Macrophage-specific promoters and uses thereof

Abstract

Described herein are compositions and methods for regulating expression of effector molecules using engineered macrophage-specific promoters. Immunoresponsive cells (such as macrophages) comprising the same are also described.

Description

MACROPHAGE-SPECIFIC PROMOTERS AND USES THEREOFCROSS REFERENCE TO RELATED APPLICATIONS.

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 554,586, filed February 16, 2024, 63 / 565,983, filed March 15, 2024, 63 / 664,122, filed June 25, 2024, 63 / 554,605, filed February 16, 2024, and 63 / 566,003, filed March 15, 2024, each of which are hereby incorporated by reference in their entirety for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on Month XX, 20XX, is named XXXXXUS_sequencelisting.xml, and is X,XXX,XXX bytes in size.BACKGROUND

[0003] Cell-based therapy platforms provide promising avenues for treating a variety of diseases. Engineering of macrophages as cell therapies and drug delivery vehicles has become prominent as a potential immunotherapy. These engineered macrophages are typically genetically modified to express therapeutic payloads, such as but not limited to, checkpoint inhibitors (e.g., PD-1 / PD-L1 binders, SIRPa or CD47 blockers, etc.), immunomodulatory cytokines (e.g., interferons or interleukins), chimeric antigen receptors and / or other immune regulatory elements.

[0004] Gene regulatory elements, such as promoters and enhancers, can possess cell-type or state specific activities which provide controlled and restricted expression of certain genes in a particular target cell, while minimizing or excluding expression of certain genes in surrounding non-target cells. Such cell-type specific regulatory elements can be adapted for use in therapies, such as cell and gene therapies. However, problems with transcriptional strength and specificity persist in known cell-specific regulatory systems. Accordingly, there is a need for improved cell-specific and / or state-selective regulatory elements for use in therapy.SUMMARY

[0005] This disclosure provides polarization-state specific promoters which enable the controlled expression of payloads only when macrophages encounter a given polarization cue. These polarization-state specific promoters not only provide selective payload expression but can also be used for controlled expression of payloads to prevent unwanted macrophage polarization plasticity, or to guide macrophages to a desired macrophage state.

[0006] The present disclosure provides for, among other things, an engineered enhancer comprising a nucleotide sequence having at least 75%, 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 one or more of SEQ ID NOs: 17-31, wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or M0 macrophage.

[0007] In some embodiments, a nucleotide sequence is at least 96% identical to any one of: SEQ ID NOs: 17-31. In some embodiments, a nucleotide sequence is 100% identical to any one of: SEQ ID NOs: 17-31.

[0008] In some embodiments, a macrophage specific promoter further comprises a spacer sequence. In some embodiments, a spacer sequence is between the engineered enhancer and a minimal promoter.

[0009] In some embodiments, an engineered macrophage specific promoter is selected from the group consisting of SEQ ID NOs: 225-239.

[0010] The present disclosure provides for, among other things, an engineered enhancer comprising: a) a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NOs: 2-16, and 192-194, wherein the nucleotide sequence does not comprise SEQ ID NO: 1; or b) a nucleotide sequence at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to any one or more of SEQ ID NOs: 2-16, and 192-194; wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or M0 macrophage.

[0011] In some embodiments, a nucleotide sequence is at least 96% identical to any one of: SEQ ID NOs: 2-16, and 192-194. In some embodiments, a nucleotide sequence is 100% identical to any one of: SEQ ID NOs: 2-16, and 192-194.

[0012] In some embodiments, an engineered enhancer comprises: a) a nucleotide sequence at least 91.5% identical to SEQ ID NO: 2, wherein the engineered enhancer does not comprise the sequence TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168); b) a nucleotide sequence at least 96.4% identical to SEQ ID NO: 3, wherein the engineered enhancer does not comprise the sequence TGTGACTCACA (SEQ ID NO: 169); c) a nucleotide sequence at least 94.8% identical to SEQ ID NO: 4, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCC (SEQ ID NO: 170); d) a nucleotide sequence at least 98.4% identical to SEQ ID NO: 5, wherein the engineered enhancer does not comprise the sequence ATAAAT (SEQ ID NO: 171); e) a nucleotide sequence at least 89.8% identical to SEQ ID NO: 6, wherein the engineered enhancer does not comprise the sequence TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172); f) a nucleotide sequence at least 92.8% identical to SEQ ID NO: 7, wherein the engineered enhancer does not comprise the sequence TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173); g) a nucleotide sequence at least 90.4% identical to SEQ ID NO: 8, wherein the engineered enhancer does not comprise the sequence AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174); h) a nucleotide sequence at least 93.5% identical to SEQ ID NO: 9, wherein the engineered enhancer does not comprise the sequenceCAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175); i) a nucleotide sequence at least 91.6% identical to SEQ ID NO: 10, wherein the engineered enhancer does not comprise the sequence CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176); j) a nucleotide sequence at least 90% identical to SEQ ID NO: 11, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177); k) a nucleotide sequence at least 91.2% identical to SEQ ID NO: 12, wherein the engineered enhancer does not comprise the sequence TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178); or 1) a nucleotide sequence at least 91.6% identical to SEQ ID NO: 13, wherein the engineered enhancer does not comprise the sequence AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179).

[0013] The present disclosure further provides for an engineered enhancer comprising a variant of SEQ ID NO: 1, wherein the variant of SEQ ID NO: 1 comprises an ablation of any one or more regions selected from: (a) positions 76-110 of SEQ ID NO: 1; (b) positions 122-132 of SEQ ID NO: 1; (c) positions 157-172 of SEQ ID NO: 1; (d) positions 1-6 of SEQ ID NO: 1; (e) positions 7-36 of SEQ ID NO: 1; (f) positions 37-66 of SEQ ID NO: 1; (g) positions 67-96 of SEQ ID NO: 1; (h) positions 97-126 of SEQ ID NO: 1; (i) positions 127-156 of SEQ ID NO: 1; (j) positions 157-186 of SEQ ID NO: 1; (k) positions 187-216 of SEQ ID NO: 1; and (1) positions 217-246 of SEQ ID NO: 1; wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or M0 macrophage.

[0014] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 76-110 of SEQ ID NO: 1, wherein the sequence corresponding to position 76-110 of SEQ ID NO: 1 is: TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168).

[0015] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 122-132 of SEQ ID NO: 1, wherein the sequence corresponding to position 122-132 of SEQ ID NO: 1 is: TGTGACTCACA (SEQ ID NO: 169).

[0016] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 157-172 of SEQ ID NO: 1, wherein the sequence corresponding to position 157-172 of SEQ ID NO: 1 is: GGGGACACAATGTTCC (SEQ ID NO: 170).

[0017] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 1-6 of SEQ ID NO: 1, wherein the sequence corresponding to position 1-6 of SEQ ID NO: 1 is: ATAAAT (SEQ ID NO: 171).

[0018] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 7-36 of SEQ ID NO: 1, wherein the sequence corresponding to position 7-36 of SEQ ID NO: 1 is: TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172).

[0019] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 37-66 of SEQ ID NO: 1, wherein the sequence corresponding to position 37-66 of SEQ ID NO: 1 is: TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173).

[0020] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 67-96 of SEQ ID NO: 1, wherein the sequence corresponding to position 67-96 of SEQ ID NO: 1 is: AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174).

[0021] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 97-126 of SEQ ID NO: 1, wherein the sequence corresponding to position 97-126 of SEQ ID NO: 1 is: CAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175).

[0022] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 127-156 of SEQ ID NO: 1, wherein the sequence corresponding to position 127-156 of SEQ ID NO: 1 is: CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176).

[0023] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 157-186 of SEQ ID NO: 1, wherein the sequence corresponding to position 157-186 of SEQ ID NO: 1 is: GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177).

[0024] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 187-216 of SEQ ID NO: 1, wherein the sequence corresponding to position 187-216 of SEQ ID NO: 1 is: TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178).

[0025] In some embodiments, an engineered enhancer comprises a sequence corresponding to position 217-246 of SEQ ID NO: 1, wherein the sequence corresponding to position 217-246 of SEQ ID NO: 1 is: AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179).

[0026] The present disclosure provides for an engineered macrophage specific promoter comprising an engineered enhancer as described herein operably linked to a minimal promoter.

[0027] In some embodiments, a minimal promoter is selected from: a hybrid YBTATA-SCP3 (“YB-SCP3”), SCP3, SCP3 containing DPR, minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB TATA, minTK, inducer molecule responsive promoters, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb, and tandem repeats thereof. In some embodiments, a minimal promoter is YB-SCP3. In some embodiments, a minimal promoter comprises the sequenceTCTAGAGGGTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGTCCGCCTGGAGACCTCGAGCCGAGTGGTCGTGCCTCCATAGAA (SEQ ID NO: 50). In some embodiments, a minimal promoter comprises the sequence CGGATCAACTTCTAGAGGGTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGTCCGC

[0028] The present disclosure also provides for a heterologous construct comprising: a) an engineered enhancer as described herein or an engineered macrophage-specific promoter as described herein; and b) a heterologous payload, wherein the engineered enhancer or engineered promoter is operably linked to the heterologous payload.

[0029] In some embodiments, a heterologous payload comprises a polynucleotide. In some embodiments, a polynucleotide comprises a nucleotide sequence encoding a polypeptide. In some embodiments, a polypeptide comprises at least one effector molecule. In some embodiments, a polypeptide comprises a first effector molecule and a second effector molecule. In some embodiments, a polynucleotide comprises a nucleotide sequence encoding the first effector molecule, a linker nucleotide sequence, and a nucleotide sequence encoding the second effector.

[0030] In some embodiments, a linker nucleotide sequence encodes one or more 2A ribosome skipping elements. In some embodiments, one or more 2A ribosome skipping elements comprise elements that are each selected from the group consisting of: P2A, T2A, E2A, and F2A.

[0031] In some embodiments, at least one effector molecule (e.g., first effector molecule, second effector molecule, etc.) or each effector molecule used in accordance with the present disclosure is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a polynucleotide molecule, a co-activation molecule, a tumor microenvironment modifier, a receptor, a ligand, a transcription factor, an antibody, a peptide, and an enzyme. In some embodiments, at least one effector molecule or each effector molecule used in accordance with the present disclosure is a human-derived effector molecule.

[0032] The present disclosure provides for a heterologous construct for inducing a macrophage to transition from an M2 state to an Ml state, comprising: a) an engineered enhancer as described herein or an engineered macrophage-specific promoter as described herein; and b) a heterologous payload encoding a master regulator of polarization to an Ml macrophage, wherein the engineered enhancer or the engineered macrophage-specific promoter of a) is operably linked to the heterologous pay load and configured to induce expression of the heterologous payload.

[0033] In some embodiments, a master regulator of polarization to an Ml macrophage is a cytokine. In some embodiments, a cytokine is IFNgamma, IFNalpha, TNF alpha, GM-CSF, IL- 12, IL-12p70, IL-12p40, IL-12p35, IL-6, IL-23, IL-lalpha, IL- 1 beta, or a derivative thereof. In some embodiments, a cytokine is modified to comprise a membrane tethering domain. In some embodiments, a membrane tethering domain is or comprises a transmembrane-intracellular domain and / or transmembrane domain of a protein selected from: PDGFR-beta, CDS, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA, or a functional portion thereof. In some embodiments, a membrane tethering domain is or comprises a transmembrane domain of B7-1 protein, or a functional portion thereof.

[0034] In some embodiments, a master regulator of polarization to an Ml macrophage is a transcription factor selected from IRF7 or a derivative thereof, or p65 / RelA or a derivative thereof.

[0035] The present disclosure further provides for a heterologous construct for stabilizing a macrophage in an M2 polarization state, comprising: a) an engineered enhancer as described herein or an engineered macrophage-specific promoter as described herein; and b) a heterologous payload encoding a master regulator of polarization to an M2 macrophage, wherein the engineered enhancer or the engineered macrophage-specific promoter of a) is operably linked to the heterologous pay load and configured to induce expression of the heterologous payload.

[0036] In some embodiments, a master regulator of polarization to an M2 macrophage is IL- 10, IL-4, IL-13, IL-21, TGF-beta, M-CSF, or a derivative thereof.

[0037] In some embodiments, a cytokine is modified to comprise a membrane tethering domain. In some embodiments, a membrane tethering domain is or comprises a transmembrane- intracellular domain and / or transmembrane domain of a protein selected from: PDGFR-beta, CDS, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA, or a functional portion thereof. In some embodiments, a membrane tethering domain is or comprises a transmembrane domain of B7-1 protein, or a functional portion thereof.

[0038] In some embodiments, an M2 state is an M2c state, an M2a state, or an M2b state.

[0039] The present disclosure also provides for a vector comprising a heterologous construct as described herein.

[0040] The present disclosure provides for a dual expression vector comprising a heterologous construct as described herein and a second construct comprising a nucleotide sequence encoding an activating immune receptor.

[0041] The present disclosure provides for an immunoresponsive cell comprising a heterologous construct as described herein, a vector as described herein, or a dual expression vector as described herein.

[0042] In some embodiments, an immunoresponsive cell is selected from the group consisting of: a macrophage, a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (HL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC- derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell. In some embodiments, an immunoresponsive cell is a macrophage. In some embodiments, a macrophage is a tumor-resident macrophage.

[0043] In some embodiments, an immunoresponsive cell expresses an activating immune receptor. In some embodiments, an activating immune receptor comprises an antigen recognizing receptor.

[0044] In some embodiments, an immunoresponsive cell is autologous. In some embodiments, an immunoresponsive cell is allogeneic.

[0045] The present disclosure further provides for a pharmaceutical composition comprising a vector as described herein, a dual expression vector as described herein, or an immunoresponsive cell as described herein, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

[0046] The present disclosure provides for a method of increasing expression of a target gene, the method comprising use of an engineered enhancer or an engineered macrophagespecific promoter as described herein, a vector as described herein, or a dual expression vector as described herein to increase expression of the target gene.

[0047] In some embodiments, a target gene is an immunomodulatory gene.

[0048] The present disclosure provides for a method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of a vector asdescribed herein, a dual expression vector as described herein, an immunoresponsive cell as described herein, or a pharmaceutical composition as described herein.

