polynucleotide encoding the engineered CD47 protein disclosed herein, and / or a vector comprising the polynucleotide.

[0295] As used herein, the term "composition" includes, but is not limited to, a pharmaceutical composition. A "pharmaceutical composition" refers to an active pharmaceutical agent formulated in pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or an animal, either alone, or in combination with one or more other modalities of therapy. It will also be understood that, if desired, the compositions of the invention may be administered in combination with other agents, such as, e.g., cytokines, growth factors, hormones, small molecules, chemotherapeutics, pro-drugs, drugs, antibodies, or other various pharmaceutically active agents. There is virtually no limit to other components that may also be included in the compositions, provided that the additional agents do not adversely affect the ability of the composition to deliver the intended therapy. The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope ofsound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0296] The compositions may also comprise a pharmaceutically acceptable carrier, diluent, or excipient. As used herein "pharmaceutically acceptable carrier, diluent, or excipient" includes, without limitation, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surfactant, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, to sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth; malt; gelatin; talc; cocoa butter; waxes; animal and vegetable fats; paraffins; silicones; bentonites; silicic acid; zinc oxide; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate, and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other compatible substances employed in pharmaceutical formulations.

[0297] The liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline; Ringers solution; isotonic sodium chloride; fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium; polyethylene glycols; glycerin; propylene glycol or other solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity, such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile.

[0298] The composition may be suitably developed for intravenous, intratumoral, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic, or another route of administration.VII. Methods of gene editing

[0299] In some embodiments, the methods for genetically modifying cells to knock out, knock down, or otherwise modify one or more genes comprise using a site-directed nuclease, including, for example, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, transposases, and clustered regularly interspaced short palindromic repeat (CRISPR) / Cas systems, as well as nickase systems, base editing systems, prime editing systems, and any other gene editing systems known in the art.

[0300] ZFNs are fusion proteins comprising an array of site-specific DNA binding domains adapted from zinc finger-containing transcription factors attached to the endonuclease domain of the bacterial FokI restriction enzyme. A ZFN may have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) DNA binding domains or zinc finger domains. See, e.g., Carroll782; Kim etai., Proc. Natl Acad. Sci. USA (1996)93: 1156-1160. The individual DNA binding domains are typically referred to as "fingers" or "zinc fingers." Each zinc finger domain is a small protein structural motif stabilized by one or more zinc ions and usually recognizes a 3- to 4-bpDNA sequence. Tandem domains can thus potentially bind to an extended nucleotide sequence that is unique within a cell's genome. Each finger binds from two to four base pairs of DNA, typically three or four base pairs of DNA. A DNA binding domain binds to a nucleic acid sequence called a target site or target segment. Each finger typically comprises an approximately 30 amino acid, zinc-chelating, DNA-binding subdomain. Studies have demonstrated that a single zinc finger of this class consists of an alpha helix containing the two invariant histidine residues coordinated with zinc along with the two cysteine residues of a single beta turn (see, e.g., Berg & Shi, Science 271: 1081-1085 (1996)).

[0301] Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15, or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one- hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Sera et al., Biochemistry (2002) 41:7074-7081; Liu eta / ., Bioinformatics (2008) 24: 1850-1857.

[0302] ZFNs containing FokI nuclease domains or other dimeric nuclease domains function as a dimer. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. See Bitinaite et ai., Proc. Natl. Acad. Sei. USA (1998) 95: 10570- 10575. To cleave a specific site in the genome, a pair of ZFNs are designed to recognize two sequences flanking the site, one on the forward strand and the other on the reverse strand. Upon binding of the ZFNs on either side of the site, the nuclease domains dimerize and cleave the DNA at the site, generating a DSB with 5' overhangs. HDR can then be utilized to introduce a specific mutation, with the help of a repair template containing the desired mutation flanked by homology arms. The repair template is usually an exogenous double-stranded DNA vector introduced to the cell. See Miller et ai., Nat. Biotechnoi. (2011) 29: 143-148; Hockemeyer et ai., Nat. Biotechnoi. (2011) 29:731-734.

[0303] TALENs are another example of an artificial nuclease which can be used to edit a target gene. A"TALE-nuclease" (TALEN) is a fusion protein consisting of a nucleic acid-binding domain typically derived from a Transcription Activator Like Effector (TALE) and one nuclease catalytic domain to cleave a nucleic acid target sequence. The catalytic domain is preferably a nuclease domain and more preferably a domain having endonuclease activity, for instance I-TevI, ColE7, NucA and Fok-I. In numerous embodiments, the TALE domain can be fused to a meganuclease, for instance I-Crel and I-Onul or functional variant thereof. In a more preferred embodiment, said nuclease is a monomeric TALE-Nuclease. A monomeric TALE-Nuclease is a TALE-Nuclease that does not require dimerization for specific recognition and cleavage, such as the fusions of engineered TAL repeats with the catalytic domain of I-TevI described in WO2012138927.

[0304] Transcription Activator like Effector (TALE) are proteins from the bacterial species Xanthomonas and comprise a plurality of repeated sequences. Each repeat is 33 to 35 amino acids in length, with two adjacent amino acids (termed the repeat-variable di-residue, or RVD) in position 12 and 13 that are specific to each nucleotide base of the nucleic acid targeted sequence. Binding domains with similar modular base-per-base nucleic acid binding properties (MBBBD) can also be derived from new modular proteins recently discovered in a different bacterial species. The new modular proteins have the advantage of displaying more sequence variability than TAL repeats. Preferably, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW forrecognizing A. In another embodiment, critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. TALEN kits are sold commercially.

[0305] TALENs are produced artificially by fusing one or more TALE DNA binding domains (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) to a nuclease domain, for example, a FokI endonuclease domain. See Zhang, Nature Biotech. (2011) 29: 149-153. Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. See Cermak et ai., Nud. Acids Res. (2011) 39:e82; Miller et ai., Nature Biotech. (2011) 29: 143-148; Hockemeyer etai., Nature Biotech. (2011) 29:731-734; Wood etai., Science (2011) 333:307; Doyon etai., Nature Methods (2010) 8:74-79; Szczepek et ai., Nature Biotech (2007) 25:786-793; Guo etai., J. Moi. Biol (2010) 200:96. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI nuclease domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et ai., Nature Biotech. (2011) 29: 143-148.

[0306] By combining engineered TALE repeats with a nuclease domain, a site-specific nuclease can be produced specific to any desired DNA sequence. Similar to ZFNs, TALENs can be introduced into a cell to generate DSBs at a desired target site in the genome, and so can be used to knock out genes or knock in mutations in similar, HDR- mediated pathways. See Boch, Nature Biotech. (2011) 29: 135-136; Boch etai., Science (2009) 326: 1509-1512; Moscou etai., Science (2009) 326:3501.

[0307] Meganucleases are sequence-specific enzymes in the endonuclease family which are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs). (Chevalier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774). They can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al., Nucleic Acids Res., 1993, 21, 5034- 5040; Rouet et aL, Mol. Cell. BioL, 1994, 14, 8096-8106; Choulika et aL, Mol. Cell. Biol., 1995, 15, 1968-1973; Puchta et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 5055-5060; Sargent et aL, Mol. Cell. Biol., 1997, 17, 267-77; Donoho et al., Mol. Cell. Biol, 1998, 18, 4070-4078; Elliott et al., Mol. Cell. Biol., 1998, 18, 93-101; Cohen-Tannoudji et al., Mol. Cell. BioL, 1998, 18, 1444-1448).

[0308] Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and / or DNA recognition. The most widespread and best known meganucleases are the proteins in the LAGLIDADG family, which owe their name to a conserved amino acid sequence. See Chevalier etai., Nucleic Acids Res. (2001) 29(18): 3757-3774. On the other hand, the GIY-YIG family members have a GIY-YIG module, which is 70-100 residues long and includes four or five conserved sequence motifs with four invariant residues, two of which are required for activity. See Van Roey etai., Nature Struct. Biol (2002) 9:806-811. The His-Cys family meganucleases are characterized by a highly conserved series of histidines and cysteines over a region encompassing several hundred amino acid residues. See Chevalier et ai., Nucleic Acids Res. (2001) 29(18):3757-3774. Members of the NHN family are defined by motifs containing two pairs of conserved histidines surrounded by asparagine residues. See Chevalier et ai., Nucleic Acids Res. (2001) 29(18):3757-3774.

[0309] Because the chance of identifying a natural meganuclease for a particular target DNA sequence is low due to the high specificity requirement, various methods including mutagenesis and high throughput screening methods have been used to create meganuclease variants that recognize unique sequences. Strategies for engineering a meganuclease with altered DNA-binding specificity, e.g, to bind to a predetermined nucleic acid sequence are known in the art. See, e.g., Chevalier etai, Moi. Ceil (2002) 10:895-905; Epinat et ai., Nucleic Acids Res (2003) 31:2952-2962;Silva et al., J Mol. Biol. (2006) 361:744-754; Seligman eta!., Nucleic Acids Res (2002) 30:3870-3879; Sussman etal, J Mol Biol (2004) 342:31-41; Doyon et al., J Am Chem Soc (2006) 128:2477-2484; Chen et al., Protein Eng Des Sei (2009) 22:249-256; Arnould etal., J Mol Biol. (2006) 355:443-458; Smith et al., Nucleic Acids Res. (2006) 363(2): 283-294.

[0310] Like ZFNs and TALENs, Meganucleases can create DSBs in the genomic DNA, which can create a frame-shift mutation if improperly repaired, e.g., via NHEJ, leading to a decrease in the expression of a target gene in a cell. Alternatively, foreign DNA can be introduced into the cell along with the meganuclease. Depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify the target gene. See Silva et al., Current Gene Therapy (2011) 11: 11-27.

[0311] Transposases are enzymes that bind to the end of a transposon and catalyze its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. By linking transposases to other systems such as the CRISPER / Cas system, new gene editing tools can be developed to enable site specific insertions or manipulations of the genomic DNA. There are two known DNA integration methods using transposons which use a catalytically inactive Cas effector protein and Tn7-like transposons. The transposase-dependent DNA integration does not provoke DSBs in the genome, which may guarantee safer and more specific DNA integration.

[0312] The CRISPR system was originally discovered in prokaryotic organisms (e.g., bacteria and archaea) as a system involved in defense against invading phages and plasmids that provides a form of acquired immunity. Now it has been adapted and used as a popular gene editing tool in research and clinical applications.

[0313] CRISPR / Cas systems generally comprise at least two components: one or more guide RNAs (gRNAs) and a Cas protein. The Cas protein is a nuclease that introduces a DSB into the target site. CRISPR-Cas systems fall into two major classes: class 1 systems use a complex of multiple Cas proteins to degrade nucleic acids; class 2 systems use a single large Cas protein for the same purpose. Class 1 is divided into types I, III, and IV; class 2 is divided into types II, V, and VI. Different Cas proteins adapted for gene editing applications include, but are not limited to, Cas3, Cas4, Cas5, Cas8a, Cas8b, Cas8c, Cas9, Casio, Casl2, Casl2a (Cpfl), Casl2b (C2cl), Casl2c (C2c3), Casl2d (CasY), Casl2e (CasX), Casl2f (C2cl0), Casl2g, Casl2h, Casl2i, Casl2k (C2c5), Casl3, Casl3a (C2c2), Casl3b, Casl3c, Casl3d, C2c4, C2c8, C2c9, Cmr5, Csel, Cse2, Csfl, Csm2, Csn2, CsxlO, Csxll, Csyl, Csy2, Csy3, and Mad7. The most widely used Cas9 is described herein as illustrative. These Cas proteins may be originated from different source species. For example, Cas9 can be derived from S. pyogenes or S. aureus.

[0314] In the original microbial genome, the type II CRISPR system incorporates sequences from invadingDNA between CRISPR repeat sequences encoded as arrays within the host genome. Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs) each harboring a variable sequence transcribed from the invading DNA, known as the "protospacer" sequence, as well as part of the CRISPR repeat. Each crRNA hybridizes with a second transactivating CRISPR RNA (tracrRNA), and these two RNAs form a complex with the Cas9 nuclease. The protospacer- encoded portion of the crRNA directs the Cas9 complex to cleave complementary target DNA sequences, provided that they are adjacent to short sequences known as "protospacer adjacent motifs" (PAMs).