[0049] The present disclosure provides for a kit for treating and / or preventing a disease or disorder, comprising an immunoresponsive cell as described herein or a pharmaceutical composition as described herein.

[0050] In some embodiments, a disease or disorder comprises a tumor.

[0051] In some embodiments, a kit further comprises written instructions for using an immunoresponsive cell as described herein or a pharmaceutical composition as described herein for treating and / or preventing the disease or disorder in a subjectBRIEF DESCRIPTION OF THE DRAWINGS

[0052] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fees.

[0053] FIG. 1 depicts macrophage polarization to Ml or M2 states, and phenotype plasticity of macrophages between Ml and M2 states.

[0054] FIG. 2 depicts exemplary promoter system designs for keeping macrophages in a stable M2 state or directing Ml macrophages from an Ml phenotype to M2 phenotype.

[0055] FIG. 3 depicts exemplary promoter system designs for keeping macrophages in a stable Ml state or directing M2 macrophages from an M2 phenotype to Ml phenotype.

[0056] FIG. 4A and FIG. 4B depict state-selective promoter activity and strength of engineered M2 promoters, derived from ATAC-Seq nominated enhancers.

[0057] FIG. 5 depicts state-selective promoter activity and strength of engineered M2 promoters, derived from re-engineered ATAC-Seq nominated enhancers.

[0058] FIG. 6 depicts promoter activity of Enhancers 1-8 paired with alternative core promoters, minPros 1-6.

[0059] FIG. 7 depicts state-selective promoter activity and strength of select enhancers (selected from Enhancers 1-8) paired with alternative core promoters (selected from minPros 1- 6).

[0060] FIG. 8 depicts state-selective promoter activity and strength of select enhancers (selected from Enhancers 1-8) paired with alternative core promoters (selected from minPros 1- 6).

[0061] FIG. 9 depicts state selective promoter activity and strength of ablation variants ofSB11760.

[0062] FIG. 10 depicts state selective promoter activity and strength of ablation variants ofSB11760.

[0063] FIG. 11 depicts state selective promoter activity and strength of first and third generation of state-selective promoters.

[0064] FIG. 12 depicts state selective promoter activity and strength of next generation enhancer constructs and native enhancer constructs in Ml, Ml, and M2c macrophages.

[0065] FIG. 13 A depicts state selective promoter activity and strength of 1* 3rdand 4thgeneration enhancers, where the radius of the datapoint corresponds to the M2c promoter strength as compared to EFS, a control promoter. FIG. 13B depicts the same data s FIG. 13A, where the data is colored to distinguish which enhancer families are used to generate the next generation promoters.DETAILED DESCRIPTIONDefinitions

[0066] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0067] The terms “macrophage-specific,” “polarization-specific,” “state-selective,” and “polarization state-specific” enhancer or promoter are used interchangeably herein to refer to an enhancer or promoter that is determined to have higher activity in one macrophage polarization state over another macrophage polarization state. Macrophages can transition between different polarization states, such as the Ml macrophage or M2 macrophage polarization state. For example, an Ml macrophage-specific enhancer or promoter can have higher activity in a macrophage in the Ml polarization state compared to a macrophage in the M2 polarization state and / or M0 polarization state. For example, an M2 macrophage-specific enhancer or promoter can have higher activity in a macrophage in the M2 polarization state compared to a macrophage in the Ml and / or M0 polarization state. Polarization of M2 macrophages can transition M2 macrophages into different M2 macrophage subtypes depending on the stimulatory cues. These can include, but are not limited to, M2a, M2b, or M2c subtypes.

[0068] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0069] The term “in vitro" refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[0070] The term “in vivo" refers to processes that occur in a living organism.

[0071] The term “mammal” as used herein includes both humans and non-human animals, and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0072] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill), or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0073] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are set. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0074] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTF1T, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0075] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0076] The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0077] The term “therapeutically effective amount’ ’ is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactically effective amount” as prophylaxis can be considered therapy.

[0078] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0079] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.Polarization-Specific Enhancers and Promoters

[0080] Macrophages are white blood cells that phagocytose and degrade cellular debris, foreign substances, microbes, cancer cells, etc.. In addition to their role in phagocytosis, these cells play an important role in development, tissue maintenance and repair, and in both innate and adaptive immunity in that they recruit and influence other cells including immune cells such as lymphocytes. Macrophages can exist in many phenotypes (also referred to herein as polarization states), including phenotypes that have been referred to as Ml and M2. Macrophages that perform primarily pro-inflammatory functions are called Ml macrophages. Such Ml macrophages can be CD86+ / CD68+. Macrophages that decrease inflammation and encourage and regulate tissue repair are called M2 macrophages. Such macrophages can be CD206+ / CD68+. Engineering of macrophages is described, e.g., in WO2017044487, BrempelisKJ et al. J Immunother Cancer. 2020;8(2):e001356, and Xia et al., Adv. Mater. 2020, 32, 2002054.

[0081] In one aspect, described herein are polarization state-specific enhancers and promoters (e.g., Ml, M2, M0) for use in engineered cells, e.g., engineered macrophages. Such state-specific enhancers and promoters are usefill, e.g., in modulating transcriptional activity and inducing expression of desired payloads in a state-selective manner, e.g., when the macrophage is in a desired polarization state. For instance, M2-specific enhancers and promoters can be useful in selectively inducing expression of desired payloads in M2 macrophages as compared to Ml or M0 macrophages.

[0082] Accordingly, in one aspect, described herein are engineered enhancers, wherein the engineered enhancers induce greater transcriptional activity in an M2 macrophage as compared to an Ml or M0 macrophage. In some embodiments, an engineered enhancer used in accordance with the present disclosure can be any engineered enhancer described herein, e.g., any of those described in Table 10. An engineered enhancer of the present disclosure may comprise a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NOs: 17-31.

[0083] In some embodiments, the nucleotide sequence is at least 96% identical to any one of: SEQ ID NOs: 17-31. In some embodiments, the nucleotide sequence is at least 97% identical to any one of: SEQ ID NOs: 17-31. In some embodiments, the nucleotide sequence is at least 98% identical to any one of: SEQ ID NOs: 17-31. In some embodiments, the nucleotide sequence is at least 99% identical to any one of: SEQ ID NOs: 17-31. In some embodiments, the nucleotide sequence is 100% identical to any one of: SEQ ID NOs: 17-31.

[0084] In some embodiments, a polarization state-specific enhancer can be an ablation variant of a reference enhancer, e.g., a variant of SEQ ID NO: 1 (see Table 1). In some embodiments, a polarization state-specific enhancer used in accordance with the present disclosure can be any engineered enhancer described herein, e.g., any of those described in Table 1.

[0085] Accordingly, in one aspect, described herein are engineered enhancers, wherein the engineered enhancers induce greater transcriptional activity in an M2 macrophage as compared to an Ml or M0 macrophage. An engineered enhancer of the present disclosure may comprise(a) a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NOs: 2-16, and 192-194, wherein the nucleotide sequence does not comprise SEQ ID NO: 1; or (b) a nucleotide sequence at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to any one or more of SEQ ID NOs: 2-16, and 192-194.

[0086] In some embodiments, the nucleotide sequence is at least 96% identical to any one of: SEQ Id NOs: 2-16, and 192-194. In some embodiments, the nucleotide sequence is at least 97% identical to any one of: SEQ ID NOs: 2-16, and 192-194. In some embodiments, the nucleotide sequence is at least 98% identical to any one of: SEQ ID NOs: 2-16, and 192-194. In some embodiments, the nucleotide sequence is at least 99% identical to any one of: SEQ ID NOs: 2- 16, and 192-194. In some embodiments, the nucleotide sequence is 100% identical to any one of: SEQ ID NOs: 2-16, and 192-194.

[0087] In some embodiments, the engineered enhancer comprises: a) a nucleotide sequence at least 91.5% identical to SEQ ID NO: 2, wherein the engineered enhancer does not comprise the sequence TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168); b) a nucleotide sequence at least 96.4% identical to SEQ ID NO: 3, wherein the engineered enhancer does not comprise the sequence TGTGACTCACA (SEQ ID NO: 169); c) a nucleotide sequence at least 94.8% identical to SEQ ID NO: 4, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCC (SEQ ID NO: 170); d) a nucleotide sequence at least 98.4% identical to SEQ ID NO: 5, wherein the engineered enhancer does not comprise the sequence ATAAAT (SEQ ID NO: 171); e) a nucleotide sequence at least 89.8% identical to SEQ ID NO: 6, wherein the engineered enhancer does not comprise the sequence TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172); f) a nucleotide sequence at least 92.8% identical to SEQ ID NO: 7, wherein the engineered enhancer does not comprise the sequence TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173);g) a nucleotide sequence at least 90.4% identical to SEQ ID NO: 8, wherein the engineered enhancer does not comprise the sequence AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174); h) a nucleotide sequence at least 93.5% identical to SEQ ID NO: 9, wherein the engineered enhancer does not comprise the sequence CAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175); i) a nucleotide sequence at least 91.6% identical to SEQ ID NO: 10, wherein the engineered enhancer does not comprise the sequence CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176); j) a nucleotide sequence at least 90% identical to SEQ ID NO: 11, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177); k) a nucleotide sequence at least 91.2% identical to SEQ ID NO: 12, wherein the engineered enhancer does not comprise the sequence TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178); or1) a nucleotide sequence at least 91.6% identical to SEQ ID NO: 13, wherein the engineered enhancer does not comprise the sequence AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179).

[0088] In some aspects, an engineered enhancer of the present disclosure that induces greater transcriptional activity in an M2 macrophage as compared to an Ml or MO macrophage may comprise a variant of SEQ ID NO: 1. The variant of SEQ ID NO: 1 may comprise an ablation of any one or more regions selected from: (a) positions 76-110 of SEQ ID NO: 1; (b) positions 122- 132 of SEQ ID NO: 1; (c) positions 157-172 of SEQ ID NO: 1; (d) positions 1-6 of SEQ ID NO: 1; (e) positions 7-36 of SEQ ID NO: 1; (f) positions 37-66 of SEQ ID NO: 1; (g) positions 67-96 of SEQ ID NO: 1; (h) positions 97-126 of SEQ ID NO: 1; (i) positions 127-156 of SEQ ID NO: 1; (j) positions 157-186 of SEQ ID NO: 1; (k) positions 187-216 of SEQ ID NO: 1; and (1) positions 217-246 of SEQ ID NO: 1. In some embodiments, the engineered enhancer comprises a sequence corresponding to position 76-110 of SEQ ID NO: 1, wherein the sequence corresponding to position 76-110 of SEQ ID NO: 1 is: TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 122-132of SEQ ID NO: 1, wherein the sequence corresponding to position 122-132 of SEQ ID NO: 1 is: TGTGACTCACA (SEQ ID NO: 169). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 157-172 of SEQ ID NO: 1, wherein the sequence corresponding to position 157-172 of SEQ ID NO: 1 is: GGGGACACAATGTTCC (SEQ ID NO: 170). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 1-6 of SEQ ID NO: 1, wherein the sequence corresponding to position 1-6 of SEQ ID NO: 1 is: ATAAAT (SEQ ID NO: 171). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 7-36 of SEQ ID NO: 1, wherein the sequence corresponding to position 7-36 of SEQ ID NO: 1 is: TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 37-66 of SEQ ID NO: 1, wherein the sequence corresponding to position 37-66 of SEQ ID NO: 1 is: TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 67-96 of SEQ ID NO: 1, wherein the sequence corresponding to position 67-96 of SEQ ID NO: 1 is: AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 97-126 of SEQ ID NO: 1, wherein the sequence corresponding to position 97-126 of SEQ ID NO: 1 is: CAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 127-156 of SEQ ID NO: 1, wherein the sequence corresponding to position 127-156 of SEQ ID NO: 1 is: CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 157-186 of SEQ ID NO: 1, wherein the sequence corresponding to position 157-186 of SEQ ID NO: 1 is: GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 187-216 of SEQ ID NO: 1, wherein the sequence corresponding to position 187-216 of SEQ ID NO: 1 is: TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178). In some embodiments, the engineered enhancer comprises a sequence corresponding to position 217-246 of SEQ ID NO: 1, wherein the sequence corresponding to position 217-246 of SEQ ID NO: 1 is: AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179).

[0089] In another aspect, described herein are engineered enhancers, wherein the engineered enhancers induce greater transcriptional activity in an M2 macrophage as compared to an Ml or MO macrophage. In some embodiments, the engineered enhancer of the present disclosure comprises a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NOs: 24-26, 194.

[0090] In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 75%, 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 SEQ ID NO: 24. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 24. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 24. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 24. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 24. In some embodiments, the engineered enhancer comprises a nucleotide sequence identical to SEQ ID NO: 24. In some embodiments, the engineered enhancer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 24.

[0091] In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 75%, 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 SEQ ID NO: 25. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 25. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 25. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 25. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 25. In some embodiments, the engineered enhancer comprises a nucleotide sequence identical to SEQ ID NO: 25. In some embodiments, the engineered enhancer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 25.

[0092] In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 75%, 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 SEQ ID NO: 26. In some embodiments, theengineered enhancer comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 24. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 26. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 26. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 26. In some embodiments, the engineered enhancer comprises a nucleotide sequence identical to SEQ ID NO: 26. In some embodiments, the engineered enhancer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 26.

[0093] In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NO: 194. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 194. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 194. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 194. In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 194. In some embodiments, the engineered enhancer comprises a nucleotide sequence identical to SEQ ID NO: 194. In some embodiments, the engineered enhancer comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 194.

[0094] In another embodiment, described herein the engineered enhancers can be combined with a minimal promoter and / or a spacer sequence, thereby forming an engineered promoter of the present disclosure. In some embodiments, the engineered promoter of the present disclosure comprises a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NOs: 232-234, and 262.

[0095] In some embodiments, the engineered enhancer comprises a nucleotide sequence at least 75%, 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 SEQ ID NO: 232. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 232. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 97%identical to SEQ ID NO: 232. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 232. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 232. In some embodiments, the engineered promoter comprises a nucleotide sequence identical to SEQ ID NO: 232. In some embodiments, the engineered promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 232.

[0096] In some embodiments, the engineered promoter comprises a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NO: 233. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 233. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 233. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 233. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 233. In some embodiments, the engineered promoter comprises a nucleotide sequence identical to SEQ ID NO: 233. In some embodiments, the engineered promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 233.

[0097] In some embodiments, the engineered promoter comprises a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NO: 234. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 234. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 234. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 234. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 234. In some embodiments, the engineered promoter comprises a nucleotide sequence identical to SEQ ID NO: 234.1n some embodiments, the engineered promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 234.

[0098] In some embodiments, the engineered promoter comprises a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NO: 262. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 96% identical to SEQ ID NO: 262. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 97% identical to SEQ ID NO: 262. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 98% identical to SEQ ID NO: 262. In some embodiments, the engineered promoter comprises a nucleotide sequence at least 99% identical to SEQ ID NO: 262. In some embodiments, the engineered promoter comprises a nucleotide sequence identical to SEQ ID NO: 262. In some embodiments, the engineered promoter comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different compared to SEQ ID NO: 262.

[0099] As used herein, an “ablation” refers to a deletion, using any means of nucleotide deletion as known in the art (e.g., molecular cloning, CRISPR, etc.). Ablation may further comprise replacement of a region of the nucleotide sequence with a transcriptionally inert segment, e.g., of the same length. In some embodiments, ablation of the region increases activity and / or selectivity of the promoter (e.g., transcriptional levels downstream of the promoter following stimulation), wherein the increased activity and / or selectivity is relative to the promoter lacking such ablation.

[0100] Methods of quantifying transcriptional levels are known in the art and include, for example and without limitation, mRNA analysis by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), fluorescent reporters, colorimetric reporters, etc. In some embodiments, the ablation increases inducibility by at least 0.5-fold, at least 1-fold, at least 2- fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, or at least 8-fold. It is contemplated herein that changes in inducibility of the engineered promoter may depend on the test system, e.g., the cell type comprising the promoter.

[0101] In some embodiments, the ablation comprises a substitution of a second nucleotide motif (also referred to herein as “replacement sequence”) at the site of ablation. In some embodiments, introduction of the second nucleotide motif in the ablation site does not introduce new regulatory sites in the engineered promoter (e.g., transcription factor binding sites). Suchengineered promoter comprising a deletion or a substitution of a nucleotide motif at the site of ablation are referred to herein as “ablation variants.”

[0102] Sequences of exemplary engineered polarization specific enhancers are shown in Table 1. Sequences of ablated regions of SEQ ID NO: 1 and the replacement sequences which, in some embodiments, may be used for substitution are depicted in Table 2. In some embodiments, a provided engineered enhancer may comprise an ablation at one or more sites shown in Table 2 and / or may comprise a replacement sequence as shown in Table 2 at one or more sites (e.g. at one or more ablation sites).

[0103] Also provided herein are polarization state-specific promoters. In some embodiments, a polarization state-specific promoter comprises an engineered enhancer described herein. In some embodiments, the polarization state-specific promoter further comprises a minimal promoter sequence. The minimal promoter sequence may be operably linked to the engineered enhancer. In some embodiments, the minimal promoter is selected from: a hybrid YBTATA-SCP3 (“YB-SCP3”), SCP3, SCP3 containing DPR, minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB TATA, minTK, inducer molecule responsive promoters, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb, and tandem repeats thereof. In some embodiments, a minimal promoter is or comprises a YBTATA promoter. In some embodiments, a minimal promoter is or comprises a minCMV promoter. In some embodiments, a minimal promoter is or comprises a minTK promoter. In some embodiments, a minimal promoter is or comprises a SCP3 promoter. In some embodiments, a minimal promoter is or comprises a YB-SCP3 promoter. In some embodiments, a minimal promoter comprises a flanking spacer sequence at the N- and / or C-terminus of the minimal promoter. In some embodiments, a flanking spacer sequence comprises the sequence CGGATCAACT (SEQ ID NO: 260). In some embodiments, a flanking spacer sequence comprises the sequence GGATCCGCGTCAAGTGGAGCAAGGCAGGTGGACAGTCCTGCAGGGGAGCTACC(SEQ ID NO: 261). Exemplary minimal promoter sequences are shown in Table 3. In some embodiments, an engineered promoter of the present disclosure comprises a minimal promoter(e.g., any minimal promoter described herein, e.g., those provided in Table 3) and an engineered polarization specific enhancer.

[0104] In some embodiments, any of the promoters described herein may further comprise a translation initiator site at a 3’ end of the promoter, e.g., a consensus Kozak sequence. An exemplary consensus Kozak sequence may be or may include the nucleotide sequence GCCACC. In some embodiments, a translation initiator site (e.g., a Kozak sequence) comprises a flanking spacer sequence at the 5’ and / or 3’ end. In some embodiments, a spacer sequence comprises a nucleotide sequence of ACGCGTACCGGTGTC (SEQ ID NO: 258). In some embodiments, a translation initiator site with a flanking spacer sequence comprises a nucleotide sequence of ACGCGTACCGGTGTCGCCACC (SEQ ID NO: 259). In some embodiments, a promoter described herein does not comprise a translation initiator site.

[0105] In some embodiments, an engineered enhancer or promoter described herein may be operably linked to a heterologous pay load. In some embodiments, the heterologous payload comprises a polynucleotide. In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a polypeptide.

[0106] In some embodiments, a polynucleotide sequence used in accordance with the present disclosure (e.g., for an enhancer, promoter, linker, heterologous payload, signal secretion sequence, etc.) may have 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides that are different from any provided polynucleotide sequence (e.g., any sequence described herein, including sequences shown in Tables 1-6, and 9-15).Macrophage polarization logic circuits

[0107] Macrophages can be polarized to Ml or M2 states by various extracellular cues. For example, when encountering inflammatory cues such as EPS, TNFa or IFNy, macrophages can be polarized to a Ml state. Alternatively, when encountering anti-inflammatory cues such as IL- 4, TGF-0 JL-10, or dexamethasone, macrophages can be polarized to a M2 state. These polarization phenotypes can be plastic depending on what the cell encounters, e.g., can transition between polarization states depending on the surrounding microenvironment The plasticity of macrophage polarization state can lead to undesired loss of macrophage activity in vivo when the cells encounter an opposing cue. For example, an Ml -polarized cell that is phagocytic may lose its inflammatory or phagocytic ability in the presence of anti-inflammatory cytokines such as IL-4, TGF-0 or IL- 10. See, e.g., FIG. 1. This plasticity can be undesirable when engineered macrophages are being used as a cell therapy with either inflammatory or anti-inflammatory activity. There is a need for technologies that enable the synthetic control of macrophage polarization logic.

[0108] In the instant disclosure, it is contemplated that Ml and / or M2 phenotype may be “locked” into a desired phenotype or undergo a phenotype switch in a manner that is controlled by a state-specific specific enhancer or promoter disclosed herein. Such lock would prevent the macrophage plasticity and result in regulated expression of the target macrophage activity.

[0109] Macrophage polarization state-specific enhancers and promoters provided herein are useful for implementing macrophage polarization logic, e.g., in a macrophage state-selective manner. Engineered macrophage-specific promoter systems described herein can beneficially provide synthetic macrophage polarization logic, for example, by keeping macrophages in a desired phenotype state (“phenotype lock”) or driving macrophages to switch from an undesired phenotype state to a desired phenotype state (“phenotype switch”).

[0110] For example, a promoter system can include a promoter having greater activity in an M2 macrophage as compared to an Ml or M0 macrophage (also referred to herein as an M2 promoter, an M2-specific promoter), operably linked to a polynucleotide encoding an effector molecule that acts as an M2 master regulator, e.g., an effector molecule that controls macrophage polarization state by directing macrophages to an M2 state. The M2 master regulator can be, e.g., an M2 transcription factor or M2 cytokine. Exemplary M2 transcription factors and M2 cytokines are disclosed herein. Without wishing to be bound by theory, such a promoter system can be used to keep M2 macrophages in a stable M2 state (“M2 Phenotype Lock”), e.g., even in M2 macrophages exposed to opposing cues from the environment. See, e.g., FIG. 2.

[0111] For other example, a promoter system can include a promoter having greater activity in an M2 macrophage as compared to an Ml or M0 macrophage (also referred to herein as an M2 promoter, an M2-specific promoter), operably linked to a polynucleotide encoding an effector molecule that act as an Ml master regulator, e.g., an Ml transcription factor or Ml cytokine. Exemplary Ml transcription factors and Ml cytokines are disclosed herein. Without wishing to be bound by theory, such a promoter system can be used to direct M2 macrophages from an M2 phenotype to a Ml phenotype (“M2 to Ml Phenotype switch”). See, e.g., FIG. 3.Effector molecules

[0112] Any suitable effector molecule known in the art can be encoded by the engineered nucleic acid or expressed by the engineered cell. In some embodiments, an engineered nucleic acid provided by the present disclosure comprises an engineered polarization state-specific enhancer or promoter operably linked to a nucleic acid sequence encoding one or more effector molecules described herein. Suitable effector molecules can be grouped into therapeutic classes based on structure similarity, sequence similarity, or function. Effector molecule therapeutic classes include, but are not limited to, cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, transcription factors, antibodies, polynucleotides, peptides, shRNAs, miRNAs, and enzymes.

[0113] In some embodiments, at least one effector molecule or each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from: a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier a, a receptor, a ligand, a transcription factor, an antibody, a polynucleotide, a peptide, and an enzyme.

[0114] In some embodiments, at least one effector molecule or each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from: a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier a, a receptor, a ligand, a transcription factor, an antibody, a peptide, and an enzyme.

[0115] In some embodiments, an effector molecule is a transcription factor. The transcription factor may be a master regulator of polarization to an Ml macrophage. Exemplary transcription factor Ml master regulators (also referred to herein as Ml transcription factors) include, e.g., IRF7, p65 / RelA, and derivatives thereof.

[0116] In some embodiments, a transcription factor as used in accordance with the present disclosure is IRF7. In some embodiments, an IRF7 comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 67. In some embodiments, an IRF7 comprises an amino acid sequence as set forth in SEQ ID NO: 67.

[0117] In some embodiments, a transcription factor as used in accordance with the present disclosure is p65 / RelA. In some embodiments, a p65 / RelA comprises an amino acid sequencehaving at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 69. In some embodiments, a p65 / RelA comprises an amino acid sequence as set forth in SEQ ID NO: 69.

[0118] In some embodiments, an effector molecule is a transcription factor. The transcription factor may be a master regulator of polarization to an M2 macrophage. Exemplary transcription factor M2 master regulators (also referred to herein as M2 transcription factors) include, e.g., STAT3, PPAR-alpha, Myc, PPAR-gamma, and / or EGR2.

[0119] In some embodiments, the transcription factor as used in accordance with the present disclosure is STAT3. In some embodiments, a STAT3 comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 200. In some embodiments, a STAT3 comprises an amino acid sequence as set forth in SEQ ID NO: 200.

[0120] In some embodiments, the transcription factor as used in accordance with the present disclosure is PPAR-alpha. In some embodiments, a PPAR-alpha comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 202. In some embodiments, a PPAR-alpha comprises an amino acid sequence as set forth in SEQ ID NO: 202.

[0121] In some embodiments, the transcription factor as used in accordance with the present disclosure is PPAR-gamma. In some embodiments, a PPAR-gamma comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 204. In some embodiments, a PPAR-gamma comprises an amino acid sequence as set forth in SEQ ID NO: 204.

[0122] In some embodiments, the transcription factor as used in accordance with the present disclosure is EGR2. In some embodiments, a EGR2 comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 206. In some embodiments, a EGR2 comprises an amino acid sequence as set forth in SEQ ID NO: 206.

[0123] In some embodiments, the transcription factor as used in accordance with the present disclosure is MYC. In some embodiments, a Myc comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or greater sequence identity to SEQ ID NO: 208. In some embodiments, a Myc comprises an amino acid sequence as set forth in SEQ ID NO: 208.

[0124] In some embodiments, a transcription factor comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 210. In some embodiments, a transcription factor comprises an amino acid sequence as set forth in SEQ ID NO: 210. In some embodiments, a transcription factor comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 212. In some embodiments, a transcription factor comprises an amino acid sequence as set forth in SEQ ID NO: 212. In some embodiments, a transcription factor comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 214. In some embodiments, a transcription factor comprises an amino acid sequence as set forth in SEQ ID NO: 214.

[0125] In some embodiments, an effector molecule is a chemokine. Chemokines are small cytokines or signaling proteins secreted by cells that can induce directed chemotaxis in cells. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C and XC, all of which exert biological effects by binding selectively to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines that may be encoded by the engineered nucleic acids of the present disclosure include: CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1, or any combination thereof. In some embodiments, the chemokine is selected from: CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, andXCLl.

[0126] In some embodiments, a effector molecule is a cytokine. Non-limiting examples of cytokines that may be encoded by the engineered nucleic acids of the present disclosure include: IL-lalpha, IL 1 -beta, IL2, IL4, IL6, IL7, IL10, IL13, IL12, an IL12p70 fusion protein, IL12p35m IL12-p40, IL15, IL 17 A, IL 18, IL21, IL22, IL-23, TGF-beta, M-CSF, Type I interferons, Interferon-alpha, GM-CSF, Interferon-gamma, and TNF-alpha, or any combination thereof. In some embodiments, the cytokine is selected from: IL 1 -beta, IL2, IL4, IL6, IL7, IL10, IL 12, an IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, Type I interferons, Interferon-gamma, and TNF-alpha.

[0127] In some embodiments, the cytokine is a master regulator of polarization to an Ml macrophage (also referred to herein as Ml cytokine). Exemplary cytokines that are master regulators of Ml polarization include, e.g., IFNgamma, IFNalpha, TNF alpha, GM-CSF, IL-12, IL-12p70, IL-12p40, IL-12p35, IL-6, IL-23, IL-lalpha, IL- 1 beta, and derivatives thereof.

[0128] In some embodiments, the cytokine is a master regulator of polarization to an M2 macrophage. Exemplary cytokines that are master regulators of M2 polarization include, e.g., IL- 10, IL-4, IL-13, IL-21, TGF-beta, M-CSF, and derivatives thereof.