[0315] Since its discovery, the CRISPR system has been adapted for inducing sequence specific DSBs and targeted genome editing in a wide range of cells and organisms spanning from bacteria to eukaryotic cells including human cells. In its use in gene editing applications, artificially designed, synthetic gRNAs have replaced the original crRNA:tracrRNA complex. For example, the gRNAs can be single guide RNAs (sgRNAs) composed of a crRNA, a tetraloop, and a tracrRNA. The crRNA usually comprises a complementary region (also called a spacer, usually about 20 nucleotides in length) that is user-designed to recognize a target DNA of interest. The tracrRNA sequence comprises a scaffold region for Cas nuclease binding. The crRNA sequence and the tracrRNA sequence are linked by the tetraloopand each have a short repeat sequence for hybridization with each other, thus generating a chimeric sgRNA. One can change the genomic target of the Cas nuclease by simply changing the spacer or complementary region sequence present in the gRNA. The complementary region will direct the Cas nuclease to the target DNA site through standard RNA-DNA complementary base pairing rules.

[0316] In order for the Cas nuclease to function, there must be a PAM immediately downstream of the target sequence in the genomic DNA. Recognition of the PAM by the Cas protein is thought to destabilize the adjacent genomic sequence, allowing interrogation of the sequence by the gRNA and resulting in gRNA-DNA pairing when a matching sequence is present. The specific sequence of PAM varies depending on the species of the Cas gene. For example, the most commonly used Cas9 nuclease derived from S. pyogenes recognizes a PAM sequence of 5'-NGG-3' or, at less efficient rates, 5'-NAG-3', where "N" can be any nucleotide. Other Cas nuclease variants with alternative PAMs have also been characterized and successfully used for genome editing, which are summarized in Table 3 below.Table 3. Exemplary Cas nuclease variants and their PAM sequencesR = A or G; Y = C or T; W = A or T; V = A or C or G; N = any base

[0317] In some embodiments, Cas nucleases may comprise one or more mutations to alter their activity, specificity, recognition, and / or other characteristics. For example, the Cas nuclease may have one or more mutations that alter its fidelity to mitigate off-target effects (e.g., eSpCas9, SpCas9-HFl, HypaSpCas9, HeFSpCas9, and evoSpCas9 high-fidelity variants of SpCas9). For another example, the Cas nuclease may have one or more mutations that alter its PAM specificity.

[0318] Nuclease domains of the Cas, in particular the Cas9, nuclease can be mutated independently to generate enzymes referred to as DNA "nickases". Nickases are capable of introducing a single-strand cut with the same specificity as a regular CRISPR / Cas nuclease system, including for example CRISPR / Cas9. Nickases can be employed to generate double-strand breaks which can find use in gene editing systems (Mali eta!., Nat Biotech, 31(9):833-838 (2013); Mali etai. Nature Methods, 10:957-963 (2013); Mali et al., Science, 339(6121) :823-826 (2013)). In some instances, when two Cas nickases are used, long overhangs are produced on each of the cleaved ends instead of blunt ends which allows for additional control over precise gene integration and insertion (Mali etal., Nat Biotech, 31(9):833- 838 (2013); Mali et ai. Nature Methods, 10:957-963 (2013); Mali et ai., Science, 339(6121):823-826 (2013)). As both nicking Cas enzymes must effectively nick their target DNA, paired nickases can have lower off-target effects compared to the double-strand-cleaving Cas-based systems (Ran et ai., Cell, 155(2):479-480(2013); Mali etal., Nat Biotech, 31(9):833-838 (2013); Mali et al. Nature Methods, 10:957-963 (2013); Mali eta!., Science, 339(6121):823-826 (2013)).

[0319] The molecular machinery of such Cas proteins that allows the CRISPR / Cas system to alter target polynucleotide sequences in cells include RNA binding proteins, endo- and exo-nucleases, helicases, and polymerases. In some embodiments, the CRISPR / Cas system includes a Cas protein and at least one to two ribonucleic acids that arecapable of directing the Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. As used herein, "protein" and "polypeptide" are used interchangeably to refer to a series of amino acid residues joined by peptide bonds (i.e., a polymer of amino acids) and include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments and other equivalents, variants, and analogs of the above.

[0320] In some embodiments, a Cas protein comprises one or more amino acid substitutions or modifications.In some embodiments, the one or more amino acid substitutions comprises a conservative amino acid substitution. In some instances, substitutions and / or modifications can prevent or reduce proteolytic degradation and / or extend the halflife of the polypeptide in a cell. In some embodiments, the Cas protein can comprise a peptide bond replacement (e.g., urea, thiourea, carbamate, sulfonyl urea, etc.). In some embodiments, the Cas protein can comprise a naturally occurring amino acid. In some embodiments, the Cas protein can comprise an alternative amino acid (e.g., D-amino acids, beta-amino acids, homocysteine, phosphoserine, etc.). In some embodiments, a Cas protein can comprise a modification to include a moiety (e.g., PEGylation, glycosylation, lipidation, acetylation, end-capping, etc.).

[0321] In some embodiments, a Cas protein comprises a core Cas protein, isoform thereof, or any Cas-like protein with similar function or activity of any Cas protein or isoform thereof. In some embodiments, a Cas protein comprises a core Cas protein. Exemplary Cas core proteins include, but are not limited to Cast, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8 and Cas9. In some embodiments, a Cas protein comprises type V Cas protein. In some embodiments, a Cas protein comprises a Cas protein of an E. coli subtype (also known as CASS2). Exemplary Cas proteins of the E. Coli subtype include, but are not limited to Csel, Cse2, Cse3, Cse4, and Cas5e. In some embodiments, a Cas protein comprises a Cas protein of the Ypest subtype (also known as CASS3). Exemplary Cas proteins of the Ypest subtype include, but are not limited to Csyl, Csy2, Csy3, and Csy4. In some embodiments, a Cas protein comprises a Cas protein of the Nmeni subtype (also known as CASS4). Exemplary Cas proteins of the Nmeni subtype include, but are not limited to Csnl and Csn2. In some embodiments, a Cas protein comprises a Cas protein of the Dvulg subtype (also known as CASS1). Exemplary Cas proteins of the Dvulg subtype include Csdl, Csd2, and Cas5d. In some embodiments, a Cas protein comprises a Cas protein of the Tneap subtype (also known as CASS7). Exemplary Cas proteins of the Tneap subtype include, but are not limited to, Cstl, Cst2, Cas5t. In some embodiments, a Cas protein comprises a Cas protein of the Hmari subtype. Exemplary Cas proteins of the Hmari subtype include, but are not limited to Cshl, Csh2, and Cas5h. In some embodiments, a Cas protein comprises a Cas protein of the Apern subtype (also known as CASS5).Exemplary Cas proteins of the Apern subtype include, but are not limited to Csal, Csa2, Csa3, Csa4, Csa5, and Cas5a. In some embodiments, a Cas protein comprises a Cas protein of the Mtube subtype (also known as CASS6). Exemplary Cas proteins of the Mtube subtype include, but are not limited to Csml, Csm2, Csm3, Csm4, and Csm5. In some embodiments, a Cas protein comprises a RAMP module Cas protein. Exemplary RAMP module Cas proteins include, but are not limited to, Cmrl, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6. See, e.g., Klompe et al., Nature 571, 219-225 (2019); Strecker et al., Science 365, 48-53 (2019). Examples of Cas proteins include, but are not limited to: Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and / or GSU0054. In some embodiments, a Cas protein comprises Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and / or GSU0054. Examples of Cas proteins include, but are not limited to: Cas9, Csn2, and / or Cas4. In some embodiments, a Cas protein comprises Cas9, Csn2, and / or Cas4. In some embodiments, Examples of Cas proteins include, but are not limited to: Casio, Csm2, Cmr5, Casio, Csxll, and / or CsxlO. In some embodiments, a Cas protein comprises a Casio, Csm2, Cmr5, Casio, Csxll, and / or CsxlO. In some embodiments, examples of Cas proteins include, but are not limited to: Csfl. In some embodiments, a Cas protein comprises Csfl. In some embodiments, examples of Cas proteins include, but are not limitedto: Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, and C2c9; as well as CasX (Casl2e) and CasY (Casl2d). Also see, e.g., Koonin et al., Curr Opin Microbiol. 2017; 37:67-78: "Diversity, classification and evolution of CRISPR-Cas systems." In some embodiments, a Cas protein comprises Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl2d, and / or Casl2e. In some embodiments, a Cas protein comprises Casl3, Casl3a, C2c2, Casl3b, Casl3c, and / or Casl3d. In some embodiments, the CRISPR / Cas system comprises a Cas effector protein selected from the group consisting of: a) Cas3, Cas8a, Cas5, Cas8b, Cas8c, CaslOd, Csel, Cse2, Csyl, Csy2, Csy3, and GSU0054; b) Cas9, Csn2, and Cas4; c) Casio, Csm2, Cmr5, CaslO, Csxll, and CsxlO; d) Csfl; e) Casl2a, Casl2b, Casl2c, C2c4, C2c8, C2c5, C2cl0, C2c9, CasX (Casl2e), and CasY (Casl2d); and f) Casl3, Casl3a, C2c2, Casl3b, Casl3c, and Casl3d.

[0322] In some embodiments, a Cas protein comprises any one of the Cas proteins described herein or a functional portion thereof. As used herein, "functional portion" refers to a portion of a peptide which retains its ability to complex with at least one ribonucleic acid {e.g., guide RNA (gRNA)) and cleave a target polynucleotide sequence. In some embodiments, the functional portion comprises a combination of operably linked Cas9 protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional portion comprises a combination of operably linked Casl2a (also known as Cpfl) protein functional domains selected from the group consisting of a DNA binding domain, at least one RNA binding domain, a helicase domain, and an endonuclease domain. In some embodiments, the functional domains form a complex. In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of a RuvC-like domain. In some embodiments, a functional portion of the Cas9 protein comprises a functional portion of the HNH nuclease domain. In some embodiments, a functional portion of the Casl2a protein comprises a functional portion of a RuvC-like domain.

[0323] In some embodiments, the exogenous Cas protein can be introduced into the cell in polypeptide form.In certain embodiments, the Cas proteins can be conjugated to or fused to a cell-penetrating polypeptide or cellpenetrating peptide. As used herein, "cell-penetrating polypeptide" and "cell-penetrating peptide" refers to a polypeptide or peptide, respectively, which facilitates the uptake of molecule into a cell. The cell-penetrating polypeptides can contain a detectable label.

[0324] In many embodiments, Cas proteins can be conjugated to or fused to a charged protein {e.g., that carries a positive, negative or overall neutral electric charge). Such linkage may be covalent. In some embodiments, the Cas protein can be fused to a superpositively charged GFP to significantly increase the ability of the Cas protein to penetrate a cell (Cronican et al. ACS Chem Biol. 2010; 5(8): 747-52). In certain embodiments, the Cas protein can be fused to a protein transduction domain (PTD) to facilitate its entry into a cell. Exemplary PTDs include Tat, oligoarginine, and penetratin. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a cell-penetrating peptide. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a PTD. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a tat domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to an oligoarginine domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a penetratin domain. In some embodiments, the Cas9 protein comprises a Cas9 polypeptide fused to a superpositively charged GFP. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a cell-penetrating peptide. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a PTD. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a tat domain. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to an oligoarginine domain. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a penetratin domain. In some embodiments, the Casl2a protein comprises a Casl2a polypeptide fused to a superpositively charged GFP.

[0325] In some embodiments, the Cas protein can be introduced into a cell containing the target polynucleotide sequence in the form of a nucleic acid encoding the Cas protein. The process of introducing the nucleic acids into cells can be achieved by any suitable technique. Suitable techniques include those described herein.

[0326] In some embodiments, the Cas protein is complexed with one to two ribonucleic acids. In some embodiments, the Cas protein is complexed with two ribonucleic acids. In some embodiments, the Cas protein is complexed with one ribonucleic acid. In some embodiments, the Cas protein is encoded by a modified nucleic acid, as described herein (e.g., a synthetic, modified mRNA).

[0327] The methods of the present disclosure contemplate the use of any ribonucleic acid that is capable of directing a Cas protein to and hybridizing to a target motif of a target polynucleotide sequence. In some embodiments, at least one of the ribonucleic acids comprises tracrRNA. In some embodiments, at least one of the ribonucleic acids comprises CRISPR RNA (crRNA). In some embodiments, a single ribonucleic acid comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. In some embodiments, at least one of the ribonucleic acids comprises a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. In some embodiments, both of the one to two ribonucleic acids comprise a guide RNA that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell. The ribonucleic acids of the present disclosure can be selected to hybridize to a variety of different target motifs, depending on the particular CRISPR / Cas system employed, and the sequence of the target polynucleotide, as will be appreciated by those skilled in the art. The one to two ribonucleic acids can also be selected to minimize hybridization with nucleic acid sequences other than the target polynucleotide sequence. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least two mismatches when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids hybridize to a target motif that contains at least one mismatch when compared with all other genomic nucleotide sequences in the cell. In some embodiments, the one to two ribonucleic acids are designed to hybridize to a target motif immediately adjacent to a deoxyribonucleic acid motif recognized by the Cas protein. In some embodiments, each of the one to two ribonucleic acids are designed to hybridize to target motifs immediately adjacent to deoxyribonucleic acid motifs recognized by the Cas protein which flank a mutant allele located between the target motifs.

[0328] In some embodiments, each of the one to two ribonucleic acids comprises guide RNAs that directs the Cas protein to and hybridizes to a target motif of the target polynucleotide sequence in a cell.

[0329] In some embodiments, one or two ribonucleic acids (e.g., guide RNAs) are complementary to and / or hybridize to sequences on the same strand of a target polynucleotide sequence. In some embodiments, one or two ribonucleic acids {e.g., guide RNAs) are complementary to and / or hybridize to sequences on the opposite strands of a target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids {e.g., guide RNAs) are not complementary to and / or do not hybridize to sequences on the opposite strands of a target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids {e.g., guide RNAs) are complementary to and / or hybridize to overlapping target motifs of a target polynucleotide sequence. In some embodiments, the one or two ribonucleic acids {e.g., guide RNAs) are complementary to and / or hybridize to offset target motifs of a target polynucleotide sequence.VIII. Methods of expressing engineered CD47 proteins

[0330] The engineered CD47 protein provided herein can be produced by any method known to those of skill in the art including in vivo and in vitro methods. Desired proteins can be expressed in any organism suitable to producethe required amounts and forms of the proteins. Expression hosts include prokaryotic and eukaryotic organisms such as f. coli, yeast, plants, insect cells, mammalian cells, including human cell lines and transgenic animals. Expression hosts can differ in their protein production levels as well as the types of post-translational modification that are present on the expressed proteins. The choice of expression host can be made based on these and other factors, such as regulatory and safety considerations, production costs and the need and methods for purification.

[0331] In some embodiments, introducing the polynucleotides encoding the engineered CD47 protein described herein into cells can be achieved by any suitable technique. Suitable techniques include calcium phosphate, lipid-mediated transfection, electroporation, fusogens, and transduction or infection using a viral vector, as discussed herein. In some embodiments, the polynucleotides are introduced into a cell via viral transduction (e.g., AAV transduction, lentiviral transduction) or otherwise delivered on a viral vector (e.g., fusogen-mediated delivery). In some embodiments, the polynucleotides are introduced into a cell via a fusogen-mediated delivery or a transposase system selected from the group consisting of conditional or inducible transposases, conditional or inducible PiggyBac transposons, conditional or inducible Sleeping Beauty (SB11) transposons, conditional or inducible Most transposons, and conditional or inducible Tol2 transposons.

[0332] Many expression vectors are available and known to those of skill in the art and can be used for expression of proteins. The choice of expression vector will be influenced by the choice of host expression system. In general, expression vectors can include transcriptional promoters and optionally enhancers, translational signals, and transcriptional and translational termination signals. Expression vectors that are used for stable transformation typically have a selectable marker which allows selection and maintenance of the transformed cells. In some cases, an origin of replication can be used to amplify the copy number of the vector.

[0333] Expression vectors can be introduced into host cells via, for example, transformation, transfection, transduction, infection, electroporation, and sonoporation. A skilled artisan is able to select methods and conditions suitable for introducing an expression vector into host cells.

[0334] In some embodiments, the engineered CD47 protein is delivered using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide.

[0335] In some embodiments, the engineered CD47 protein is delivered using one or more gene editing systems. In some embodiments, the gene editing system is CRISPR / Cas. In some embodiments, the gene editing system includes a TALEN. In some embodiments, the gene editing system includes a zinc finger nuclease. In some embodiments, the gene editing system includes a meganuclease.

[0336] Following the introduction of a vector comprising a selectable marker, cells can be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells that successfully express the introduced sequences. Resistant cells of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell types. In some embodiments, the engineered CD47 protein is expressed in a mammalian expression system. Expression constructs can be transferred to mammalian cells by viral infection, such as by adenovirus constructs, or by direct DNA transfer, such as liposomes, calcium phosphate, DEAE-dextran, and by physical means such as electroporation and microinjection. In some embodiments, the engineered CD47 protein is delivered using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector that carries the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vectorpseudotyped with a vesicular stomatitis VSV-G envelope, and which carries the exogenous polynucleotide. In some embodiments, the engineered CD47 protein is delivered using one or more gene editing systems. In some embodiments, the gene editing system is CRISPR / Cas. In some embodiments, the gene editing system includes a TALEN. In some embodiments, the gene editing system includes a zinc finger nuclease. In some embodiments, the gene editing system includes a meganuclease.

[0337] In some embodiments, the expression system used in the context of this disclosure is disclosed in theUS application 63 / 270,454.

[0338] Expression vectors for mammalian cells typically include an mRNA cap site, a TATA box, a translational initiation sequence (Kozak consensus sequence), and polyadenylation elements. IRES elements also can be added to permit bicistronic expression with another gene, such as a selectable marker. Such vectors often include transcriptional promoter-enhancers for high-level expression, for example the SV40 promoter-enhancer, the human cytomegalovirus (CMV) promoter and the long terminal repeat of Rous sarcoma virus (RSV). These promoter-enhancers are active in many cell types. Tissue and cell-type promoters and enhancer regions also can be used for expression. Exemplary promoter / enhancer regions include, but are not limited to, those from genes such as elastase I, insulin, immunoglobulin, mouse mammary tumor virus, albumin, alpha fetoprotein, alpha 1 antitrypsin, beta globin, myelin basic protein, myosin light chain 2, and gonadotropic releasing hormone gene control. Selectable markers can be used to select for and maintain cells with the expression construct. Examples of selectable marker genes include, but are not limited to, hygromycin B phosphotransferase, adenosine deaminase, xanthine-guanine phosphoribosyl transferase, aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR) and thymidine kinase. For example, expression can be performed in the presence of methotrexate to select for only those cells expressing the DHFR gene.

[0339] Once the vector has been incorporated into the appropriate host cell, the host cell is maintained under conditions suitable for expression of the engineered CD47 proteins encoded by the incorporated polynucleotides. A skilled artisan is able to select conditions suitable for expression of the engineered CD47 proteins.IX. Other Features

[0340] It will be understood that, in the process of manufacturing a cell therapy, certain modifications may be introduced to the cell that are considered desirable for the cell therapy product. Cells that are profiled for donor capability can be edited or unedited cells. Profiling cells can take place before or after cell editing. Edited cells include one or more modifications such as HIP modifications (hypoimmune gene modifications that enable immune evasion). When transplanted in vivo without immunosuppression, HIP-modified cells have reduced expression of one or more molecules of the MHC class I and / or MHC class II molecules and thus there may be no evidence of a systemic immune response, such as no T cell activation, antibody production, or NK cell activity. Disclosure relating to edited cells is provided herein. Such disclosure is applicable to the methods, uses and cells described herein. The methods for profiling a population of cells for donor capability disclosed herein are advantageous for many cell types as described herein. In some embodiments, the cells are T cells (e.g., CAR-T cells).Engineered Cells and Methods of Engineering Cells

[0341] One modification considered desirable for the cell therapy product is that the engineered cells and populations thereof exhibit reduced expression of one or more molecules of the MHC class I and / or MHC class II molecules. A further modification considered desirable for the cell therapy product is that the engineered cells and populations thereof exhibit increased expression of at least one tolerogenic factor, such as tolerogenic factors described herein.

[0342] Provided herein are methods and compositions for alleviating and / or evading the effects of immune system reactions to allogenic transplants. To overcome the problem of immune rejection of cell-derived and / or tissue transplants, disclosed herein is an engineered immune-evasive cell (e.g., an engineered primary hypo-immunogenic cell), or population or pharmaceutical composition thereof, that represents a viable source for any transplantable cell type. The engineered cells disclosed herein provide for reduced recognition the recipient subject's immune system, regardless of the subject's genetic make-up, or any existing response within the subject to one or more previous allogeneic transplants, previous autologous chimeric antigen receptor (CAR) T rejection, and / or other autologous or allogenic therapies wherein a transgene is expressed. The engineered cells may include, but are not limited to, beta islet cells, B cells, T cells, NK cells, retinal pigmented epithelium cells, glial progenitor cells, endothelial cells, hepatocytes, thyroid cells, skin cells, and blood cells (e.g., plasma cells or platelets).

[0343] In some embodiments, the engineered cells described herein further comprise increased expression and / or overexpression of one or more complement inhibitors. In some embodiments, the one or more complement inhibitors are selected from CD46, CD59, and DAF / CD55. In some embodiments, the engineered cells comprise increased expression of two or more complement inhibitors in combination, such as increased expression of CD46 and CD59 or increased expression of CD46, CD59, and CD55.

[0344] The engineered cells provided herein utilize expression of tolerogenic factors and can also modulate(e.g., reduce or eliminate) one or more MHC class I molecules and / or one or more MHC class II molecules expression (e.g., surface expression). In some embodiments, genome editing technologies utilizing rare-cutting endonucleases (e.g., the CRISPR / Cas, TALEN, zinc finger nuclease, meganuclease, and homing endonuclease systems) are also used to reduce or eliminate expression of critical immune genes (e.g., by deleting genomic DNA of critical immune genes) in human cells. In certain embodiments, genome editing technologies or other gene modulation technologies are used to insert tolerance-inducing (tolerogenic) factors in human cells, (e.g., CD47), thus producing engineered cells that can evade immune recognition upon engrafting into a recipient subject. Therefore, the engineered cells provided herein exhibit modulated expression (e.g., reduced or eliminated expression) of one or more genes and factors that affect one or more MHC class I molecules and / or one or more MHC class II molecules, modulated expression (e.g., reduced or and modulated expression (e.g., overexpression) of tolerogenic factors, such as CD47, and provide for reduced recognition by the recipient subject's immune system. In some embodiments, the engineered cells provided herein exhibit modulated expression (e.g., reduced expression) of CD142. In some embodiments, the engineered cells provided herein exhibit modulated expression (e.g., increased expression) of one or more complement inhibitors selected from CD46, CD59, and DAF / CD55.

[0345] In some aspects, engineered cells provided herein exhibit reduced innate immune cell rejection and / or adaptive immune cell rejection (e.g., hypo-immunogenic cells). For example, in some embodiments, the engineered cells exhibit reduced susceptibility to NK cell-mediated lysis and / or macrophage engulfment. In some embodiments, the engineered cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that are transplanted into a recipient subject with little to no immunosuppressant agent needed. Such hypo- immunogenic cells retain cell-specific characteristics and features upon transplantation.

[0346] Also provided herein are methods for treating a disorder comprising administering the engineered cells(e.g., engineered primary cells) that evade immune rejection in an MHC-mismatched allogenic recipient. In some embodiments, the engineered cells produced from any one of the methods described herein evade immune rejection when repeatedly administered (e.g., transplanted or grafted) to MHC-mismatched allogenic recipient.

[0347] The practice of the embodiments described herein will employ, unless indicated specifically to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology that are within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in journals such as Advances in Immunology.

[0348] All publications, including patent documents, scientific articles, and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0349] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of the present disclosure. The following description illustrates the disclosure and, of course, should not be construed in any way as limiting the scope of contemplated embodiments described herein.

[0350] Described here are engineered cells that comprise one or more modifications. In some embodiments, the provided engineered cells also contain a modification of one or more target polynucleotide sequences that regulates the expression of one or more MHC class I molecules, one or more MHC class II molecules, or one or more MHC class I molecules and one or more MHC class II molecules.

[0351] In some embodiments, the provided engineered cells also include a modification to increase expression of one or more tolerogenic factor. In some embodiments, the tolerogenic factor is one or more of A20 / TNFAIP3, B2M- HLA-E, CD16, CD16 Fc receptor, CD24, CD27, CD35, CD39, CD46, CD47, CD52, CD55, CD59, CD64, CD200, CCL21, CCL22, CTLA4-Ig, Cl inhibitor, CR1, DUX4, FASL, HLA-C, HLA-E, HLA-E heavy chain, HLA-F, HLA-G, H2-M3, IDO1, IL-10, IL15-RF, IL-35, IL-39, MANF, Mfge8, PD-L1, Serpinb9, or any combination thereof. In some embodiments, the modification to increase expression of one or more tolerogenic factor is or includes increased expression of CD47. In some embodiments, the modification to increase expression of one or more tolerogenic factor is or includes increased expression of PD-L1. In some embodiments, the modification to increase expression of one or more tolerogenic factor is or includes increased expression of HLA-E. In some embodiments, the modification to increase expression of one or more tolerogenic factor is or includes increased expression of HLA-G. In some embodiments, the modification to increase expression of one or more tolerogenic factor is or includes increased expression of CCL21, PD-L1, FasL, Serpinb9, H2-M3 (HLA-G), CD47, CD200, and Mfge8.