[0129] In some embodiments, a cytokine as used in accordance with the present disclosure is IFN-gamma. In some embodiments, an IFN-gamma comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 61. In some embodiments, an IFN- gamma comprises an amino acid sequence as set forth in SEQ ID NO: 61.

[0130] In some embodiments, a cytokine as used in accordance with the present disclosure is TNF-alpha. In some embodiments, a TNF-alpha comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 63. In some embodiments, a TNF-alpha comprises an amino acid sequence as set forth in SEQ ID NO: 63.

[0131] In some embodiments, a cytokine as used in accordance with the present disclosure is IL-12p70. In some embodiments, an IL-12p70 comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 65. In some embodiments, an IL-12p70 comprises an amino acid sequence as set forth in SEQ ID NO: 65.

[0132] In some embodiments, a cytokine as used in accordance with the present disclosure is IL-10. In some embodiments, an IL-10 comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 71. In some embodiments, an IL-10 comprises an amino acid sequence as set forth in SEQ ID NO: 71.

[0133] In some embodiments, a cytokine as used in accordance with the present disclosure is IL-4. In some embodiments, an IL-4 comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orgreater sequence identity to SEQ ID NO: 73. In some embodiments, an IL-4 comprises an amino acid sequence as set forth in SEQ ID NO: 73.

[0134] In some embodiments, an effector molecule (e.g., a cytokine, or any effector molecule described herein) is engineered so that it is tethered to the membrane of a cell. In some embodiments, a tethered effector molecule (e.g., a tethered cytokine) is tethered to the membrane of a cell by operably linking the tethered effector molecule to a transmembrane domain (e.g., a transmembrane domain of a membrane protein).

[0135] In some embodiments, effector molecules provided for herein contain a cellmembrane tethering domain (referred to as “MT’ in the formula E - L - MT or MT - L - E, where “E” is an effector molecule (e.g., a cytokine) and “L” is a linker, e.g., a linker comprising a cleavable linker). In general, the cell-membrane tethering domain can be any amino acid sequence motif capable of directing a polypeptide of interest (e.g., an effector molecule, e.g., a cytokine) to be localized to (e.g., inserted into), or otherwise associated with, the cell membrane of the cell expressing the polypeptide of interest. The cell-membrane tethering domain can be a transmembrane-intracellular domain. The cell-membrane tethering domain can be a transmembrane domain. The cell-membrane tethering domain can be an integral membrane protein domain (e.g., a transmembrane domain). The cell-membrane tethering domain can be derived from a Type I, Type n, or Type in transmembrane protein. The cell-membrane tethering domain can include post-translational modification tag, or motif capable of post-translational modification to modify the polypeptide of interest to include a post-translational modification tag, where the post-translational modification tag allows association with a cell membrane. Examples of post-translational modification tags include, but are not limited to, lipid-anchor domains (e.g., a GPI lipid-anchor, a myristoylation tag, or palmitoylation tag). Examples of cellmembrane tethering domains include, but are not limited to, a transmembrane-intracellular domain and / or transmembrane domain derived from PDGFR-beta, CDS, CD28, CD3zeta-chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7- 1, or BTLA. The cell membrane tethering domain can be a cell surface receptor or a cell membrane-bound portion thereof.

[0136] In some embodiments, the cell-membrane tethering domain is or comprises a transmembrane domain of a B7-1 protein, or a functional portion thereof. In some embodiments,the transmembrane domain comprises the sequence LLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV (SEQ ID NO: 95)

[0137] In some embodiments, the cell-membrane tethering domain comprises a transmembrane domain of a CDS polypeptide, or a functional portion thereof. Any suitable CDS polypeptide may be used. Exemplary CDS polypeptides include, without limitation, NCBI Reference Nos. NP 001139345 and AAA92533.1. Examples of CDS transmembrane domains include IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 161), IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 162), and IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 163). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 161). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHR (SEQ ID NO: 162). In some embodiments, the transmembrane domain is encoded by a nucleotide sequence at least 75%, 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(SEQ ID NO: 270). In some embodiments, thetransmembrane domain is encoded by a secretion signal at least 75%, 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(SEQ ID NO: 271). In some embodiments, the transmembrane domain isencoded by a secretion signal at least 75%, 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(SEQ ID NO: 272). In some embodiments, the transmembrane domain comprises the sequence IYIWAPLAGTCGVLLLSLVITLYCNHRN (SEQ ID NO: 163).In some embodiments, the cellmembrane tethering domain comprises a hinge and transmembrane domain derived from CDS. In some embodiments, the CDS hinge comprises the sequence (SEQ ID NO: 157). Insome embodiments, the CDS hinge comprises the sequence(SEQ ID NO: 156).

[0138] In some embodiments, the cell membrane tethering domain is either: (1) C- terminal of a linker and N-terminal of any intracellular domain, if present (in other words, the cell membrane tethering domain is in between a linker and, if present, an intracellular domain); or (2) N-terminal of a linker and C-terminal of any intracellular domain, if present (also between the linker and, if present, an intracellular domain with domain orientation inverted). The cell membrane tethering domain can be connected to an intracellular domain, if present, by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of the cell membrane tethering domain or the intracellular domain. A polypeptide linker can be any amino acid sequence that connects a first polypeptide sequence and a second polypeptide sequence. A polypeptide linker can be a flexible linker (e.g., a Gly-Ser-Gly sequence). Examples of polypeptide linkers include, but are not limited to, GSG linkers (e.g., [GS]4GG [SEQ ID NO: 164]), A(EAAAK)3A (SEQ ID NO: 165), and Whitlow linkers (e.g., a “KEGS” linker such as the amino acid sequence KESGSVSSEQLAQFRSLD (SEQ ID NO: 166), an eGK linker such as the amino acid sequence EGKSSGSGSESKST (SEQ ID NO: 167), and linkers described in more detail in U.S. Pat No. 5,990,275 herein incorporated by reference). Other polypeptide linkers may be selected based on desired properties (e.g., length, flexibility, amino acid composition etc.) and are known to those skilled in the art

[0139] In some embodiments, a linker is a cleavable linker, e.g., comprising a protease cleavage site. In some embodiments, the cell membrane tethering domain can be connected to a protease cleavage site by a polypeptide linker, i.e., a polypeptide sequence not generally considered to be part of cell membrane tethering domain or protease cleavage site.

[0140] In some embodiments, the cell-membrane tethering domain is oriented such that the effector molecule and optionally the protease cleavage site are extracellularly exposed following insertion into, or association with, the cell membrane. In some embodiments wherein the linker comprises a protease cleavage site, the effector molecule and the protease cleavage site are extracellularly exposed such that the protease cleavage site is capable of being cleaved by its respective protease and releasing (“secreting”) the effector molecule into the extracellular space.

[0141] In some embodiments a transmembrane domain comprises a B7-1 transmembrane domain. In some embodiments a B7-1 transmembrane domain comprises an amino acid sequence as set forth in SEQ ID NO: 95. In some embodiments, an effector molecule is operably linked to a transmembrane domain via a linker. In some embodiments, a linker comprises an amino acid sequence as set forth in SEQ ID NO: 93.

[0142] In some embodiments, a tethered effector molecule is a tethered cytokine. In some embodiments, a tethered cytokine is a tethered IL- 10 cytokine. In some embodiments, a tethered IL-10 cytokine comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 81. In some embodiments, a tethered IL-10 cytokine comprises an amino acid sequence as set forth in SEQ ID NO: 81.

[0143] In some embodiments, a tethered cytokine is a tethered IFNg cytokine. In some embodiments, a tethered IFNg cytokine comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 83. In some embodiments, a tethered IFNg cytokine comprises an amino acid sequence as set forth in SEQ ID NO: 83. In some embodiments, a tethered IFNg cytokine comprises a truncated IFNg. In some embodiments, a truncated IFNg comprises an amino acid sequence as set forth in SEQ ID NO: 91.

[0144] In some embodiments, a tethered cytokine is a tethered IL-4. In some embodiments, a tethered IL-4 comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 216. In some embodiments, a tethered IL-4 comprises an amino acid sequence as set forth in SEQ ID NO: 216.

[0145] In some embodiments, a tethered cytokine comprises at least 2 cytokines. In some embodiments, the at least 2 cytokines comprise IL4 and IL10. In some embodiments, the tethered cytokine comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 218. In some embodiments, the tethered cytokine comprises an amino acid sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 220. In some embodiments, a tethered IL-4 comprises an amino acid sequence as set forth in SEQ ID NO: 218.In some embodiments, a tethered IL-4 comprises an amino acid sequence as set forth in SEQ ID NO: 220.

[0146] In some embodiments, engineered nucleic acids are configured to produce at least one homing molecule. “Homing,” refers to active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., tumor), or organ). A “homing molecule” refers to a molecule that directs cells to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate interaction of an engineered cell to a target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR IL6R CXCR1, CSCR7, PDGFR anti-integrin alpha4,beta7; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15, or any combination thereof. In some embodiments, the homing molecule is selected from: anti-integrin alpha4,beta7; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15.

[0147] In some embodiments, engineered nucleic acids are configured to produce at least one growth factor. Suitable growth factors for use as an effector molecule include, but are not limited to, FLT3L and GM-CSF, or any combination thereof. In some embodiments, the growth factor is selected from: FLT3L and GM-CSF.

[0148] In some embodiments, engineered nucleic acids are configured to produce at least one co-activation molecule. Suitable co-activation molecules for use as an effector molecule include, but are not limited to, c-Jun, 4-1BBL and CD40L, or any combination thereof. In some embodiments, the co-activation molecule is selected from: c-Jun, 4-1 BBL and CD40L.

[0149] A “tumor microenvironment” is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R & Weinberg, RA. Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F.R etal JCellSci 125, 5591-5596, 2012; and Li, H. etal JCellBiochem 101(4), 805-15, 2007). Suitable tumor microenvironment modifiers for use as an effector molecule include, but are not limited to, adenosine deaminase, TGFbeta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, or any combination thereof. In some embodiments, the tumor microenvironment modifier is selected from: adenosine deaminase, TGFbeta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2.

[0150] In some embodiments, engineered nucleic acids are configured to produce at least one TGFbeta inhibitor. Suitable TGFbeta inhibitors for use as an effector molecule include, but are not limited to, an anti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, or combinations thereof. In some embodiments, the TGFbeta inhibitors are selected from: an anti- TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, and combinations thereof.

[0151] In some embodiments, engineered nucleic acids are configured to produce at least one immune checkpoint inhibitor. Suitable immune checkpoint inhibitors for use as an effector molecule include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD- L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TTM-3 antibodies, anti- TI GIT antibodies, anti- VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7- H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti- TREM1 antibodies, and anti-TREM2 antibodies, or any combination thereof. In some embodiments, the immune checkpoint inhibitors are selected from: anti-PD-1 antibodies, anti- PD-Ll antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti- TIM-3 antibodies, anti-TIGIT antibodies, anti- VISTA antibodies, anti-KIR antibodies, anti-B7- H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREMl antibodies, and anti-TREM2 antibodies.

[0152] Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK- 3475 / Keytruda® - Merck), nivolumamb (anti-PD-1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-Ll; Bavencio® - Pfizer), durvalumab (anti-PD-Ll; MEDI4736 / Imfinzi® - Medimmune / AstraZeneca), atezolizumab (anti-PD-Ll; Tecentriq® - Roche / Genentech), BMS- 936559 (anti-PD-Ll - BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti- NKG2A; Innate Pharma / AstraZeneca).

[0153] In some embodiments, engineered nucleic acids are configured to produce at least one VEGF inhibitor. Suitable VEGF inhibitors for use as an effector molecule include, but are not limited to, anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof. In someembodiments, the VEGF inhibitors comprise anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof.

[0154] In some embodiments, each effector molecule is a human-derived effector molecule.

[0155] In some embodiments, engineered nucleic acids are configured to produce at least one reporter molecule. In some embodiments, a reporter molecule is a fluorescent protein (e.g., luciferase, nanoluciferase, GFP, or variants or derivatives thereof). In some embodiments, a reporter molecule is an EGFP. In some embodiments, an EGFP comprises an amino acid sequence as set forth in SEQ ID NO: 75. In some embodiments, a reporter molecule comprises mCherry. In some embodiments, an mCherry comprises an amino acid sequence as set forth in SEQ ID NO: 77. In some embodiments, a reporter molecule comprises a nanoLuc. In some embodiments, a nanoLuc comprises an amino acid sequence as set forth in SEQ ID NO: 79.

[0156] Sequences of exemplary effector molecules and other payloads (e.g., reporter molecules) are in Table 4. Any effector molecule, reporter molecule, or other payload sequence as described herein, e.g., as shown in Table 4, may be used in accordance with the present disclosure.Engineered Multiristronic and Multiple Promoter Systems

[0157] In some embodiments, engineered nucleic acids are configured to produce multiple effector molecules. For example, engineered nucleic acids may be configured to produce 2-20 different effector molecules. In some embodiments, nucleic acids are configured to produce 2- 20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3- 20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6- 15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7- 12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11- 12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16- 20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 effector molecules. In someembodiments, nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 effector molecules.

[0158] In some embodiments, engineered nucleic acids can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple exogenous polynucleotides or effector molecules) can be produced from a single mRNA transcript Engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first exogenous polynucleotide or effector molecule can be linked to a nucleotide sequence encoding a second exogenous polynucleotide or effector molecule, such as in a first gene: linker: second gene 5’ to 3’ orientation. A linker polynucleotide sequence can encode a 2A ribosome skipping element, such as T2A. Other 2A ribosome skipping elements include, but are not limited to, E2A, P2A, and F2A. 2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. A cleavable linker can include a polypeptide sequence, such as such a flexible linker (e.g., a Gly-Ser-Gly sequence), that further promotes cleavage. In some embodiments of the present disclosure, a 2A peptide encoding nucleotide sequences is at least 75%, 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(SEQ ID NO: 267). In some embodiments, a 2A peptideencoding nucleotide sequences comprises a nucleotide sequence

[0159] In some embodiments, an expression cassette comprises one or more polarization state-specific enhancers or promoters that are operably linked to one or more nucleic acid sequences that each encode for one or more payload molecules (e.g., any payload molecule described herein, e.g., polypeptides). In some embodiments, when an expression cassette provided herein (e.g., a first expression cassette, a second expression cassette, etc.) comprisestwo or more units of (Li - E)x, e.g., where X is 2 or more, each Li linker polynucleotide sequence is operably associated with the translation of each effector molecule (E) as a separate polypeptide.