[0352] In some embodiments, the cells include one or more genomic modifications that reduce expression of one or more MHC class I molecules and a modification that increases expression of CD47. In other words, the engineered cells comprise exogenous CD47 proteins and exhibit reduced or silenced surface expression of one or more MHC class I molecules. In some embodiments, the cells include one or more genomic modifications that reduce expression of one or more MHC class II molecules and a modification that increases expression of CD47. In some instances, the engineered cells comprise exogenous CD47 nucleic acids and proteins, and exhibit reduced or silenced surface expression of one or more MHC class I molecules. In some embodiments, the cells include one or more genomic modifications that reduce or eliminate expression of one or more MHC class II molecules, one or more genomic modifications that reduce or eliminate expression of one or more MHC class II molecules, and a modification that increases expression of CD47. In some embodiments, the engineered cells comprise exogenous CD47 proteins, exhibit reduced or silenced surface expression of one or more MHC class I molecules and exhibit reduced or lack surface expression of one or more MHC class II molecules. In many embodiments, the cells are B2MQ^^dei / Mei, CD47ft / cells.1. Chimeric Antigen Receptor

[0353] In some embodiments, a provided engineered cell is further modified to express a chimeric antigen receptor (CAR). It will be understood that embodiments concerning CAR modified cells may be readily applied to any suitable cell type as described herein, as well as HIP cells, safety switches and other modified / gene edited cells as described herein.

[0354] In some embodiments, a provided cell contains a genetic modification of one or more target polynucleotide sequences that regulates the expression of one or more MHC class I molecules, one or more MHC class II molecules, or one or more MHC class I molecules and one or more MHC class II molecules overexpresses a tolerogenic factor as described herein (e.g., CD47), and expresses a CAR. In some embodiments, the cell is one in which: B2M is reduced or eliminated (e.g., knocked out), CIITA is reduced or eliminated (e.g., knocked out), CD47 is overexpressed, and a CAR is expressed. In some embodiments, the cell is B2M~ / ~, CIITA / ', CD47tg, CAR+. In some embodiments, the cell (e.g., T cell) may additional be one in which TRAC is reduced or eliminated (e.g., knocked out). In some embodiments, the cell is B2~ / ', CIITA, CD47tg, TRAC' / CAR+ .

[0355] In some embodiments, a polynucleotide encoding a CAR is introduced into the cell. In some embodiments, the cell is a T cell, such as a primary T cell or a T cell differentiated from a pluripotent cell (e.g., iPSC). In some embodiments, the cell is a Natural Killer (NK) cell, such as a primary NK cell or an NK cell differentiated from a pluripotent cell (e.g., iPSC).

[0356] In some embodiments, the CAR is selected from the group consisting of a first generation CAR, a second generation CAR, a third generation CAR, and a fourth generation CAR. In some embodiments, the CAR is or comprises a first generation CAR comprising an antigen binding domain, a transmembrane domain, and at least one signaling domain (e.g., one, two or three signaling domains). In some embodiments, the CAR comprises a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains. In some embodiments, the CAR comprises a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, the antigen binding domain is or comprises an antibody, an antibody fragment, an scFv or a Fab.

[0357] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a first generation CAR. In some embodiments, a first generation CAR comprises an antigen binding domain, a transmembrane domain, and signaling domain. In some embodiments, a signaling domain mediates downstream signaling during T cell activation.

[0358] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a second generation CAR. In some embodiments, a second generation CAR comprises an antigen binding domain, a transmembrane domain, and two signaling domains. In some embodiments, a signaling domain mediates downstream signaling during T cell activation. In some embodiments, a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CART cell proliferation, and / or CART cell persistence during T cell activation.

[0359] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a third generation CAR. In some embodiments, a third generation CAR comprises an antigen binding domain, a transmembrane domain, and at least three signaling domains. In some embodiments, a signaling domain mediates downstream signaling during T cell activation. In some embodiments, a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation. In some embodiments, a third generation CAR comprises at least two costimulatory domains. In some embodiments, the at least two costimulatory domains are not the same.

[0360] In some embodiments, any one of the cells described herein comprises a nucleic acid encoding a CAR or a fourth generation CAR. In some embodiments, a fourth generation CAR comprises an antigen binding domain, a transmembrane domain, and at least two, three, or four signaling domains. In some embodiments, a signaling domain mediates downstream signaling during T cell activation. In some embodiments, a signaling domain is a costimulatory domain. In some embodiments, a costimulatory domain enhances cytokine production, CAR T cell proliferation, and or CAR T cell persistence during T cell activation.

[0361] In some embodiments, an engineered cell provided herein (e.g., primary or iPSC-derived T cell or primary or iPSC-derived NK cell) includes a polynucleotide encoding a CAR, wherein the polynucleotide is inserted in a genomic locus. In some embodiments, the polynucleotide is inserted into a safe harbor locus, such as but not limited to, an AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91), HMGB1, ABO, RHD, FUT1, or KDM5D gene locus. In some embodiments, the polynucleotide is inserted in a B2M, CIITA, TRAC, TRB, PD1 or CTLA4 gene. Any suitable method can be used to insert the CAR into the genomic locus of the hypoimmunogenic cell including the gene editing methods described herein (e.g., a CRISPR / Cas system).

[0362] In some embodiments, a first, second, third, or fourth generation CAR further comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, a cytokine gene is endogenous or exogenous to a target cell comprising a CAR which comprises a domain which upon successful signaling of the CAR induces expression of a cytokine gene. In some embodiments, a cytokine gene encodes a pro-inflammatory cytokine. In some embodiments, a cytokine gene encodes IL-1, IL-2, IL-9, IL-12, IL-18, TNF, or IFN-gamma, or functional fragment thereof. In some embodiments, a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments, a domain which upon successful signaling of the CAR induces expression of a cytokine gene is or comprises a transcription factor or functional domain or fragment thereof. In some embodiments, a transcription factor or functional domain or fragment thereof is or comprises a nuclear factor of activated T cells (NFAT), an NF-kB, or functional domain or fragmentthereof. See, e.g., Zhang. C. et al., Engineering CAR-T cells. Biomarker Research. 5:22 (2017); WO 2016126608; Sha, H. et al. Chimaeric antigen receptor T-cell therapy for tumour immunotherapy. Bioscience Reports Jan 27, 2017, 37 (1).

[0363] A skilled artisan is familiar with CARs and different components and configurations of CARs. Any known CAR can be employed in connection with the provided embodiments. In addition to the CARs described herein, various CARs and nucleotide sequences encoding the same are known in the art and would be suitable for engineering cells as described herein. See, e.g., W02013040557; W02012079000; W02016030414; Smith T, et al., Nature Nanotechnology. 2017. DOI: 10.1038 / NNAN0.2017.57, the disclosures of which are herein incorporated by reference. Exemplary features and components of a CAR are described in the following subsections. a. Antigen Binding Domain

[0364] In some embodiments, a CAR antigen binding domain (ABD) is or comprises an antibody or antigenbinding portion thereof. In some embodiments, a CAR antigen binding domain is or comprises an scFv or Fab.

[0365] In some embodiments, an antigen binding domain binds to a cell surface antigen of a cell. In some embodiments, a cell surface antigen is characteristic of (e.g., expressed by) a particular or specific cell type. In some embodiments, a cell surface antigen is characteristic of more than one type of cell.

[0366] In some embodiments, the antigen may be an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen that is characteristic of an autoimmune or inflammatory disease. In some embodiments, the antigen binding domain (ABD) targets an antigen characteristic of a neoplastic cell. For instance, the antigen binding domain targets an antigen expressed by a neoplastic or cancer cell. In some embodiments, the ABD binds a tumor associated antigen. In some embodiments, the antigen characteristic of a neoplastic cell (e.g., antigen associated with a neoplastic or cancer cell) or a tumor associated antigen is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine / threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor.

[0367] In some embodiments, the target antigen is an antigen that includes, but is not limited to, EpidermalGrowth Factor Receptors (EGFR) (including ErbBl / EGFR, ErbB2 / HER2, ErbB3 / HER3, and ErbB4 / HER4), Fibroblast Growth Factor Receptors (FGFR) (including FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF18, and FGF21) Vascular Endothelial Growth Factor Receptors (VEGFR) (including VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF), RET Receptor and the Eph Receptor Family (including EphAl, EphA2, EphA3, EphA4, EphA5, EphA6, EphA7, EphA8, EphA9, EphAlO, EphBl, EphB2. EphB3, EphB4, and EphB6), CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR8, CFTR, CIC-1, CIC-2, CIC-4, CIC-5, CIC-7, CIC-Ka, CIC-Kb, Bestrophins, TMEM16A, GABA receptor, glycin receptor, ABC transporters, NAV1.1, NAV1.2, NAV1.3, NAV1.4, NAVI.5, NAV1.6, NAV1.7, NAV1.8, NAV1.9, sphingosin-l-phosphate receptor (S1P1R), NMDA channel, transmembrane protein, multispan transmembrane protein, T-cell receptor motifs; T- cell alpha chains; T-cell 0 chains; T-cell y chains; T-cell 6 chains, CCR7, CD3, CD4, CD5, CD7, CD8, CDllb, CDllc, CD16, CD19, CD20, CD21, CD22, CD25, CD28, CD34, CD35, CD40, CD45RA, CD45RO, CD52, CD56, CD62L, CD68, CD80, CD95, CD117, CD127, CD133, CD137 (4-1 BB), CD163, F4 / 80, IL-4Ra, Sca-1 , CTLA-4, GITR, GARP, LAP, granzyme B, LFA-1, transferrin receptor, NKp46, perforin, CD4+, Thl, Th2, Thl7, Th40, Th22, Th9, Tfh, Canonical Treg, FoxP3+, Tri, Th3, Tregl7, TREG, CDCP, NT5E, EpCAM, CEA, gpA33, Mucins, TAG-72, Carbonic anhydrase IX, PSMA, Folate binding protein, Gangliosides (e.g., CD2, CD3, GM2), Lewis-y2, VEGF, VEGFR 1 / 2 / 3, oV03, o501, ErbBl / EGFR, ErbBl / HER2, ErB3, c-MET, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, Tenascin, PDL-1, BAFF, HDAC, ABL, FLT3, KIT, MET, RET, IL-10, ALK, RANKL, mTOR, CTLA-4, IL-6, IL-6R, JAK3, BRAF, PTCH, Smoothened, PIGF, ANPEP, TIMP1, PLAUR, PTPRJ, LTBR, orANTXR1, Folate receptor alpha (FRa), ERBB2 (Her2 / neu), EphA2, IL-13Ra2, epidermal growth factor receptor (EGFR), Mesothelin, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII , GD2, GD3, BCMA, MUC16 (CA125), L1CAM, LeY, MSLN, IL13Ral, Ll-CAM, Tn Ag, prostate specific membrane antigen (PSMA), R0R1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, interleukin-11 receptor a (IL-llRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, MUC1, NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-1 receptor, CAIX, LMP2, gplOO, bcr-abl, tyrosinase, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, Polysialic acid, PLACI, GloboH, NY- BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Major histocompatibility complex class I-related gene protein (MR1), urokinase-type plasminogen activator receptor (uPAR), Fos-related antigen 1, p53, p53 mutant, prostein, survivin, telomerase, PCTA-l / Galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYPIB I, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, a neoantigen, CD133, CD15, CD184, CD24, CD56, CD26, CD29, CD44, HLA-A, HLA-B, HLA-C, (HLA- A,B,C) CD49f, CD151 CD340, CD200, tkrA, trkB, or trkC, or an antigenic fragment or antigenic portion thereof.

[0368] In some embodiments, exemplary target antigens include, but are not limited to, CDS, CD19, CD20, CD22, CD23, CD30, CD70, Kappa, Lambda, and B cell maturation agent (BCMA) (associated with leukemias); CS1 / SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myelomas); GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRa, IL-13Ra, Mesothelin, MUC1, MUC16, and R0R1 (associated with solid tumors).

[0369] In some embodiments, the CAR is a CD19 CAR. In some embodiments, the extracellular binding domain of the CD19 CAR comprises an antibody that specifically binds to CD19, for example, human CD19. In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv antibody fragment derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of FMC63 connected by a linker peptide. In some embodiments, the linker peptide is a "Whitlow" linker peptide. FMC63 and the derived scFv have been described in Nicholson et al., Mai. Immun. 34(16-17): 1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337 A 1, the entire content of each of which is incorporated by reference herein.

[0370] In some embodiments, the extracellular binding domain of the CD19 CAR comprises an antibody derived from one of the CD19-specific antibodies including, for example, SJ25C1 (Bejcek et al., Cancer Res. 55:2346-2351 (1995)), HD37 (Pezutto et al., J. Immunol. 138(9): 2793-2799 (1987)), 4G7 (Meeker et aL, Hybridoma 3:305-320 (1984)), B43 (Bejcek (1995)), BLY3 (Bejcek (1995)), B4 (Freedman et al., 70:418-427 (1987)), B4 HB12b (Kansas & Tedder, J. Immunol. 147:4094-4102 (1991); Yazawa et al., Proc. Natl. Acad. Sci. USA 102: 15178-15183 (2005); Herbst et al., J. Pharmacol. Exp. Ther. 335:213-222 (2010)), BU12 (Gallard et al., J. Immunology, 148(10): 2983-2987 (1992)), and CLB-CD19 (De Rie Cell. Immunol. 118:368-381(1989)).

[0371] In some embodiments, the CAR is CD22 CAR. CD22, which is a transmembrane protein found mostly on the surface of mature B cells that functions as an inhibitory receptor for B cell receptor (BCR) signaling. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g., B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's lymphoma) and is not present on the cell surface in early stages of B cell development or on stem cells. In some embodiments, the CD22 CAR comprises an extracellular binding domain that specifically binds CD22, a transmembrane domain, an intracellular signaling domain, and / or an intracellular costimulatorydomain. In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv antibody fragment derived from the m971 monoclonal antibody (m971 ), which comprises the heavy chain variable region (VH) and the light chain variable region (VL) of m971 connected by a linker. In some embodiments, the extracellular binding domain of the CD22 CAR comprises an scFv antibody fragment derived from m971-L7, which an affinity matured variant of m971 with significantly improved CD22 binding affinity compared to the parental antibody m971 (improved from about 2 nM to less than 50 pM). In some embodiments, the scFv antibody fragment derived from m971-L7 comprises the VH and the VL of m971-L7 connected by a 3xG4S linker. In some embodiments, the extracellular binding domain of the CD22 CAR comprises immunotoxins HA22 or BL22. Immunotoxins BL22 and HA22 are therapeutic agents that comprise an scFv specific for CD22 fused to a bacterial toxin, and thus can bind to the surface of the cancer cells that express CD22 and kill the cancer cells. BL22 comprises a dsFv of an anti-CD22 antibody, RFB4, fused to a 38-kDa truncated form of Pseudomonas exotoxin A (Bang et al., Clin. Cancer Res., 11: 1545-50 (2005)). HA22 (CAT8015, moxetumomab pasudotox) is a mutated, higher affinity version of BL22 (Ho et al., J. Biol. Chem., 280(1): 607-17 (2005)). Suitable sequences of antigen binding domains of HA22 and BL22 specific to CD22 are disclosed in, for example, U.S. Patent Nos. 7,541,034; 7,355,012; and 7,982,011, which are hereby incorporated by reference in their entirety.

[0372] In some embodiments, the CAR is BCMA CAR. BCMA is a tumor necrosis family receptor (TNFR) member expressed on cells of the B cell lineage, with the highest expression on terminally differentiated B cells or mature B lymphocytes. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity. The expression of BCMA has been recently linked to a number of cancers, such as multiple myeloma, Hodgkin's and non-Hodgkin's lymphoma, various leukemias, and glioblastoma. In some embodiments, the BCMA CAR comprises an extracellular binding domain that specifically binds BCMA, a transmembrane domain, an intracellular signaling domain, and / or an intracellular costimulatory domain. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an antibody that specifically binds to BCMA, for example, human BCMA. CARs directed to BCMA have been described in PCT Application Publication Nos. WO2016 / 014789, WO2016 / 014565, WO2013 / 154760, and WO 2015 / 128653. BCMA-binding antibodies are also disclosed in PCT Application Publication Nos. WO2015 / 166073 and W02014 / 068079. In some embodiments, the extracellular binding domain of the BCMA CAR comprises an scFv antibody fragment derived from a murine monoclonal antibody as described in Carpenter et al., Clin. Cancer Res. 19(8): 2048-2060 (2013). In some embodiments, the scFv antibody fragment is a humanized version of the murine monoclonal antibody (Sommermeyer et al., Leukemia 31:2191-2199 (2017)). In some embodiments, the extracellular binding domain of the BCMA CAR comprises single variable fragments of two heavy chains (VHH) that can bind to two epitopes of BCMA as described in Zhao et al., J. Hematol. Oneal. 11(1): 141 (2018). In some embodiments, the extracellular binding domain of the BCMA CAR comprises a fully human heavy-chain variable domain (FHVH) as described in Lam et aL, Nat. Commun. 11(1): 283 (2020).

[0373] In some embodiments, the antigen binding domain targets an antigen characteristic of an autoimmune or inflammatory disorder. In some embodiments, the ABD binds an antigen associated with an autoimmune or inflammatory disorder. In some instances, the antigen is expressed by a cell associated with an autoimmune or inflammatory disorder. In some embodiments, the autoimmune or inflammatory disorder is selected from chronic graft- vs-host disease (GVHD), lupus, arthritis, immune complex glomerulonephritis, goodpasture, uveitis, hepatitis, systemic sclerosis or scleroderma, type I diabetes, multiple sclerosis, cold agglutinin disease, Pemphigus vulgaris, Grave's disease, autoimmune hemolytic anemia, Hemophilia A, Primary Sjogren's Syndrome, thrombotic thrombocytopenia purrpura, neuromyelits optica, Evan's syndrome, IgM mediated neuropathy, cyroglobulinemia, dermatomyositis, idiopathic thrombocytopenia, ankylosing spondylitis, bullous pemphigoid, acquired angioedema, chronic urticarial, antiphospholipid demyelinating polyneuropathy, and autoimmune thrombocytopenia or neutropenia or pure red cell aplasias, whileexemplary non-limiting examples of alloimmune diseases include allosensitization (see, for example, Blazar et al., 2015, Am. J. Transplant, 15(4):931-41) or xenosensitization from hematopoietic or solid organ transplantation, blood transfusions, pregnancy with fetal allosensitization, neonatal alloimmune thrombocytopenia, hemolytic disease of the newborn, sensitization to foreign antigens such as can occur with replacement of inherited or acquired deficiency disorders treated with enzyme or protein replacement therapy, blood products, and gene therapy. Allosensitization, in some instances, refers to the development of an immune response (such as circulating antibodies) against human leukocyte antigens that the immune system of the recipient subject or pregnant subject considers to be non-self antigens. In some embodiments, the antigen characteristic of an autoimmune or inflammatory disorder is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine / threonine kinase, receptor guanylyl cyclase, or histidine kinase associated receptor.

[0374] In some embodiments, an antigen binding domain of a CAR binds to a ligand expressed on B cells, plasma cells, or plasmablasts. In some embodiments, an antigen binding domain of a CAR binds to CD10, CD19, CD20, CD22, CD24, CD27, CD38, CD45R, CD138, CD319, BCMA, GPRC5D, CD28, TNF, interferon receptors, GM-CSF, ZAP-70, LFA-1, CD3 gamma, CD5 or CD2. See, US 2003 / 0077249; WO 2017 / 058753; WO 2017 / 058850, the contents of which are herein incorporated by reference. In some embodiments, the CAR is an anti-CD19 CAR. In some embodiments, the CAR is an anti-BCMA CAR.

[0375] In some embodiments, the antigen binding domain targets an antigen characteristic of senescent cells, e.g., urokinase-type plasminogen activator receptor (uPAR). In some embodiments, the ABD binds an antigen associated with a senescent cell. In some instances, the antigen is expressed by a senescent cell. In some embodiments, the CAR may be used for treatment or prophylaxis of disorders characterized by the aberrant accumulation of senescent cells, e.g., liver and lung fibrosis, atherosclerosis, diabetes and osteoarthritis.

[0376] In some embodiments, the antigen binding domain targets an antigen characteristic of an infectious disease. In some embodiments, the ABD binds an antigen associated with an infectious disease. In some instances, the antigen is expressed by a cell affected by an infectious disease. In some embodiments, wherein the infectious disease is selected from HIV, hepatitis B virus, hepatitis C virus, Human herpes virus, Human herpes virus 8 (HHV-8, Kaposi sarcoma- associated herpes virus (KSHV)), Human T-lymphotrophic virus-1 (HTLV-1), Merkel cell polyomavirus (MCV), Simian virus 40 (SV40), Epstein-Barr virus, CMV, human papillomavirus. In some embodiments, the antigen characteristic of an infectious disease is selected from a cell surface receptor, an ion channel-linked receptor, an enzyme-linked receptor, a G protein-coupled receptor, receptor tyrosine kinase, tyrosine kinase associated receptor, receptor-like tyrosine phosphatase, receptor serine / threonine kinase, receptor guanylyl cyclase, histidine kinase associated receptor, HIV Env, gpl20, or CD4- induced epitope on HIV-1 Env.

[0377] In any of these embodiments, the extracellular binding domain of the CAR can be codon-optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain.

[0378] In some embodiments, the CAR is bispecific to two target antigens. In some embodiments, the target antigens are different target antigens. In some of any such embodiments, the two different target antigens are any two different antigens described above. In some embodiments, the extracellular binding domains are different and bind two different antigens from (i) CD19 and CD20, (ii) CD20 and Ll-CAM, (iii) Ll-CAM and GD2, (iv) EGFR and Ll-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1. In some embodiments, each of the two different antigen binding domains is an scFv. In some embodiments, the C-terminus of one variable domain (VH or VL) of a first scFv is tethered to the N-terminus of the second scFv (VL or VH, respectively)via a polypeptide linker. In some embodiments, the linker connects the N-terminus of the VH with the C-terminus of VL or the C-terminus of VH with the N-terminus of VL. These scFvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody. The scFvs, specific for at least two different antigens, are arranged in tandem and linked to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In some embodiments, an extracelluar spacer domain may be linked between the antigen-specific binding region and the transmembrane domain.

[0379] In some embodiments, each antigen-specific targeting region of the CAR comprises a divalent (or bivalent) single-chain variable fragment (di-scFvs, bi-scFvs). In CARs comprising di-scFVs, two scFvs specific for each antigen are linked together by producing a single peptide chain with two VH and two VL regions, yielding tandem scFvs. (Xiong, Cheng-Yi; Natarajan, A; Shi, X B; Denardo, G L; Denardo, S J (2006). "Development of tumor targeting anti-MUC- 1 multimer: effects of di-scFv unpaired cysteine location on PEGylation and tumor binding". Protein Engineering Design and Selection 19 (8): 359-367; Kufer, Peter; Lutterbuse, Ralf; Baeuerle, Patrick A. (2004). "A revival of bispecific antibodies". Trends in Biotechnology 22 (5): 238-244). CARs comprising at least two antigen-specific targeting regions would express two scFvs specific for each of the two antigens. The resulting antigen-specific targeting region, specific for at least two different antigens, is joined to the co-stimulatory domain and the intracellular signaling domain via a transmembrane domain. In some embodiments, an extracelluar spacer domain may be linked between the antigen-specific binding domain and the transmembrane domain.

[0380] In some embodiments, each antigen-specific targeting region of the CAR comprises a diabody. In a diabody, the scFvs are created with linker peptides that are too short for the two variable regions to fold together, driving the scFvs to dimerize. Still shorter linkers (one or two amino acids) lead to the formation of trimers, the so-called triabodies or tribodies. Tetrabodies may also be used.

[0381] In some embodiments, the cell is engineered to express more than one CAR, such as two different CARs, in which each CAR has an antigen-binding domain directed to a different target antigen. In some of any such embodiments, the two different target antigens are any two different antigens described above. In some embodiments, the extracellular binding domains are different and bind two different antigens from (i) CD19 and CD20, (ii) CD20 and Ll-CAM, (iii) Ll-CAM and GD2, (iv) EGFR and Ll-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1.