[0160] A linker can encode an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation. A linker can encode a splice acceptor, such as a viral splice acceptor.

[0161] A linker can be a combination of linkers, such as a Furin-2A linker that can produce separate polypeptides through 2A ribosome skipping followed by further cleavage of the Furin site to allow for complete removal of 2A residues. In some embodiments, a combination of linkers can include a Furin sequence, a flexible linker, and 2A linker. Accordingly, in some embodiments, the linker is a Furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, a linker is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0162] In general, a multicistronic system can use any number or combination of linkers, to express any number of genes or portions thereof (e.g., An engineered nucleic acid can encode a first, a second, and a third effector molecule, each separated by linkers such that separate polypeptides encoded by the first, second, and third effector molecules are produced).

[0163] “Linkers,” as used herein can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence or the multicistronic linkers described above.Secretion Signals

[0164] In general, the one or more effector molecules comprise a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the effector molecule’s N-terminus that direct newly synthesized proteins destined for secretion or membrane insertion to the proper protein processing pathways. In some embodiments with two or more effector molecules, each effector molecule can comprise a secretion signal (S). In some embodiments with two or more effector molecules, each effector molecule can comprise a secretion signal such that each effector molecule is secreted from an engineered cell. In some embodiments, an expression cassette as provided herein (e.g., a first expression cassette, a second expression cassette, etc.) comprises one or more units of (L - E)x further comprising a polynucleotide sequence encoding a secretion signal peptide (S). In some embodiments, for each X the corresponding secretion signal peptide is operably associated with the effector molecule (E). In some embodiments, anexpression cassette as provided herein (e.g., a first expression cassette, a second expression cassette, etc.) comprises a promoter and a second exogenous polynucleotide sequence having the formula: (L -S- E)x.

[0165] The secretion signal peptide operably associated with a effector molecule can be a native secretion signal peptide (e.g., the secretion signal peptide generally endogenously associated with the given effector molecule). The secretion signal peptide operably associated with a effector molecule can be a non-native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments, such as tumor microenvironments. Non-limiting examples of non-native secretion signal peptide are shown in Table 5.Antigen recognizing receptors

[0166] Certain aspects of the present disclosure relate to an engineered nucleic comprising an antigen recognizing receptor. In some embodiments, an engineered nucleic acid of the present disclosure comprises an expression cassette (e.g., a first expression cassette, a second expression cassette, etc.) that comprises an antigen recognizing receptor. In some embodiments, the expression cassette comprises a polynucleotide sequence encoding the antigen recognizing receptor that is operably linked to a provided promoter (e.g., a polarization state-specific promoter) or engineered enhancer. Suitable antigen recognizing receptors for use as an effector molecule recognize antigens that include, but are not limited to, 5T4, ADAM9, AFP, AXL, B7- H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, Claudinl8.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRalpha, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra2, IL-8, IL-15, IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, Mesothelin (MSLN), MUC1, MUC16, MUCIC, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, R0R1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIMl, TROP2, and WT1, or any combination thereof.

[0167] In some embodiments, the antigen recognizing receptor recognizes an antigen selected from: 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, Cadherin 3, Cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, Claudinl8.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, ENPP3, EpCAM, EphA2, Ephrin A4, ETBR, FGFR2, FGFR3, FRalpha, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra2, IL-8, IL-15, IL1RAP, Integrin aV, KIT, LI CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MUC1, MUC16, MUCIC, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY ESO 1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, Survivin, TDGF1, TIMl, TROP2, and WT1.

[0168] In some embodiments, the antigen recognizing receptor comprises an antigen-binding domain. In some embodiments, the antigen-binding domain comprises an antibody, an antigenbinding fragment of an antibody, a F(ab) fragment, a F(ab') fragment, a single chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker.

[0169] An scFv has a variable domain of light chain (VL) connected from its C-terminus to the N-terminal end of a variable domain of heavy chain (VH) by a polypeptide chain. Alternately the scFv comprises of polypeptide chain where in the C -terminal end of the VH is connected to the N-terminal end of VL by a polypeptide chain. In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is the peptide linker, and VL is the light chain variable domain.

[0170] An sdAb is a molecule in which one variable domain of an antibody specifically binds to an antigen without the presence of the other variable domain.

[0171] A F(ab) fragment contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. F(ab') fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CHI domain including one or morecysteines from the antibody hinge region. F(ab’)2 fragments contain two Fab’ fragments joined, near the hinge region, by disulfide bonds.

[0172] In some embodiments, the antigen recognizing receptor is a chimeric antigen receptor (CAR) or T cell receptor (TCR). In some embodiments, the antigen recognizing receptor is a CAR In some embodiments, the CAR comprises one or more intracellular signaling domains, and the one or more intracellular signaling domains are selected from: a CD3zeta-chain intracellular signaling domain, a CD97 intracellular signaling domain, a CDlla-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CDS intracellular signaling domain, an 0X40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyDSS intracellular signaling domain. In some embodiments, the CAR comprises a CD3zeta-chain intracellular signaling domain and one or more additional intracellular signaling domains (e.g., co-stimulatory domains) selected from a CD97 intracellular signaling domain, a CDlla-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD 154 intracellular signaling domain, a CDS intracellular signaling domain, an 0X40 intracellular signaling domain, a 4- IBB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyDSS intracellular signaling domain, a 2B4 intracellular signaling domain, a CD 16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, a NKp44 intracellular signaling domain, a NKp46 intracellular signaling domain, a FceRlg intracellular signaling domain, a NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0173] In some embodiments, the CAR further comprises a transmembrane domain, and the transmembrane domain is selected from: a CDS transmembrane domain, a CD28 transmembrane domain a CD3zeta-chain transmembrane domain, a CD4 transmembrane domain, a 4-1BBtransmembrane domain, an 0X40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an 0X40 transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD 16a transmembrane domain, a DNAM-1 transmembrane domain, a KER2DS1 transmembrane domain, a KIR3DS1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D transmembrane domain.

[0174] In some embodiments, the CAR further comprises a spacer region (e.g., hinge domain) between the antigen-binding domain and the transmembrane domain. A spacer or hinge domain is any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and / or the intracellular signaling domain in the polypeptide chain. Spacer or hinge domains provide flexibility to the inhibitory chimeric receptor or tumor-targeting chimeric receptor, or domains thereof, or prevent steric hindrance of the inhibitory chimeric receptor or tumor-targeting chimeric receptor, or domains thereof. In some embodiments, a spacer domain or hinge domain may comprise up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more spacer domain(s) may be included in other regions of an inhibitory chimeric receptor or tumor-targeting chimeric receptor.

[0175] Exemplary spacer or hinge domains may include, without limitation an IgG domain (such as an IgGl hinge, an IgG2 hinge, an IgG3 hinge, or an IgG4 hinge), an IgD hinge domain, a CD8a hinge domain, and a CD28 hinge domain. In some embodiments, the spacer or hinge domain is an IgG domain, an IgD domain, a CD8a hinge domain, or a CD28 hinge domain.

[0176] Exemplary spacer or hinge domain protein sequences are shown in Table 6. Any spacer or hinge sequences described herein, e.g., as shown in Table 6, may be used in accordance with the present disclosure.

[0177] Suitable transmembrane domains, spacer or hinge domains, and intracellular domains for use in a CAR are generally described in Stoiber et al, Cells 2019, 8(5), 472; Guedan et al, Mol Therapy: Met & Clinic Dev, 2019 12:145-156; and Sadelain eta / , Cancer Discov; 2013, 3(4); 388-98, each of which are hereby incorporated by reference in their entirety.

[0178] In some embodiments, the CAR further comprises a secretion signal peptide. Any suitable secretion signal peptide of the present disclosure may be used.Post-Transcriptional Regulatory Elements

[0179] In some embodiments, an engineered nucleic acid of the present disclosure comprises a post-transcriptional regulatory element (PRE). PREs can enhance gene expression via enabling tertiary RNA structure stability and 3’ end formation. Non-limiting examples of PREs include the Hepatitis B virus PRE (HPRE) and the Woodchuck Hepatitis Virus PRE (WPRE). In some embodiments, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the WPRE comprises the alpha, beta, and gamma components of the WPRE element. In some embodiments, the WPRE comprises the alpha component of the WPRE element.Immunoresponsive Cells

[0180] Certain aspects of the present disclosure relate to a cell, such as an immunoresponsive cell, that has been genetically engineered to comprise one or more nucleic acids of the present disclosure, and to methods of using such cells for treating solid tumors.

[0181] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a primary cell. In some embodiments, the mammalian cell is a cell line. In some embodiments, the mammalian cell, a bone marrow cell, a blood cell, a skin cell, bone cell, a muscle cell, a neuronal cell, a fat cell, a liver cell, or a heart cell. In some embodiments, the cell is a stem cell. Exemplary stem cells include, without limitation embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), adult stem cells, and tissue-specific stem cells, such as hematopoietic stem cells (blood stem cells), mesenchymal stem cells (MSC), neural stem cells, epithelial stem cells, or skin stem cells. In some embodiments, the cell is a cell that is derived or differentiated from a stem cell of the present disclosure. In some embodiments, the cell is an immune cell. Immune cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary immune cells include, without limitation, T cells (e g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, alpha beta T cells, and gamma delta T cells), B cells, natural killer (NK) cells, dendritic cells, myeloid cells, macrophages, and monocytes. In some embodiments, the cell is a neuronal cell. Neuronal cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary neuronal cells include, without limitation, neural progenitor cells, neurons (e.g., sensory neurons,motor neurons, cholinergic neurons, GAB Aergic neurons, glutamatergic neurons, dopaminergic neurons, or serotonergic neurons), astrocytes, oligodendrocytes, and microglia.

[0182] In some embodiments, the cell is an immunoresponsive cell. Immunoresponsive cells of the present disclosure may be isolated or differentiated from a stem cell of the present disclosure (e.g., from an ESC or iPSC). Exemplary immunoresponsive cells of the present disclosure include, without limitation, cells of the lymphoid lineage. The lymphoid lineage, comprising B cells, T cells, and natural killer (NK) cells, provides for the production of antibodies, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, and the like. Examples of immunoresponsive cells of the lymphoid lineage include, without limitation, T cells, Natural Killer (NK) cells, embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells (e.g., those from which lymphoid cells may be derived or differentiated).

[0183] T cells can be lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. In some embodiments, T cells of the present disclosure can be any type of T cells, including, without limitation, T helper cells, cytotoxic T cells, memory T cells (including central memory T cells, stem-cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells: e.g., TEM cells and TEMRA cells, regulatory T cells (also known as suppressor T cells), natural killer T cells, mucosal associated invariant T cells, and y5 T cells. Cytotoxic T cells (CTL or killer T cells) are a subset of T lymphocytes capable of inducing the death of infected somatic or tumor cells. A patient's own T cells may be genetically modified to target specific antigens through the introduction of one or more chimeric receptors, such as a chimeric TCRs or CARs.

[0184] Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation in order to perform their cytotoxic effect on target cells.

[0185] In some embodiments, an immunoresponsive cell of the present disclosure is a T cell. T cells of the present disclosure may be autologous, allogeneic, or derived in vitro from engineered progenitor or stem cells.

[0186] In some embodiments, an immunoresponsive cell of the present disclosure is a universal T cell with deficient TCR-ap. Methods of developing universal T cells are described inthe art, for example, in Valton etal., Molecular Therapy (2015); 23 9, 1507-1518, and Torikai et al., Blood 2012 119:5697-5705.

[0187] In some embodiments, an immimoresponsive cell of the present disclosure is an isolated immimoresponsive cell comprising one or more chimeric receptors of the present disclosure. In some embodiments, the immimoresponsive cell comprises one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more chimeric receptors of the present disclosure.

[0188] In some embodiments, an immimoresponsive cell is a T cell. In some embodiments, an immimoresponsive cell is a Natural Killer (NK) cell. In some embodiments, an immimoresponsive cell is a macrophage. In some embodiments, an immimoresponsive cell is an Ml macrophage. In some embodiments, an immimoresponsive cell is an M2 macrophage. In some embodiments, the M2 macrophage is selected from the group consisting of M2a, M2b, and M2c subtypes.

[0189] In some embodiments, an immimoresponsive cell expresses or is capable of expressing an immune receptor. Immune receptors generally are capable of inducing signal transduction or changes in protein expression in the immune receptor-expressing cell that results in the modulation of an immune response upon binding to a cognate ligand (e.g., regulate, activate, initiate, stimulate, increase, prevent, attenuate, inhibit, reduce, decrease, inhibit, or suppress an immune response). For example, when CD3 chains present in a TCR / CAR cluster in response to ligand binding, an immunoreceptor tyrosine-based activation motifs (ITAMs)- meditated signal transduction cascade is produced. Specifically, in certain embodiments, when an endogenous TCR, exogenous TCR, chimeric TCR, or a CAR (specifically an activating CAR) binds their respective antigen, a formation of an immunological synapse occurs that includes clustering of many molecules near the bound receptor (e.g. CD4 or CDS, CDSy / S / e / ^, etc.). This clustering of membrane bound signaling molecules allows for ITAM motifs contained within the CD3 chains to become phosphorylated that in turn can initiate a T cell activation pathway and ultimately activates transcription factors, such as NF-KB and AP-1. These transcription factors are capable of inducing global gene expression of the T cell to increase IL-2 production for proliferation and expression of master regulator T cell proteins in order to initiate a T cell mediated immune response, such as cytokine production and / or T cell mediated killing.Cells Expressing Chimeric Antigen Receptors

[0190] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises two or more chimeric receptors. In some embodiments, the cell comprises two or more chimeric receptors, wherein a first of the two or more chimeric receptors is an activating chimeric receptor and a second of the two or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises a first activating chimeric receptor and a second activating chimeric receptor. In some embodiments, the cell comprises three or more chimeric receptors, wherein at least one of the three or more chimeric receptors is an activating chimeric receptor. In some embodiments, the cell comprises three or more chimeric receptors, wherein at least one of the three or more chimeric receptors is a chimeric inhibitory receptor. In some embodiments, the cell comprises four or more chimeric receptors. In some embodiments, the cell comprises five or more chimeric receptors.