[0382] In some embodiments, two different engineered cells are prepared that contain the provided modifications with each engineered with a different CAR. In some embodiments, each of the two different CARs has an antigen-binding domain directed to a different target antigen. In some of any such embodiments, the two different target antigens are any two different antigens described above. In some embodiments, the extracellular binding domains are different and bind two different antigens from (i) CD19 and CD20, (ii) CD20 and Ll-CAM, (iii) Ll-CAM and GD2, (iv) EGFR and Ll-CAM, (v) CD19 and CD22, (vi) EGFR and C-MET, (vii) EGFR and HER2, (viii) C-MET and HER2, or (ix) EGFR and ROR1. In some embodiments, a population of engineered cells (e.g., hypoimmunogenic) expressing a first CAR directed against a first target antigen and a population of engineered cells (e.g., hypoimmunogenic) expressing a second CAR directed against a second target antigen are separately administered to the subject. In some embodiments, the first and second population of cells are administered sequentially in any order. For instance, the population of cells expressing the second CAR is administered a after administration of the population of cells expressing the first CAR. b. Spacer

[0383] In some embodiments, the CAR further comprises one or more spacers, e.g., wherein the spacer is a first spacer between the antigen binding domain and the transmembrane domain. In some embodiments, the first spacerincludes at least a portion of an immunoglobulin constant region or variant or modified version thereof. In some embodiments, the spacer is a second spacer between the transmembrane domain and a signaling domain. In some embodiments, the second spacer is an oligopeptide, e.g., wherein the oligopeptide comprises glycine and serine residues such as but not limited to glycine-serine doublets. In some embodiments, the CAR comprises two or more spacers, e.g., a spacer between the antigen binding domain and the transmembrane domain and a spacer between the transmembrane domain and a signaling domain. c. Transmembrane Domain

[0384] In some embodiments, the CAR transmembrane domain comprises at least a transmembrane region of the alpha, beta or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional variant thereof. In some embodiments, the transmembrane domain comprises at least a transmembrane region(s) of CD8a, CD8p, 4-1BB / CD137, CD28, CD34, CD4, FccRIy, CD16, OX40 / CD134, CD3<, CD3c, CD3y, CD36, TCRa, TCR0, TCR(, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or functional variant thereof. d. Signaling Domain(s)

[0385] In some embodiments, a CAR described herein comprises one or at least one signaling domain selected from one or more of B7-1 / CD80; B7-2 / CD86; B7-H1 / PD-L1; B7-H2; B7-H3; B7-H4; B7-H6; B7-H7; BTLA / CD272; CD28; CTLA-4; GI24 / VISTA / B7-H5; ICOS / CD278; PD-1; PD-L2 / B7-DC; PDCD6); 4-1BB / TNFSF9 / CD137; 4-1BB Ligand / TNFSF9; BAFF / BLyS / TNFSF13B; BAFF R / TNFRSF13C; CD27 / TNFRSF7; CD27 Ligand / TNFSF7; CD30 / TNFRSF8; CD30 Ligand / TNFSF8; CD40 / TNFRSF5; CD40 / TNFSF5; CD40 Ligand / TNFSF5; DR3 / TNFRSF25; GITR / TNFRSF18; GITR Ligand / TNFSF18; HVEM / TNFRSF14; LIGHT / TNFSF14; Lymphotoxin-alpha / TNF-beta; OX40 / TNFRSF4; 0X40 Ligand / TNFSF4; RELT / TNFRSF19L; TACI / TNFRSF13B; TL1A / TNFSF15; TNF-alpha; TNF RII / TNFRSF1B); 2B4 / CD244 / SLAMF4; BLAME / SLAMF8; CD2; CD2F-10 / SLAMF9; CD48 / SLAMF2; CD58 / LFA-3; CD84 / SLAMF5; CD229 / SLAMF3; CRACC / SLAMF7; NTB-A / SLAMF6; SLAM / CD150); CD2; CD7; CD53; CD82 / Kai-1; CD90 / Thyl; CD96; CD160; CD200; CD300a / LMIRl; HLA Class I; H LA-DR; Ikaros; Integrin alpha 4 / CD49d; Integrin alpha 4 beta 1; Integrin alpha 4 beta 7 / LPAM-l; LAG-3; TCL1A; TCL1B; CRTAM; DAP12; Dectin-1 / CLEC7A; DPPIV / CD26; EphB6; TIM-l / KIM- 1 / HAVCR; TIM-4; TSLP; TSLP R; lymphocyte function associated antigen-1 (LFA-1); NKG2C, a CD3 zeta domain, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or functional fragment thereof.

[0386] In some embodiments, the at least one signaling domain comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof.

[0387] In some embodiments, a CAR comprises a signaling domain which is a costimulatory domain. In some embodiments, a CAR comprises a second costimulatory domain. In some embodiments, a CAR comprises at least two costimulatory domains. In some embodiments, a CAR comprises at least three costimulatory domains. In some embodiments, a CAR comprises a costimulatory domain selected from one or more of CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83. In some embodiments, if a CAR comprises two or more costimulatory domains, two costimulatory domains are different. In some embodiments, if a CAR comprises two or more costimulatory domains, two costimulatory domains are the same.

[0388] In other embodiments, the at least one signaling domain comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BBdomain, or functional variant thereof. In yet other embodiments, the at least one signaling domain comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the at least one signaling domain comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0389] In some embodiments, the at least two signaling domains comprise a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof. In other embodiments, the at least two signaling domains comprise (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof. In yet other embodiments, the at least one signaling domain comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the at least two signaling domains comprise a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0390] In some embodiments, the at least three signaling domains comprise a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof. In other embodiments, the at least three signaling domains comprise (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof. In yet other embodiments, the least three signaling domains comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof. In some embodiments, the at least three signaling domains comprise a (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene.

[0391] In some embodiments, the CAR comprises a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine -based activation motif (ITAM), or functional variant thereof; and (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof.

[0392] In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosinebased activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; and (iii) a 4- 1BB domain, or a CD134 domain, or functional variant thereof.

[0393] In some embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain, or a 4-1BB domain, or functional variant thereof, and / or (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof.

[0394] In some embodiments, the CAR comprises a (i) a CD3 zeta domain, or an immunoreceptor tyrosinebased activation motif (ITAM), or functional variant thereof; (ii) a CD28 domain or functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene. e. Exemplary CARs

[0395] In some embodiments, the CAR comprises an extracellular antigen binding domain (e.g., antibody or antibody fragment, such as an scFv) that binds to an antigen (e.g., tumor antigen), a spacer (e.g., containing a hinge domain, such as any as described herein), a transmembrane domain (e.g., any as described herein), and an intracellular signaling domain (e.g., any intracellular signaling domain, such as a primary signaling domain or costimulatory signaling domain as described herein). In some embodiments, the intracellular signaling domain is or includes a primary cytoplasmic signaling domain. In some embodiments, the intracellular signaling domain additionally includes an intracellular signaling domain of a costimulatory molecule (e.g., a costimulatory domain). Any of such components can be any as described above.

[0396] Examples of exemplary components of a CAR are described in Table 4. In provided aspects, the sequences of each component in a CAR can include any combination listed in Table 4.Table 4. CAR Components and Exemplary Sequences

[0397] In certain embodiments, the CAR may comprise a signal peptide at the N-terminus. Non-limiting examples of signal peptides include CD8a signal peptide, IgK signal peptide, and granulocyte-macrophage colonystimulating factor receptor subunit alpha (GMCSFR-a, also known as colony stimulating factor 2 receptor subunit alpha (CSF2RA)) signal peptide, and variants thereof, the amino acid sequences of which are provided in Table 5 below.Table 5. Exemplary Sequences of Signal Peptides

[0398] In certain embodiments, the extracellular binding domain of the CAR may comprise one or more antibodies specific to one target antigen or multiple target antigens. The antibody may be an antibody fragment, for example, an scFv, or a single-domain antibody fragment, for example, a VHH. In certain embodiments, the scFv may comprise a heavy chain variable region (VH) and a light chain variable region (VL) of an antibody connected by a linker. The VHand the VL may be connected in either order, i.e., VH-linker-Vi or VL-linker-Vn. Non-limiting examples of linkers include Whitlow linker, (G4S)n(n can be a positive integer, e.g., 1, 2, 3, 4, 5, 6, etc.) linker, and variants thereof. In certain embodiments, the antigen may be an antigen that is exclusively or preferentially expressed on tumor cells, or an antigen that is characteristic of an autoimmune or inflammatory disease. Exemplary target antigens include, but are not limited to, CD5, CD19, CD20, CD22, CD23, CD30, CD33, CD70, Kappa, Lambda, B cell maturation agent (BCMA), and G-protein coupled receptor family C group 5 member D (GPRC5D) (associated with leukemias); CS1 / SLAMF7, CD38, CD138, GPRC5D, TACI, and BCMA (associated with myelomas); CD123, LeY, NKG2D ligand, and WT1 (associated with other hematological cancers); GD2, HER2, EGFR, EGFRvIII, B7H3, PSMA, PSCA, CAIX, CD171, CEA, CSPG4, EPHA2, FAP, FRo, IL-13R0, Mesothelin, MUC1, MUC16, R0R1, C-Met, CD133, Ep-CAM, GPC3, HPV16-E6, IL13Ra2, MAGEA3, MAGEA4, MARTI, NY- ESO-1, VEGFR2, a-Folate receptor, CD24, CD44v7 / 8, EGP-2, EGP-40, erb-B2, erb-B 2,3,4, FBP, Fetal acethylcholine e receptor, GD2, GDS, HMW-MAA, IL-llRa, KDR, Lewis Y, Ll-cell adhesion molecule, MAGE-A1, Oncofetal antigen (h5T4), and TAG-72 (associated with solid tumors); A*02 (associated with organ transplantation); fibroblast activation protein (FAP)(associated with fibrosis); urokinase-type plasminogen activator receptor (uPAR) (associated with senescence). In certain embodiments, the CAR can be re-engineered as a chimeric autoantibody receptor (CAAR) to selectively deplete autoreactive immune cells. In certain embodiments, CAARs are engineered to target autoantibodies present on immune cells. Exemplary target antigens for CAARs include, but are not limited to, DSG3 (associated with pemphigus volgaris); factor VIII (FVIII)(associated with haemophilia). In any of these embodiments, the extracellular binding domain of the CAR can be codon-optimized for expression in a host cell or have variant sequences to increase functions of the extracellular binding domain.

[0399] In certain embodiments, the CAR may comprise a hinge domain, also referred to as a spacer. The terms "hinge" and "spacer" may be used interchangeably in the present disclosure. Non-limiting examples of hinge domains include CD8a hinge domain, CD28 hinge domain, IgG4 hinge domain, IgG4 hinge-CH2-CH3 domain, and variants thereof, the amino acid sequences of which are provided in Table 6 below.Table 6. Exemplary Sequences of Hinge Domains

[0400] In certain embodiments, the transmembrane domain of the CAR may comprise a transmembrane region of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3E, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a functional variant thereof, including the human versions of each of these sequences. In other embodiments, the transmembrane domain may comprise a transmembrane region of CD8a, CD80, 4-1BB / CD137, CD28, CD34, CD4, FceRIy, CD16, OX40 / CD134, CD3<, CD3s, CD3y, CD36, TCRa, TCR0, TCR<, CD32, CD64, CD64, CD45, CD5, CD9, CD22, CD37, CD80, CD86, CD40, CD40L / CD154, VEGFR2, FAS, and FGFR2B, or a functional variant thereof, including the human versions of each of these sequences. Table 7 provides the amino acid sequences of a few exemplary transmembrane domains.Table 7. Exemplary Sequences of Transmembrane Domains

[0401] In certain embodiments, the intracellular signaling domain and / or intracellular costimulatory domain of the CAR may comprise one or more signaling domains selected from B7-1 / CD80, B7-2 / CD86, B7-H1 / PD-L1, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, BTLA / CD272, CD28, CTLA-4, Gi24 / VISTA / B7-H5, ICOS / CD278, PD-1, PD-L2 / B7-DC, PDCD6, 4- 1BB / TNFSF9 / CD137, 4-1BB Ligand / TNFSF9, BAFF / BLyS / TNFSF13B, BAFF R / TNFRSF13C, CD27 / TNFRSF7, CD27 Ligand / TNFSF7, CD30 / TNFRSF8, CD30 Ligand / TNFSF8, CD40 / TNFRSF5, CD40 / TNFSF5, CD40 Ligand / TNFSF5, DR3 / TNFRSF25, GITR / TNFRSF18, GITR Ligand / TNFSF18, HVEM / TNFRSF14, LIGHT / TNFSF14, Lymphotoxin-alpha / TNFP, OX40 / TNFRSF4, 0X40 Ligand / TNFSF4, RELT / TNFRSF19L, TACI / TNFRSF13B, TL1A / TNFSF15, TNFa, TNF RII / TNFRSF1B, 2B4 / CD244 / SLAMF4, BLAME / SLAMF8, CD2, CD2F-10 / SLAMF9, CD48 / SLAMF2, CD58 / LFA-3, CD84 / SLAMF5, CD229 / SLAMF3, CRACC / SLAMF7, NTB-A / SLAMF6, SLAM / CD150, CD2, CD7, CD53, CD82 / Kai-1, CD90 / Thyl, CD96, CD160, CD200, CD300a / LMIRl, HLA Class I, HLA-DR, Ikaros, Integrin alpha 4 / CD49d, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7 / LPAM-l, LAG-3, TCL1A, TCL1B, CRTAM, DAP12, Dectin-1 / CLEC7A, DPPIV / CD26, EphB6, TIM-l / KIM-l / HAVCR, TIM- 4, TSLP, TSLP R, lymphocyte function associated antigen-1 (LFA-1), NKG2C, CD3(, an immunoreceptor tyrosine-based activation motif (ITAM), CD27, CD28, 4-1BB, CD134 / OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and a functional variant thereof including the human versions of each of these sequences. In some embodiments, the intracellular signaling domain and / or intracellular costimulatory domain comprises one or more signaling domains selected from a CD3( domain, an ITAM, a CD28 domain, 4-1BB domain, or a functional variant thereof. Table 8 provides the amino acid sequences of a few exemplary intracellular costimulatory and / or signaling domains. In certain embodiments, as in the case of tisagenlecleucel as described below, the CD3( signaling domain of SEQ ID NO:233 may have a mutation, e.g., a glutamine (Q) to lysine (K) mutation, at amino acid position 14 (see SEQ ID NO:234).Table 8. Exemplary Sequences of Intracellular Co-Stimulatory and / or Signaling Domainsf. CD19 CAR

[0402] In some embodiments, the CAR is a CD19 CAR, and in these embodiments, the second transgene comprises a nucleotide sequence encoding a CD19 CAR. In some embodiments, the CD19 CAR may comprise a signal peptide, an extracellular binding domain that specifically binds CD19, a hinge domain, a transmembrane domain, an intracellular costimulatory domain, and / or an intracellular signaling domain in tandem.