[0191] In some embodiments, each of the two or more chimeric receptors comprise a different antigen-binding domain, e.g., that binds to the same antigen or to a different antigen. In some embodiments each antigen bound by the two or more chimeric receptors are expressed on the same cell, such as an epithelial cell type (e.g., same epithelial cell type).

[0192] In embodiments where a cell of the present disclosure (e.g., an immunoresponsive cell) expresses two or more distinct chimeric receptors, the antigen-binding domain of each of the different chimeric receptors may be designed such that the antigen-binding domains do not interact with one another. For example, a cell of the present disclosure (e.g., an immunoresponsive cell) expressing a first chimeric receptor and a second chimeric receptor may comprise a first chimeric receptor that comprises an antigen-binding domain that does not form an association with the antigen-binding domain of the second chimeric receptor. For example, the antigen-binding domain of the first chimeric receptor may comprise an antibody fragment, such as an scFv, while the antigen-binding domain of the second chimeric receptor may comprise a VHH.

[0193] Without wishing to be bound by theory, it is believed that in cells having a plurality of chimeric membrane embedded receptors that each comprise an antigen-binding domain, interactions between the antigen-binding domains of each of the receptors can be undesirable, because such interactions may inhibit the ability of one or more of the antigen-binding domains to bind their cognate antigens. Accordingly, in embodiments where cells of the presentdisclosure (e.g., immunoresponsive cells) express two or more chimeric receptors, the chimeric receptors comprise antigen-binding domains that minimize such inhibitory interactions. In one embodiment, the antigen-binding domain of one chimeric receptor comprises an scFv and the antigen-binding domain of the second chimeric receptor comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence.

[0194] In some embodiments, when present on the surface of a cell, binding of the antigenbinding domain of the first chimeric receptor to its cognate antigen is not substantially reduced by the presence of the second chimeric receptor. In some embodiments, binding of the antigenbinding domain of the first chimeric receptor to its cognate antigen in the presence of the second chimeric receptor is 85%, 90%, 95%, 96%, 97%, 98%, or 99% of binding of the antigen-binding domain of the first chimeric receptor to its cognate antigen in the absence of the second chimeric receptor. In some embodiments, when present on the surface of a cell, the antigen-binding domains of the first chimeric receptor and the second chimeric receptor associate with one another less than if both were scFv antigen-binding domains. In some embodiments, the antigenbinding domains of the first chimeric receptor and the second chimeric receptor associate with one another 85%, 90%, 95%, 96%, 97%, 98%, or 99% less than if both were scFv antigenbinding domains.Co-stimulatory ligands

[0195] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) can further include one or more recombinant or exogenous co-stimulatory ligands. For example, the cell can be further transduced with one or more co-stimulatory ligands, such that the cell co-expresses or is induced to co-express one or more chimeric receptors and one or more co-stimulatory ligands. Without wishing to be bound by theory, it is believed that the interaction between the one or more chimeric receptors and the one or more co-stimulatory ligands may provide a non-antigen-specific signal important for full activation of the cell. Examples of suitable co-stimulatory ligands include, without limitation, members of the tumor necrosis factor (TNF) superfamily, and immunoglobulin (Ig) superfamily ligands. TNF is a cytokine involved in systemic inflammation and stimulates the acute phase reaction. Its primary role is in the regulation of immune cells. Members of TNF superfamily share a number of common features.The majority of TNF superfamily members are synthesized as type II transmembrane proteins (extracellular C-terminus) containing a short cytoplasmic segment and a relatively long extracellular region. Examples of suitable TNF superfamily members include, without limitation, nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4-1BBL, TNF-o, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin- alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell-activating factor (BAFF) / BLYS / THANK / Tall-1, glucocorticoid-induced TNF Receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF 14). The immunoglobulin (Ig) superfamily is a large group of cell surface and soluble proteins that are involved in the recognition, binding, or adhesion processes of cells. These proteins share structural features with immunoglobulins and possess an immunoglobulin domain (fold). Examples of suitable immunoglobulin superfamily ligands include, without limitation, CD80 and CD86, both ligands for CD28, PD-L1 (B7-H1) that are ligands for PD-1. In certain embodiments, the one or more co-stimulatory ligands are selected from 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof.

[0196] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises one or more recombinant or exogenous co-stimulatory ligands regulated by an engineered enhancer or promoter described herein, e.g., as described in Table 1, Table 10, or Table 11. In certain embodiments, a TNF superfamily member (e.g., nerve growth factor (NGF), CD40L (CD40L) / CD 154, CD137L / 4-1BBL, TNF-a, CD134L / OX40L / CD252, CD27L / CD70, Fas ligand (FasL), CD30L / CD153, tumor necrosis factor beta (TNFP) / lymphotoxin- alpha (LTa), lymphotoxin-beta (LTP), CD257 / B cell-activating factor (BAFF) / BLYS / THANK / Tall-1, glucocorticoid-induced TNF Receptor ligand (GITRL), and TNF-related apoptosis-inducing ligand (TRAIL), LIGHT (TNFSF 14)) is regulated by an engineered enhancer or promoter described herein, e.g., as described in Table 1, Table 10, or Table 11. In certain embodiments, an Ig superfamily member ligand (e.g., CD80, CD86, PD-L1, and B7-H1) is regulated by an engineered enhancer or promoter described herein, e.g., as described in Table 1, Table 10, or Table 11. In certain embodiments, a co-stimulatory ligand (e.g., 4-1BBL, CD80, CD86, CD70, OX40L, CD48, TNFRSF14, PD-L1, and combinations thereof) is regulated by engineered enhancer or promoter described herein, e.g., as described in Table 1, Table 10, or Table 11.Chemokine receptors

[0197] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises one or more chimeric receptors and may further include one or more chemokine receptors. For example, transgenic expression of chemokine receptor CCR2b or CXCR2 in cells, such as T cells, enhances trafficking to CCL2-secreting or CXCL1 -secreting solid tumors (Craddock et al, J Immunother. 2010 Oct; 33(8):780-8 and Kershaw etal. Hum Gene Then 2002 Nov 1; 13(16): 1971 -80). Without wishing to be bound by theory, it is believed that chemokine receptors expressed on chimeric receptor-expressing cells of the present disclosure may recognize chemokines secreted by tumors and improve targeting of the cell to the tumor, which may facilitate the infiltration of the cell to the tumor and enhance the antitumor efficacy of the cell. Chemokine receptors of the present disclosure may include a naturally occurring chemokine receptor, a recombinant chemokine receptor, or a chemokine-binding fragment thereof. Examples of suitable chemokine receptors that may expressed on a cell of the present disclosure include, without limitation, a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7; a CC chemokine receptor, such as CCR1 , CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11; a CX3C chemokine receptor, such as CX3CR1; an XC chemokine receptor, such as XCR1; and chemokine-binding fragments thereof. In some embodiments, the chemokine receptor to be expressed on the cell is chosen based on the chemokines secreted by the tumor.

[0198] In some embodiments, a cell of the present disclosure (e.g., an immunoresponsive cell) comprises one or more chemokine receptors regulated by an engineered enhancer or promoter described herein, e.g., as described in Table 1, Table 10, or Table 11. Examples of such chemokine receptors include, for example and without limitation, a CXC chemokine receptor, such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, or CXCR7; a CC chemokine receptor, such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, or CCR11; a CX3C chemokine receptor, such as CX3CR1; anXC chemokine receptor, such as XCR1 ; and chemokine-binding fragments thereof. In certain embodiments, the chemokine receptor regulated by an engineered enhancer or promoter described herein, e.g., as described in Table 1, Table 10, or Table 11, is chosen based on the chemokines secreted by the tumor.Chimeric Receptor Regulation

[0199] Some embodiments of the present disclosure relate to regulating one or more chimeric receptor activities of chimeric receptor-expressing cells of the present disclosure. There are several ways chimeric receptor activities can be regulated. In some embodiments, a regulatable chimeric receptor, wherein one or more chimeric receptor activities can be controlled, may be desirable to optimize the safety and / or efficacy of the chimeric receptor therapy. For example, inducing apoptosis using a caspase fused to a dimerization domain (see, e.g., Di etal., N Engl. J. Med. 2011 Nov. 3; 365(18): 1673-1683) can be used as a safety switch in the chimeric receptor therapy. In some embodiments, a chimeric receptor-expressing cell of the present disclosure can also express an inducible Caspase-9 (iCaspase-9) that, upon administration of a dimerizer drug, such as rimiducid (IUPAC name: [(lR)-3-(3,4- dimethoxyphenyl)-l-[3-[2-[2-[[2-[3-[(lR)-3-(3,4-dimethoxyphenyl)-l-[(2S)-l-[(2S)-2-(3,4,5- trimethoxyphenyl)butanoyl]piperidine-2- carbonyl]oxypropyl]phenoxy]acetyl]amino]ethylamino]-2-oxoethoxy]phenyl]propyl] (2S)-1- [(2S)-2-(3,4,5-trimethoxyphenyl)butanoyl]piperidine-2-carboxylate), induces activation of the Caspase-9 and results in apoptosis of the cells. In some embodiments, the iCaspase-9 contains a binding domain that comprises a chemical inducer of dimerization (CID) that mediates dimerization in the presence of the CID, which results in inducible and selective depletion of the chimeric receptor-expressing cells.

[0200] Alternatively, in some embodiments a chimeric receptor of the present disclosure may be regulated by utilizing a small molecule or an antibody that deactivates or otherwise inhibits chimeric receptor activity. For example, an antibody may delete the chimeric receptor-expressing cells by inducing antibody dependent cell-mediated cytotoxicity (ADCC). In some embodiments, a chimeric receptor-expressing cell of the present disclosure may further express an antigen that is recognized by a molecule that is capable of inducing cell death by ADCC or complement- induced cell death. For example, a chimeric receptor-expressing cell of the present disclosure may further express a receptor capable of being targeted by an antibody or antibody fragment. Examples of suitable receptors that may be targeted by an antibody or antibody fragment include, without limitation, EpCAM, VEGFR, integrins (e.g., avβ3, a4, aI¾ β3, a4β7, a5β1, avβ3, av), members of the TNF receptor superfamily (e.g., TRAIL-R1 and TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1 , HLA-DR, CEA, CA-125,MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GD3, CD2, CD3, CD4, CDS, CD11, CDlla / LFA-1, GDIS, CD18 / ITGB2, CD19, CD20, CD22, CD23 / IgE Receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41 , CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147 / basigin, CD152 / CTLA-4, CD154 / CD40L, CD195 / CCR5, CD319 / SLAMF7, andEGFR, and truncated versions thereof.

[0201] In some embodiments, a chimeric receptor-expressing cell of the present disclosure may also express a truncated epidermal growth factor receptor (EGFR) that lacks signaling capacity but retains an epitope that is recognized by molecules capable of inducing ADCC (e.g., WO2011 / 056894).

[0202] In some embodiments, a chimeric receptor-expressing cell of the present disclosure further includes a highly expressing compact marker / suicide gene that combines target epitopes from both CD32 and CD20 antigens in the chimeric receptor-expressing cell, which binds an anti-CD20 antibody (e.g., rituximab) resulting in selective depletion of the chimeric receptorexpressing cell by ADCC. Other methods for depleting chimeric receptor-expressing cells of the present disclosure my include, without limitation, administration of a monoclonal anti-CD52 antibody that selectively binds and targets the chimeric receptor-expressing cell for destruction by inducing ADCC. In some embodiments, the chimeric receptor-expressing cell can be selectively targeted using a chimeric receptor ligand, such as an anti-idiotypic antibody. In some embodiments, the anti-idiotypic antibody can cause effector cell activity, such as ADCC or ADC activity. In some embodiments, the chimeric receptor ligand can be further coupled to an agent that induces cell killing, such as a toxin. In some embodiments, a chimeric receptor-expressing cell of the present disclosure may further express a target protein recognized by a cell depleting agent of the present disclosure. In some embodiments, the target protein is CD20 and the cell depleting agent is an anti-CD20 antibody. In such embodiments, the cell depleting agent is administered once it is desirable to reduce or eliminate the chimeric receptor-expressing cell. In some embodiments, the cell depleting agent is an anti-CD52 antibody.

[0203] In some embodiments, a regulated chimeric receptor comprises a set of polypeptides, in which the components of a chimeric receptor of the present disclosure are partitioned on separate polypeptides or members. For example, the set of polypeptides may include a dimerization switch that, when in the presence of a dimerization molecule, can couple the polypeptides to one another to form a functional chimeric receptor.Chimeric Receptor-Encoding Polynucleotide Constructs

[0204] Certain aspects of the present disclosure relate to polynucleotides (e.g., isolated polynucleotides) encoding one or more chimeric receptors of the present disclosure. In some embodiments, the polynucleotide is an RNA construct, such as a messenger RNA (mRNA) transcript or a modified RNA. In some embodiments, the polynucleotide is a DNA construct

[0205] In some embodiments, a polynucleotide of the present disclosure encodes a chimeric receptor that comprises one or more antigen-binding domain, where each domain binds to a target antigen, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the polynucleotide encodes a chimeric receptor that comprises an antigenbinding domain, a transmembrane domain, a primary signaling domain (e.g., CD 3 -zeta domain), and one or more costimulatory signaling domains. In some embodiments, the polynucleotide further comprises a nucleic acid sequence encoding a spacer region. In some embodiments, the antigen-binding domain is connected to the transmembrane domain by the spacer region.. In some embodiments, the nucleic acid further comprises a nucleotide sequence encoding a leader sequence.

[0206] The polynucleotides of the present disclosure may be obtained using any suitable recombinant methods known in the art, including, without limitation, by screening libraries from cells expressing the gene of interest, by deriving the gene of interest from a vector known to include the gene, or by isolating the gene of interest directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest may be produced using any suitable method.

[0207] In some embodiments, a polynucleotide of the present disclosure in comprised within a vector. In some embodiments, a polynucleotide of the present disclosure is expressed in a cell via transposons, a CRISPR / Cas9 system, a TALEN, or a zinc finger nuclease.

[0208] In some embodiments, expression of a polynucleotide encoding a chimeric receptor of the present disclosure may be achieved by operably linking the nucleic acid to a promoter and incorporating the construct into an expression vector. A suitable vector can replicate and integrate in eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid.

[0209] In some embodiments, expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols (e.g., US5399346, US5580859, and US5589466). In some embodiments, a vector of the present disclosure is a gene therapy vector.

[0210] A polynucleotide of the present disclosure can be cloned into a number of types of vectors. For example, the polynucleotide can be cloned into a vector including, without limitation, a plasmid, a phagemid, a phage derivative, an animal virus, or a cosmid. In some embodiments, the vector may be an expression vector, a replication vector, a probe generation vector, or a sequencing vector.

[0211] In some embodiments, the plasmid vector comprises a transposon / transposase system to incorporate the polynucleotides of the present disclosure into the host cell genome. Methods of expressing proteins in immune cells using a transposon and transposase plasmid system are generally described in Chicaybam L, Hum Gene Then 2019 Apr;30(4): 511-522. doi: 10.1089 / hum.2018.218; and Ptadkova P, Cytotherapy. 2018 Apr;20(4): 507-520. doi: 10.1016 / j.jcyt.2017.10.001, each of which is hereby incorporated by reference in their entirety. In some embodiments, the transposon system is the Sleeping Beauty transposon / transposase or the piggyBac transposon / transposase.

[0212] In some embodiments, an expression vector of the present disclosure may be provided to a cell in the form of a viral vector. Suitable viral vector systems are well known in the art. For example, viral vectors may be derived from retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses. In some embodiments, a vector of the present disclosure is a lentiviral vector. Lentiviral vectors are suitable for long-term gene transfer as such vectors allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors are also advantageous over vectors derived from onco-retroviruses (e.g., murine leukemia viruses) in that lentiviral vectors can transduce non-proliferating cells. In some embodiments, a vector of the present disclosure is an adenoviral vector (A5 / 35). In some embodiments, a vector of the present disclosure contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WOOl / 96584; W001 / 29058; and US6326193). A number of viral based systems have been developed for gene transfer into mammalian cells. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.The recombinant virus can then be isolated and delivered to mammalian cells either in vivo or ex vivo. A number of retroviral systems are known in the art.

[0213] In some embodiments, vectors of the present disclosure include additional promoter elements, such as enhancers that regulate the frequency of transcriptional initiation. Enhancers are typically located in a region that is 30 bp to 110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements may be flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. For example, in the thymidine kinase (tk) promoter the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements may function either cooperatively or independently to activate transcription. Exemplary promoters may include, without limitation, the SFFV gene promoter, the EFS gene promoter, the CMV IE gene promoter, the EFla promoter, the ubiquitin C promoter, and the phosphoglycerokinase (PGK) promoter.

[0214] In some embodiments, a promoter that is capable of expressing a polynucleotide of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is the EFla promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving chimeric receptor expression from polynucleotide cloned into a lentiviral vector.

[0215] In some embodiments, a promoter that is capable of expressing a polynucleotide of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is a constitutive promoter. For example, a suitable constitutive promoter is the spleen focus forming virus (SFFV) promoter. Another example of a suitable constitutive promoter is the immediate early cytomegalovirus (CMV) promoter. The CMV promoter is a strong constitutive promoter that is capable of driving high levels of expression of any polynucleotide sequence operatively linked to the promoter. Other suitable constitutive promoters include, without limitation, a ubiquitin C (UbiC) promoter, a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, anEpstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, an elongation factor-la promoter, a hemoglobin promoter, and a creatine kinase promoter.

[0216] In some embodiments, a promoter that is capable of expressing a polynucleotide of the present disclosure in a mammalian cell, such as an immunoresponsive cell of the present disclosure, is an inducible promoter. Use of an inducible promoter may provide a molecular switch that is capable of inducing or repressing expression of a polynucleotide of the present disclosure when the promoter is operatively linked to the polynucleotide. Examples of inducible promoters include, without limitation, a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0217] In some embodiments, a vector of the present disclosure may further comprise a signal sequence to facilitate secretion, a polyadenylation signal and transcription terminator, an element allowing episomal replication, and / or elements allowing for selection.

[0218] In some embodiments, a vector of the present disclosure can further comprise a selectable marker gene and / or reporter gene to facilitate identification and selection of chimeric receptor-expressing cells from a population of cells that have been transduced with the vector. In some embodiments, the selectable marker may be encoded by a polynucleotide that is separate from the vector and used in a co-transfection procedure. Either selectable marker or reporter gene may be flanked with appropriate regulator sequences to allow expression in host cells. Examples of selectable markers include, without limitation, antibiotic-resistance genes, such as neo and the like.

[0219] In some embodiments, reporter genes may be used for identifying transduced cells and for evaluating the functionality of regulatory sequences. As disclosed herein, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression results in an easily detectable property, such as enzymatic activity. Expression of the reporter gene can be assayed at a suitable time after the polynucleotide has been introduced into the recipient cells. Examples of reporter genes include, without limitation, genes encoding for luciferase, genes encoding for beta- galactosidase, genes encoding for chloramphenicol acetyl transferase, genes encoding for secreted alkaline phosphatase, and genes encoding for green fluorescent protein. Suitable expression systems are well known in the art and may be prepared using known techniques or obtained commercially. In some embodiments, aconstruct with a minimal 5' flanking region showing the highest level of expression of the reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0220] In some embodiments, a vector comprising a polynucleotide sequence encoding a chimeric receptor of the present disclosure further comprises a second polynucleotide encoding a polypeptide that increases the activity of the chimeric receptor.

[0221] In embodiments where a chimeric receptor-expressing cell comprises two or more chimeric receptors, a single polynucleotide may encode the two or more chimeric receptors under a single regulatory control element (e.g., promoter) or under separate regulatory control elements for each chimeric receptor-encoding nucleotide sequence comprised in the polynucleotide. In some embodiments where a chimeric receptor-expressing cell comprises two or more chimeric receptors, each chimeric receptor may be encoded by a separate polynucleotide. In some embodiments, each separate polynucleotide comprises its own control element (e.g., promoter). In some embodiments, a single polynucleotide encodes the two or more chimeric receptors and the chimeric receptor-encoding nucleotide sequences are in the same reading frame and are expressed as a single polypeptide chain. In such embodiments, the two or more chimeric receptors may be separated by one or more peptide cleavage sites, such as autocleavage sites or substrates for an intracellular protease. Suitable peptide cleavage sites may include, without limitation, a T2A peptide cleavage site, a P2A peptide cleavage site, an E2A peptide cleavage sire, and an F2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise a T2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise an E2A peptide cleavage site. In some embodiments, the two or more chimeric receptors comprise a T2A and an E2A peptide cleavage site.

[0222] Methods of introducing and expressing genes into a cell are well known in the art For example, in some embodiments, an expression vector can be transferred into a host cell by physical, chemical, or biological means. Examples of physical means for introducing a polynucleotide into a host cell include, without limitation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. Examples of chemical means for introducing a polynucleotide into a host cell include, without limitation, colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in- water emulsions, micelles, mixed micelles, and liposomes.Examples of biological means for introducing a polynucleotide into a host cell include, without limitation, the use of DNA and RNA vectors.

[0223] In some embodiments, liposomes may be used as a non-viral delivery system to introduce a polynucleotide or vector of the present disclosure into a host cell in vitro, ex vivo, or in vivo. In some embodiments, the polynucleotide may be associated with a lipid, for example by being encapsulated in the aqueous interior of a liposome, being interspersed within the lipid bilayer of a liposome, being attached to a liposome via a linking molecule that is associated with both the liposome and the polynucleotide, being entrapped in a liposome, being complexed with a liposome, being dispersed in a solution containing a lipid, being mixed with a lipid, being combined with a lipid, being contained as a suspension in a lipid, being contained or complexed with a micelle, or otherwise being associated with a lipid. As disclosed herein, lipid-associated polynucleotide or vector compositions are not limited to any particular structure in solution. In some embodiments, such compositions may be present in a bilayer structure, as micelles or with a "collapsed" structure. Such compositions may also be interspersed in a solution, forming aggregates that are not uniform in size or shape. As disclosed herein, lipids are fatty substances that may be naturally occurring or engineered. In some embodiments, lipids can include the fatty droplets that naturally occur in the cytoplasm or the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Suitable lipids may be obtained from commercial sources and include, without limitation, dimyristyl phosphatidylcholine ("DMPC"), dicetylphosphate ("DCP"), cholesterol, and dimyristylphosphatidylglycerol ("DMPG"). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about - 20°C. Chloroform is used as the solvent, as it is more readily evaporated than methanol. As used herein, a "liposome" may encompass a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. In some embodiments, liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. In some embodiments, multilamellar liposomes may have multiple lipid layers separated by aqueous medium. Multilamellar liposomes can form spontaneously when phospholipids are suspended in an excess of aqueous solution. In some embodiments, lipid components may undergo self-rearrangement before the formation of closed structures and can entrap water anddissolved solutes between the lipid bilayers. In some embodiments, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.

[0224] In some embodiments, a polynucleotide or vector of the present disclosure is introduced into a mammalian host cell, such as an immunoresponsive cell of the present disclosure. In some embodiments, the presence of a polynucleotide or vector of the present disclosure in a host cell may be confirmed by any suitable assay known in the art, including without limitation Southern blot assays, Northern blot assays, RT-PCR, PCR, ELISA assays, and Western blot assays.

[0225] In some embodiments, a polynucleotide or vector of the present disclosure is stably transduced into an immunoresponsive cell of the present disclosure. In some embodiments, cells that exhibit stable expression of the polynucleotide or vector express the encoded chimeric receptor for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 3 months, at least 6 months, at least 9 months, or at least 12 months after transduction.

[0226] In embodiments where a chimeric receptor of the present disclosure is transiently expressed in a cell, a chimeric receptor-encoding polynucleotide or vector of the present disclosure is transfected into an immunoresponsive cell of the present disclosure. In some embodiments the immunoresponsive cell expresses the chimeric receptor for about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, or about 15 days after transfection.Methods of Engineering Cells

[0227] Also provided herein are compositions and methods for engineering cells to produce one or more effectors molecules encoded by any effector molecule nucleic acid sequence described herein or otherwise known in the art. In some embodiments, the present disclosure provides for engineered cells comprising at least one engineered nucleic acid comprising one or more polarization state-specific enhancers or promoters that are operably linked to one or more nucleic acid sequences that encode for one or more polypeptides (e.g., a payload molecule, such as an effector molecule).

[0228] In general, as set forth herein, cells are engineered to produce effector molecules through introduction (i.e., delivery) of one or more polynucleotides of the present disclosure (e.g., any engineered nucleic acid described herein) comprising a provided engineeredpolarization state-specific promoter or enhancer, and an exogenous polynucleotide sequence encoding at least one effector molecule. In some embodiments, an engineered cell comprises one or more polynucleotides comprising an engineered polarization state-specific promoter or enhancer and an exogenous polynucleotide sequence encoding one or more effector molecules into the cell’s cytosol and / or nucleus. For example, a polynucleotide expression cassette encoding the one or more effector molecules can be any of the engineered nucleic acids described herein (e.g., an engineered nucleic acid comprising an engineered polarization statespecific promoter or enhancer operably linked to a nucleic acid encoding at least one payload molecule such as an effector molecule). Delivery methods include, but are not limited to, viral- mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

[0229] In some embodiments, the engineered cell is transduced using an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. In some embodiments, the oncolytic virus is a recombinant oncolytic virus comprising at least one engineered nucleic acid provided herein, e.g., at least one expression cassette (e.g., a first expression cassette, a second expression cassette, etc.) comprising an engineered promoter or enhancer operably linked to a nucleic acid encoding a payload molecule, such as a polypeptide (e.g., any suitable polypeptide described herein).

[0230] The virus, including any of the oncolytic viruses described herein, can be a recombinant virus that encodes one or more transgenes encoding one or more effector molecules, such as any of the engineered nucleic acids described herein. The virus, including any of the oncolytic viruses described herein, can be a recombinant virus that encodes one or moretransgenes encoding one or more of the two or more effector molecules, such as any of the engineered nucleic acids described herein. In some embodiments, the cell is engineered via transduction with an oncolytic virus.Viral-Mediated Delivery

[0231] Viral vector-based delivery platforms can be used to engineer cells. In general, a viral vector-based delivery platform engineers a cell through introducing (i.e., delivering) into a host cell. For example, a viral vector-based delivery platform can engineer a cell through introducing any of the engineered nucleic acids described herein. A viral vector-based delivery platform can be a nucleic acid, and as such, a engineered nucleic acid can also encompass a engineered virally-derived nucleic acid. Such engineered virally-derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0232] A viral vector-based delivery platform can encode more than one engineered nucleic acid, gene, or transgene within the same nucleic acid. For example, a engineered virally-derived nucleic acid, e.g., a recombinant virus or a engineered virus, can encode one or more transgenes, including, but not limited to, any of the engineered nucleic acids described herein that encode one or more effector molecules. The one or more transgenes encoding the one or more effector molecules can be configured to express the one or more effector molecules. A viral vector-based delivery platform can encode one or more genes in addition to the one or more transgenes (e.g., transgenes encoding the one or more effector molecules), such as viral genes needed for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), referred to as cis-acting elements or genes.

[0233] A viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding the engineered nucleic acids, genes, or transgenes described herein, and referred to as trans-acting elements or genes. For example, a helper-dependent viral vector-based delivery platform can provide additional genes needed for viral infectivity and / or viral production on one or more additional separate vectors in addition to the vector encoding the one or more effector molecules. One viral vector can deliver more than one engineered nucleic acids, such as one vector that delivers engineered nucleic acids that are configured to produce two or more effector molecules. More than one viral vector can deliver more than one engineered nucleic acids, such as more than one vector that delivers one or more engineered nucleic acidconfigured to produce one or more effector molecules. The number of viral vectors used can depend on the packaging capacity of the above-mentioned viral vector-based vaccine platforms, and one skilled in the art can select the appropriate number of viral vectors.