[0403] In some embodiments, the signal peptide of the CD19 CAR comprises a CD8a signal peptide. In some embodiments, the CD8a signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:219 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 219. In some embodiments, the signal peptide comprises an IgK signal peptide. In some embodiments, the IgK signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 220 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO:220. In some embodiments, the signal peptide comprises a GMCSFR-a or CSF2RA signal peptide. In some embodiments, the GMCSFR-a or CSF2RA signal peptide comprises or consists of an amino acid sequence set forth in SEQ ID NO:221 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in of SEQ ID NO: 221.

[0404] In some embodiments, the extracellular binding domain of the CD19 CAR is specific to CD19, for example, human CD19. The extracellular binding domain of the CD19 CAR can be codon-optimized for expression in a host cell or to have variant sequences to increase functions of the extracellular binding domain. In some embodiments, the extracellular binding domain comprises an immunogenically active portion of an immunoglobulin molecule, for example, an scFv.

[0405] In some embodiments, the extracellular binding domain of the CD19 CAR comprises an scFv derived from the FMC63 monoclonal antibody (FMC63), which comprises the heavy chain variable region (VH) and the light chain variable region (VQ of FMC63 connected by a linker. FMC63 and the derived scFv have been described in Nicholson et al., Mol. Immun. 34(16-17): 1157-1165 (1997) and PCT Application Publication No. WO2018 / 213337, the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequences of the entire FMC63- derived scFv (also referred to as FMC63 scFv) and its different portions are provided in Table 9 below. In some embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO: 235, 236, or 241, or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid sequence set forth in SEQ ID NO: 235, 236, or 241. In some embodiments, the CD19-specific scFv may comprise one or more CDRs havingamino acid sequences set forth in SEQ ID NOs: 237, 238, 239 and 241, 243, 244. In some embodiments, the CD19- specific scFv may comprise a light chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 237, 238, 239. In some embodiments, the CD19-specific scFv may comprise a heavy chain with one or more CDRs having amino acid sequences set forth in SEQ ID NOs: 242, 243, 244. In any of these embodiments, the CD19-specific scFv may comprise one or more CDRs comprising one or more amino acid substitutions, or comprising a sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical), to any of the sequences identified. In some embodiments, the extracellular binding domain of the CD19 CAR comprises or consists of the one or more CDRs as described herein.

[0406] In some embodiments, the linker linking the VH and the VL portions of the scFv is a Whitlow linker having an amino acid sequence set forth in SEQ ID NO: 240. In some embodiments, the Whitlow linker may be replaced by a different linker, for example, a 3xG«S linker having an amino acid sequence set forth in SEQ ID NO: 246, which gives rise to a different FMC63-derived scFv having an amino acid sequence set forth in SEQ ID NO:245. In certain of these embodiments, the CD19-specific scFv comprises or consists of an amino acid sequence set forth in SEQ ID NO:245 or an amino acid sequence that is at least 80% identical (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical) to the amino acid s...

Claims

CLAIMSWe claim:

1. A nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered protein, wherein the one or more nucleic acid sequences comprise a nucleic acid sequence at least 80% identical to SEQID NO: 17, and wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full- length CD47 intracellular domains.

2. A nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered protein, wherein the one or more nucleic acid sequences comprise a nucleic acid sequence at least 80% identical to SEQID NO: 16, and wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full- length CD47 intracellular domains.

3. A nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered protein, wherein the one or more nucleic acid sequences comprise a nucleic acid sequence at least 80% identical to SEQID NO: 15, and wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full- length CD47 intracellular domains.

4. A nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered CD47 protein comprising:(a) one or more extracellular domains; and (b) one or more membrane tethers; wherein the one or more extracellular domains comprise: an amino acid substitution at position S82 of SEQ ID NO: 2, an amino acid substitution at position S64 of SEQ ID NO: 3, an amino acid substitution at position S82 of SEQ ID NO: 1, or an amino acid substitution at position S37 of SEQ ID NO: 6; and / or an amino acid substitution at position S83 of SEQ ID NO: 2, an amino acid substitution at position S65 of SEQ ID NO: 3, an amino acid substitution at position S83 of SEQ ID NO: 1, or an amino acid substitution at position S38 of SEQ ID NO: 6; and / or an insertion of one or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 3 SEQ ID NO: 1 or SEQ ID NO: 6; and wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full-length CD47 intracellular domains.

5. The nucleic acid construct of claim 4, wherein the one or more extracellular domains comprise: an amino acid substitution of S82A relative to SEQ ID NO: 2, an amino acid substitution of S64A relative to SEQ ID NO: 3, an amino acid substitution of S82A relative to SEQ ID NO: 1, or an amino acid substitution of S37A relative to SEQ ID NO: 6.

6. The nucleic acid construct of claim 4, wherein the one or more extracellular domains comprise: an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 2, an amino acid substitution of S64V, S64I, S64L, S64F, S64W, S64G, or S64P relative to SEQ ID NO: 3, an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 1, oran amino acid substitution of S37V, S37I, S37L, S37F, S37W, S37G, or S37P relative to SEQ ID NO: 6.

7. The nucleic acid construct of any one of claims 4-6, wherein the one or more extracellular domains comprise: an amino acid substitution of S83A relative to SEQ ID NO: 2, an amino acid substitution of S65A relative to SEQ ID NO: 3, an amino acid substitution of S83A relative to SEQ ID NO: 1, or an amino acid substitution of S38A relative to SEQ ID NO: 6.

8. The nucleic acid construct of any one of claims 4-6, wherein the one or more extracellular domains comprise: an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 2, an amino acid substitution of S65V, S65I, S65L, S65F, S65W, S65G, or S65P relative to SEQ ID NO: 3, an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 1, or an amino acid substitution of S38V, S38I, S38L, S38F, S38W, S38G, or S38P relative to SEQ ID NO: 6.

9. The nucleic acid construct of any one of claims 4-8, wherein the engineered CD47 protein comprises an insertion of three amino acids relative to SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 6.

10. The nucleic acid construct of any one of claims 4-9, wherein the engineered CD47 protein comprises an insertion of the amino acids WQP relative to SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 6.

11. The nucleic acid construct of any one of claims 4-10, wherein the engineered CD47 protein comprises an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 2 or SEQ ID NO: 1.

12. A nucleic acid construct comprising one or more nucleic acid sequences encoding an engineered CD47 protein, wherein the engineered CD47 protein comprises an insertion of three amino acids relative to SEQ ID NO: 4 or SEQ ID NO: 5.

13. The nucleic acid construct of any one of claims 1-3 and 12, wherein the engineered protein comprises one or more membrane tethers.

14. The nucleic acid construct of any one of claims 4 11 and 13, wherein the one or more membrane tethers are or comprise a transmembrane domain.

15. The nucleic acid construct of claim 14, wherein the transmembrane domain is or comprises a CD3zeta, CD8a, CD16a, CD28, CD32a, CD32c, CD40, CD47, CD64, ICOS, Dectin-1, DNGR1, EGFR, GPCR, MyD88, PDGFR, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, or VEGFR transmembrane domain.

16. The nucleic acid construct of any one of claims 4-11 and 13, wherein the one or more membrane tethers are or comprise a glycosylphosphatidylinositol (GPI) anchor.

17. The nucleic acid construct of claim 16, wherein the GPI anchor is or comprises a DAF / CD55 GPI anchor, a TRAILR3 GPI anchor, or a CD59 GPI anchor.

18. The nucleic acid construct of any one of claims 4-11, wherein the one or more extracellular domains further comprise an extracellular hinge domain.

19. The nucleic acid construct of claim 18, wherein the extracellular hinge domain is or comprises a CD47 hinge, a CD8a hinge, a CD28 hinge, a PDGFR hinge, or an IgG4 hinge.

20. A vector comprising the nucleic acid construct of any one of the preceding claims.

21. An engineered protein encoded by the nucleic acid construct of any one of claims 1-19.

22. An engineered protein: wherein the engineered protein is or comprises an amino acid sequence at least 80% identical to SEQ ID NO: 5, and wherein the engineered protein does not comprise one or more full-length CD47 intracellular domains.

23. An engineered protein: wherein the engineered protein is or comprises an amino acid sequence at least 80% identical to SEQ ID NO: 4 and wherein the engineered protein does not comprise one or more full-length CD47 intracellular domains.

24. An engineered CD47 protein comprising:(a) one or more extracellular domains; and (b) one or more membrane tethers; wherein the one or more extracellular domains comprise: an amino acid substitution at position S82 of SEQ ID NO: 2, an amino acid substitution at position S64 of SEQ ID NO: 3, an amino acid substitution at position S82 of SEQ ID NO: 1, or an amino acid substitution at position S37 of SEQ ID NO: 6; and / or an amino acid substitution at position S83 of SEQ ID NO: 2, an amino acid substitution at position S65 of SEQ ID NO: 3, an amino acid substitution at position S83 of SEQ ID NO: 1, or an amino acid substitution at position S38 of SEQ ID NO: 6; and / or an insertion of one or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 3 SEQ ID NO: 1 or SEQ ID NO: 6; and wherein the nucleic acid construct does not comprise a nucleic acid sequence encoding one or more full-length CD47 intracellular domains.

25. The engineered CD47 protein of claim 24, wherein the one or more extracellular domains comprise: an amino acid substitution of S82A relative to SEQ ID NO: 2, an amino acid substitution of S64A relative to SEQ ID NO: 3, an amino acid substitution of S82A relative to SEQ ID NO: 1, oran amino acid substitution of S37A relative to SEQ ID NO: 6.

26. The engineered CD47 protein of claim 24, wherein the one or more extracellular domains comprise: an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 2, an amino acid substitution of S64V, S64I, S64L, S64F, S64W, S64G, or S64P relative to SEQ ID NO: 3, an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 1, or an amino acid substitution of S37V, S37I, S37L, S37F, S37W, S37G, or S37P relative to SEQ ID NO: 6.

27. The engineered CD47 protein of any one of claims 24-26, wherein the one or more extracellular domains comprise: an amino acid substitution of S83A relative to SEQ ID NO: 2, an amino acid substitution of S65A relative to SEQ ID NO: 3, an amino acid substitution of S83A relative to SEQ ID NO: 1, or an amino acid substitution of S38A relative to SEQ ID NO: 6.

28. The engineered CD47 protein of any one of claims 24-26, wherein the one or more extracellular domains comprise: an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 2, an amino acid substitution of S65V, S65I, S65L, S65F, S65W, S65G, or S65P relative to SEQ ID NO: 3, an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 1, or an amino acid substitution of S38V, S38I, S38L, S38F, S38W, S38G, or S38P relative to SEQ ID NO: 6.

29. The engineered CD47 protein of any one of claims 24, 25, or 27, wherein the one or more extracellular domains comprise: a first amino acid substitution selected from the group consisting of: an amino acid substitution of S82A relative to SEQ ID NO: 2, an amino acid substitution of S64A relative to SEQ ID NO: 3, an amino acid substitution of S82A relative to SEQ ID NO: 1, and an amino acid substitution of S37A relative to SEQ ID NO: 6; and a second amino acid substitution selected from the group consisting of: an amino acid substitution of S83A relative to SEQ ID NO: 2, an amino acid substitution of S65A relative to SEQ ID NO: 3, an amino acid substitution of S83A relative to SEQ ID NO: 1, and an amino acid substitution of S38A relative to SEQ ID NO: 6.

30. The engineered CD47 protein of any one of claims 24, 26, or 28, wherein the one or more extracellular domains comprise: a first amino acid substitution selected from the group consisting of: an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 2, an amino acid substitution of S64V, S64I, S64L, S64F, S64W, S64G, or S64P relative to SEQ ID NO: 3, an amino acid substitution of S82V, S82I, S82L, S82F, S82W, S82G, or S82P relative to SEQ ID NO: 1, and an amino acid substitution of S37V, S37I, S37L, S37F, S37W, S37G, or S37P relative to SEQ ID NO: 6; and a second amino acid substitution selected from the group consisting of: an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, or S83P relative to SEQ ID NO: 2, an amino acid substitution of S65V, S65I, S65L, S65F, S65W, S65G, or S65P relative to SEQ ID NO: 3, an amino acid substitution of S83V, S83I, S83L, S83F, S83W, S83G, orS83P relative to SEQ ID NO: 1, and an amino acid substitution of S38V, S38I, S38L, S38F, S38W, S38G, or S38P relative to SEQ ID NO: 6.

31. The engineered CD47 protein of any one of claims 24-30, wherein the engineered CD47 protein comprises: an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 2 and an amino acid substitution of Q19P relative to SEQ ID NO: 2, or an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 1 and an amino acid substitution of Q19P relative to SEQ ID NO: 1.

32. The engineered CD47 protein of any one of claims 24-31, wherein the engineered CD47 protein comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO: 2, SEQ ID NO: 1 or SEQ ID NO: 6.

33. The engineered CD47 protein of any one of claims 22-23, wherein the engineered protein comprises one or more membrane tethers.

34. The engineered CD47 protein of any one of claims 24-33, wherein the one or more membrane tethers are or comprise a transmembrane domain.

35. The engineered CD47 protein of claim 34, wherein the transmembrane domain is or comprises a CD3zeta, CD8a, CD16a, CD28, CD32a, CD32c, CD40, CD47, CD64, ICOS, Dectin-1, DNGR1, EGFR, GPCR, MyD88, PDGFR, SLAMF7, TRL1, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, TLR9, or VEGFR transmembrane domain.

36. The engineered CD47 protein of any one of claims 24-33, wherein the one or more membrane tethers are or comprise a glycosylphosphatidylinositol (GPI) anchor.

37. The engineered CD47 protein of claim 36, wherein the GPI anchor is or comprises a DAF / CD55 GPI anchor, a TRAILR3 GPI anchor, or a CD59 GPI anchor.

38. A genetically engineered cell comprising the engineered protein of any one of claims 21-37.

39. A genetically engineered cell comprising an engineered CD47 protein, wherein the engineered CD47 protein exhibits: decreased binding with TSP-1 relative to a wild-type CD47 protein; and / or decreased CD47 / TSP-l-mediated signaling relative to a wild-type CD47 protein.

40. The genetically engineered cell of claim 39, wherein the control cell comprises a wild-type cell, an unmodified cell, a cell that has not been engineered to express CD47 or a cell that has been engineered to express wild-type CD47.

41. The genetically engineered cell of claim 39 or 40, wherein the engineered CD47 protein is expressed at a level 1.25x, 1.5x, 1.75x, 2x, 2.25x, 2.5x, 2.75x, 3x, 3.25x, 3.5x, 3.75x, 4x, 4.5x, 5x, 5.5x, 6x, 6.5x, 7x, 7.5x, 8x, 8.5x, 9x,9.5x, lOx, 15x, 2Ox, 25x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, or lOOx the level of wild-type CD47 protein in an unmodified cell.

42. The genetically engineered cell of any one of claims 39-41, wherein the engineered CD47 protein comprises: an amino acid substitution at position S82 of SEQ ID NO: 2, an amino acid substitution at position S64 of SEQ ID NO: 3, an amino acid substitution at position S82 of SEQ ID NO: 1, or an amino acid substitution at position S37 of SEQ ID NO: 6.

43. The genetically engineered cell of any one of claims 39-42, wherein the engineered CD47 protein comprises: an amino acid substitution at position S83 of SEQ ID NO: 2, an amino acid substitution at position S65 of SEQ ID NO: 3, an amino acid substitution at position S83 of SEQ ID NO: 1, or an amino acid substitution at position S38 of SEQ ID NO: 6.

44. The genetically engineered cell of any one of claims 39-43, wherein the engineered CD47 protein comprises: amino acid substitutions at positions S82 and S83 of SEQ ID NO: 2, amino acid substitutions at positions S64 and S65 of SEQ ID NO: 3, amino acid substitutions at positions S82 and S83 of SEQ ID NO: 1, or amino acid substitutions at positions S37 and S38 of SEQ ID NO: 6.

45. The genetically engineered cell of any one of claims 39-44, wherein the engineered CD47 protein comprises: an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6; and an insertion of one or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

46. The genetically engineered cell of claim 45, wherein the engineered CD47 protein comprises an insertion of the amino acids WQP relative to SEQ ID NO: 2, SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.

47. The genetically engineered cell of claim 45 or 46, wherein the engineered CD47 protein comprises an insertion of the amino acids WQP after A18 relative to SEQ ID NO: 2 or SEQ ID NO: 1.

48. The genetically engineered cell of any one of claims 45-47, wherein the engineered CD47 protein comprises an amino acid substitution at position Q19 relative to SEQ ID NO: 2 or SEQ ID NO: 1.

49. The genetically engineered cell of any one of claims 39-48, wherein the engineered CD47 protein comprises an amino acid sequence comprising: an amino acid substitution of S82A relative to SEQ ID NO: 2, an amino acid substitution of S64A relative to SEQ ID NO: 3, an amino acid substitution of S82A relative to SEQ ID NO: 1, or an amino acid substitution of S37A relative to SEQ ID NO: 6;an insertion of the amino acids WQP between A18 and Q19 relative to SEQ ID NO: 2 or SEQ ID: 1; and an amino acid substitution of Q19P relative to SEQ ID NO: 2 or SEQ ID NO: 1.

50. The genetically engineered cell of any one of claims 39-49, wherein the engineered CD47 protein comprises an amino acid sequence comprising: an amino acid substitution of S82A relative to SEQ ID NO: 2; an insertion of the amino acids WQP between A18 and Q19 relative to SEQ ID NO: 2; and an amino acid substitution of Q19P relative to SEQ ID NO: 2.

51. The genetically engineered cell of any one of claims 38-50, wherein the genetically engineered cell comprises a modification at a TCR locus, B2M locus, a TAP I locus, a NLRC5 locus, a CIITA locus, an HLA-A locus, an HLA-B locus, an HLA-C locus, an HLA-DP locus, an HLA-DM locus, an HLA-DOA locus, an HLA-DOB locus, an HLA-DQ locus, an HLA-DR locus, a RFX5 locus, a RFXANK locus, a RFXAP locus, an NFY-A locus, an NFY-B locus, an NFY-C locus, or a combination thereof.

52. The genetically engineered cell of any one of claims 38-51, wherein the cell comprises one or more modifications that inactivate or disrupt one or more alleles of:(a) one or more major histocompatibility complex (MHC) class I molecules or one or more molecules that regulate expression of the one or more MHC class I molecules, and / or(b) one or more MHC class II molecules or one or more molecules that regulate expression of the one or more MHC class II molecules.

53. The genetically engineered cell of any one of claims 38-52, wherein the genetically engineered cell has been genetically engineered to knock-out a B2M locus.

54. The genetically engineered cell of any one of claims 38-53, wherein the genetically engineered cell has been genetically engineered to knock-out a CIITA locus.

55. The genetically engineered cell of any one of claims 38-54, wherein the cell is an islet cell, beta islet cell, pancreatic islet cell, immune cell, B cell, T cell, natural killer (NK) cell, natural killer T (NKT) cell, macrophage cell, endothelial cell, muscle cell, cardiac muscle cell, smooth muscle cell, skeletal muscle cell, dopaminergic neuron, retinal pigmented epithelium cell, optic cell, hepatocyte, thyroid cell, skin cell, glial progenitor cell, neural cell, cardiac cell, stem cell, hematopoietic stem cell, induced pluripotent stem cell (iPSC), mesenchymal stem cell (MSC), embryonic stem cell (ESC), pluripotent stem cell (PSC), blood cell, or a combination thereof.

56. The genetically engineered cell of any one of claims 38-55, wherein the genetically engineered cell is a differentiated cell derived from a pluripotent stem cell.

57. The genetically engineered cell of claim 56, wherein the pluripotent stem cell is an induced pluripotent stem cell (iPSC) or an embryonic stem cell (ESC).

58. The genetically engineered cell of any one of claims 38-57, wherein the cell is a primary cell isolated from a donor.

59. The genetically engineered cell of any one of claims 38-58, wherein the cell is an islet cell, beta islet cell, or pancreatic islet cell.

60. The genetically engineered cell of any one of claims 38-58, wherein the cell is an allogeneic T-cell.

61. The genetically engineered cell of claim 60, wherein the allogeneic T-cell is a primary T cell.

62. The genetically engineered cell of claim 60 or 61, wherein the genetically engineered cell has decreased cell surface expression of a TCR as compared to a comparable cell that has not been genetically engineered.

63. The genetically engineered cell of any one of claims 38-62, further comprising a transgene encoding a chimeric antigen receptor (CAR).

64. The genetically engineered cell of claim 63, wherein the CAR is or comprises a CD5-specific CAR, a CD19- specific CAR, a CD20-specific CAR, a CD22-specific CAR, a CD23-specific CAR, a CD30-specific CAR, a CD33-specific CAR, CD38-specific CAR, a CD70-specific CAR, a CD123-specific CAR, a CD138-specific CAR, a Kappa, Lambda, B cell maturation agent (BCMA)-specific CAR, a G-protein coupled receptor family C group 5 member D (GPRC5D)-specific CAR, a CD123-specific CAR, a LeY-specific CAR, a NKG2D ligand-specific CAR, a WTl-specific CAR, a GD2-specific CAR, a HER2-specific CAR, a EGFR-specific CAR, a EGFRvIII-specific CAR, a B7H3-specific CAR, a PSMA-specific CAR, a PSCA- specific CAR, a CAIX-specific CAR, a CD171-specific CAR, a CEA-specific CAR, a CSPG4-specific CAR, a EPHA2 -specific CAR, a FAP-specific CAR, a FRa-specific CAR, a IL-13Ra-specific CAR, a Mesothelin-specific CAR, a MUCl-specific CAR, a MUC16-specific CAR, a RORl-specific CAR, a C-Met-specific CAR, a CD133-specific CAR, a Ep-CAM-specific CAR, a GPC3- specific CAR, a HPV16-E6-specific CAR, a IL13Ra2-specific CAR, a MAGEA3-specific CAR, a MAGEA4-specific CAR, a MARTl-specific CAR, a NY-ESO-l-specific CAR, a VEGFR2-specific CAR, a a-Folate receptor-specific CAR, a CD24-specific CAR, a CD44v7 / 8-specific CAR, a EGP-2-specific CAR, a EGP-40-specific CAR, a erb-B2-specific CAR, a erb-B 2,3,4- specific CAR, a FBP-specific CAR, a Fetal acethylcholine e receptor-specific CAR, a Goz-specific CAR, a Goa-specific CAR, a HMW-MAA-specific CAR, a IL-llRa-specific CAR, a KDR-specific CAR, a Lewis Y-specific CAR, a Ll-cell adhesion molecule-specific CAR, a MAGE-Al-specific CAR, a Oncofetal antigen (h5T4)-specific CAR, a TAG-72-specific CAR, or a CD19 / CD22-bispecific CAR.

65. A composition comprising the genetically engineered cell of any one of claims 38-64.

66. A pharmaceutical composition comprising (i) the genetically engineered cell of any one of claims 38-64, and (ii) a pharmaceutically acceptable excipient.

67. A method comprising administering to a subject the genetically engineered cell of any one of claims 38-64, the composition of claim 65, or the pharmaceutical composition of claim 66.

68. A method of treating a disease in a subject, the method comprising administering to the subject the genetically engineered cell of any one of claims 38-64, the composition of claim 65, or the pharmaceutical composition of claim 66.

69. A population of cells comprising the genetically engineered cell of any one of claims 38-64 for use in treating a disease in a subject.

70. Use of the genetically modified cell of any one of claims 38-64, the population of cells of claim 69, the composition of claim 65, or the pharmaceutical composition of claim 66 for use in treating a disease in a subject.

71. Use of the genetically modified cell of any one of claims 38-64, the population of cells of claim 69, the composition of claim 65, or the pharmaceutical composition of claim 66 in the manufacture of a medicament for the treatment of a disease.

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