[0234] In general, any of the viral vector-based systems can be used for the in vitro production of molecules, such as effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more effector molecules. The selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo / payload size, immunogenicity of the viral system, target cell of interest, gene expression strength and timing, and other factors appreciated by one skilled in the art

[0235] Viral vector-based delivery platforms can be RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, a herpes simplex virus, a adenovirus, a measles virus, an influenza virus, a Indiana vesiculovirus, a Newcastle disease virus, a vaccinia virus, a poliovirus, a myxoma virus, a reovirus, a mumps virus, a Maraba virus, a rabies virus, a rotavirus, a hepatitis virus, a rubella virus, a dengue virus, a chikungunya virus, a respiratory syncytial virus, a lymphocytic choriomeningitis virus, a morbillivirus, a lentivirus, a replicating retrovirus, a rhabdovirus, a Seneca Valley virus, a sindbis virus, and any variant or derivative thereof. Other exemplary viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus, including but not limited to second, third or hybrid second / third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper etal., Rescue of splicing- mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey etal., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880).

[0236] The sequences may be preceded with one or more sequences targeting a subcellular compartment Upon introduction (i.e. delivery) into a host cell, infected cells (i.e., a engineered cell) can express, and in some case secrete, the one or more effector molecules. Vaccinia vectorsand methods useful in immunization protocols are described in, e.g., U.S. Pat No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.

[0237] The viral vector-based delivery platforms can be a virus that targets a tumor cell, herein referred to as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. Any of the oncolytic viruses described herein can be a recombinant oncolytic virus comprising one or more transgenes (e.g., a engineered nucleic acid) encoding one or more effector molecules. The transgenes encoding the one or more effector molecules can be configured to express the one or more effector molecules.

[0238] In some embodiments, the virus is selected from: a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a virus-like particle (VLP).

[0239] The viral vector-based delivery platform can be retrovirus-based. In general, retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., transgenes encoding the one or more effector molecules) into the target cell to provide permanent transgene expression. Retroviral-based delivery systems include, but are not limited to, those based upon murine leukemia, virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency vims (SIV), human immuno deficiency vims (HIV), and combinations thereof (see, e.g., Buchscher etal, J. Virol. 66:2731-2739 (1992); Johann et ah, J. Virol. 66:1635-1640 (1992); Sommnerfelt etal., Virol. 176:58-59 (1990); Wilson et ah, J. Virol.63:2374-2378 (1989); Miller et al, J, Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retrovirus system.

[0240] The viral vector-based delivery platform can be lentivirus-based. In general, lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV-based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs). . Lentiviral-based delivery platforms can be SIV, or FlV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 7,311,907; 7,262,049; 7,250,299; 7,226,780;7,220,578; 7,211,247; 7,160,721; 7,078,031; 7,070,993; 7,056,699; 6,955,919, each herein incorporated by reference for all purposes.

[0241] The viral vector-based delivery platform can be adenovirus-based. In general, adenoviral based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titer and levels of expression, and can be produced in large quantities in a relatively simple system. In general, adenoviruses can be used for transient expression of a transgene within an infected cell since adenoviruses do not typically integrate into a host’s genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras etal., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto etal., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655, each herein incorporated by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 5585362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793 and International Patent Application WO96 / 13597, each herein incorporated by reference for all purposes.

[0242] The viral vector-based delivery platform can be adeno-associated virus (AAV)-based. Adeno-associated virus (“AAV”) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used for the in vitro production of effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more effector molecules (see, e.g., West etal, Virology 160:38-47 (1987); U.S. Pat Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; US patent publications US 2003-0138772, US 2007 / 0036760, and US 2009 / 0197338; Gao, etal., J. Virol,78(12):6381-6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent applications WO 2010 / 138263 and WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each herein incorporated by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat No, 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:64666470 (1984); and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each herein incorporated by reference for all purposes. In general, an AAV-based vector comprises a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVRhlO, AAV11 and variants thereof.

[0243] The viral vector-based delivery platform can be a virus-like particle (VLP) platform. In general, VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated within the purified particle ex vivo. Accordingly, production of VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids encoding the cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art Production of VLPs are described in more detail in Seow et al.(Mol Then 2009 May; 17(5): 767-777), herein incorporated by reference for all purposes.

[0244] The viral vector-based delivery platform can be engineered to target (i.e., infect) a range of cells, target a narrow subset of cells, or target a specific cell. In general, the envelope protein chosen for the viral vector-based delivery platform will determine the viral tropism. The virus used in the viral vector-based delivery platform can be pseudotyped to target a specific cell of interest. The viral vector-based delivery platform can be pantropic and infect a range of cells. For example, pantropic viral vector-based delivery platforms can include the VSV-G envelope. The viral vector-based delivery platform can be amphotropic and infect mammalian cells. Accordingly, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein for targeting a desired cell type.Lipid Structure Delivery Systems

[0245] Engineered nucleic acids of the present disclosure (e.g., any of the engineered nucleic acids described herein) can be introduced into a cell using a lipid-mediated delivery system. In general, a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue. Lipid structure delivery systems can deliver a cargo / payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0246] A lipid-based nanoparticle can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, and a lipid preparation. As used herein, a “liposome” is a generic term encompassing in vitro preparations of lipid vehicles formed by enclosing a desired cargo, e.g., a engineered nucleic acid, such as any of the engineered nucleic acids described herein, within a lipid shell or a lipid aggregate. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner medium that generally comprises an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes can be unilamellar liposomes. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, the liposomes are neutral in charge. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of a desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Pat Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each herein incorporated by reference for all purposes.

[0247] A multilamellar liposome is generated spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution such that multiple lipid layers are separated by an aqueous medium. Water and dissolved solutes are entrapped in closed structuresbetween the lipid bilayers following the lipid components undergoing self-rearrangement A desired cargo (e.g., a polypeptide, a nucleic acid, a small molecule drug, a engineered nucleic acid, such as any of the engineered nucleic acids described herein, a viral vector, a viral-based delivery system, etc.) can be encapsulated in the aqueous interior of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of a liposome, entrapped in a liposome, complexed with a liposome, or otherwise associated with the liposome such that it can be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

[0248] A liposome used according to the present embodiments can be made by different methods, as would be known to one of ordinary skill in the art. Preparations of liposomes are described in further detail in WO 2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each herein incorporated by reference for all purposes.

[0249] Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent No. 5,962,016; 5,030,453; 6,680,068, U.S. Application 2004 / 0208921, and International Patent Applications W003 / 015757A1, WO04029213A2, and W002 / 100435A1, each hereby incorporated by reference in their entirety.

[0250] Lipid-mediated gene delivery methods are described, for instance, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat No. 5,279,833; W091 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each herein incorporated by reference for all purposes.

[0251] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. The size of exosomes ranges between 30 and 100 run in diameter. Their surface consists of a lipid bilayer from the donor cell's cell membrane, and they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface. Exosomes useful for the delivery of nucleic acids are known to those skilled in the art, e.g., the exosomes described in more detail in U.S. Pat. No. 9,889,210, herein incorporated by reference for all purposes.

[0252] As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. In general, extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally extracellular vesicles range in diameter from 20 nm to 1000 run, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. The cargo can comprise nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example and without limitation, extracellular vesicles include apoptotic bodies, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane).Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and / or cultured cells.

[0253] As used herein the term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a species of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producer cell. Exosomes and preparation of exosomes are described in further detail in WO 2016 / 201323, which is hereby incorporated by reference in its entirety.

[0254] As used herein, the term “nanovesicle” (also referred to as a “microvesicle”) refers to a cell-derived small (between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct or indirect manipulation such that said nanovesicle would not be produced by saidproducer cell without said manipulation. In general, a nanovesicle is a sub-species of an extracellular vesicle. Appropriate manipulations of the producer cell include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane. The nanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.

[0255] Lipid nanoparticles (LNPs), in general, are engineered lipid structures that rely on the amphiphilic nature of lipids to form membranes and vesicle like structures (Riley 2017). In general, these vesicles deliver cargo / payloads, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of target cells and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be engineered or naturally derived, and in some instances biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as the cationic, neutral, anionic, and amphipathic lipids. In some instances, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that facilitate attachment of additional moieties. Lipid composition can influence overall LNP size and stability. In an example, the lipid composition comprises dilinoleylmethyl- 4- dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as a PEG or PEG-conjugated lipid, a sterol, or neutral lipids. In addition, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0256] Micelles, in general, are spherical engineered lipid structures that are formed using single-chain lipids, where the single-chain lipid’s hydrophilic head forms an outer layer or membrane and the single-chain lipid’s hydrophobic tails form the micelle center. Micelles typically refer to lipid structures only containing a lipid mono-layer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), herein incorporated by reference for all purposes.

[0257] Nucleic-acid vectors, such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by the free nucleic acids. Similarly, viral delivery systems exposed directly to serum can trigger an undesired immune response and / or neutralization of the viral delivery system. Therefore, encapsulation of a engineered nucleic acid and / or viral delivery system can be used to avoid degradation, while also avoiding potential off- target affects. In certain examples, a engineered nucleic acid and / or viral delivery system is fully encapsulated within the delivery vehicle, such as within the aqueous interior of an LNP. Encapsulation of a engineered nucleic acid and / or viral delivery system within an LNP can be carried out by techniques well-known to those skilled in the art, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In an example, the desired lipid formulation, such as MC3 or MC3-like containing compositions, is provided to the droplet generating device in parallel with a engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like based LNP. In an example, the droplet generating device can control the size range and size distribution of the LNPs produced. For example, the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery vehicles encapsulating the cargo / payload (e.g., a engineered nucleic acid and / or viral delivery system) can be further treated or engineered to prepare them for administration.Nanonartide Delivery

[0258] Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Nanomaterial vehicles, importantly, can be made ofnon-immunogenic materials and generally avoid eliciting immunity to the delivery vector itself. These materials can include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery — A Review. Nanomaterials 2017, 7(5), 94), herein incorporated by reference for all purposes.Genomic Editing Systems

[0259] A genomic editing systems can be used to engineer a host genome to encode a engineered nucleic acid, such as a engineered nucleic acid of the present disclosure. In general, a “genomic editing system” refers to any system for integrating an exogenous gene into a host cell’s genome. Genomic editing systems include, but are not limited to, a transposon system, a nuclease genomic editing system, and a viral vector-based delivery platform.

[0260] A transposon system can be used to integrate a engineered nucleic acid, such as a engineered nucleic acid of the present disclosure, into a host genome. Transposons generally comprise terminal inverted repeats (TIR) that flank a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or in trans with the HR- flanked cargo. A transposon system can be a retrotransposon system or a DNA transposon system. In general, transposon systems integrate a cargo / payload (e.g., a engineered nucleic acid) randomly into a host genome. Examples of transposon systems include systems using a transposon of the Tcl / mariner transposon superfamily, such as a Sleeping Beauty transposon system, described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380), and U.S. Patent Nos. 6,489,458, 6,613,752 and 7,985,739, each of which is herein incorporated by reference for all purposes. Another example of a transposon system includes a PiggyBac transposon system, described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is herein incorporated by reference for all purposes.

[0261] A nuclease genomic editing system can be used to engineer a host genome to encode a engineered nucleic acid, such as a engineered nucleic acid of the present disclosure. Without wishing to be bound by theory, in general, the nuclease-mediated gene editing systems used to introduce an exogenous gene take advantage of a cell’s natural DNA repair mechanisms, particularly homologous recombination (HR) repair pathways. Briefly, following an insult to genomic DNA (typically a double-stranded break), a cell can resolve the insult by using anotherDNA source that has identical, or substantially identical, sequences at both its 5’ and 3’ ends as a template during DNA synthesis to repair the lesion. In a natural context, HDR can use the other chromosome present in a cell as a template. In gene editing systems, exogenous polynucleotides are introduced into the cell to be used as a homologous recombination template (HRT or HR template). In general, any additional exogenous sequence not originally found in the chromosome with the lesion that is included between the 5’ and 3’ complimentary ends within the HRT (e.g., a gene or a portion of a gene) can be incorporated (i.e., “integrated”) into the given genomic locus during templated HDR Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of an endogenous genomic target locus, a nucleotide sequence identical to a second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids encoding one or more effector molecules).

[0262] In some examples, a HR template can be linear. Examples of linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA. In particular examples, a HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.

[0263] The identical, or substantially identical, sequences found at the 5’ and 3’ ends of the HR template, with respect to the exogenous sequence to be introduced, are generally referred to as arms (HR arms). HR arms can be identical to regions of the endogenous genomic target locus (i.e., 100% identical). HR arms in some examples can be substantially identical to regions of the endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for HR arms to be identical as the efficiency of the HDR pathway may be impacted by HR arms having less than 100% identity.

[0264] Each HR arm, i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can each be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. Although HR arms can, in general, be of any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account. An HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical to, or substantially identical to, regions of an endogenous genomic target locus immediatelyadjacent to a cleavage site. Each HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus within a certain distance of a cleavage site, such as 1 base-pair, less than or equal to 10 base-pairs, less than or equal to 50 base-pairs, or less than or equal to 100 base-pairs of each other.

[0265] A nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus, including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activatorlike effector nuclease (TALEN) or derivative thereof, a zine-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.

[0266] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode a engineered nucleic acid, such as a engineered nucleic acid encoding one or more of the effector molecules described herein. CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), herein incorporated by reference for all that it teaches. In general, a CRISPR-mediated gene editing system comprises a CRISPR-associated (Cas) nuclease and a RNA(s) that directs cleavage to a particular target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 systems comprised of a Cas9 nuclease and a RNA(s) that has a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity through base-pair hybridization to a target sequence (“a defined nucleotide sequence”), e.g., a genomic sequence; and an RNA domain that hybridizes to a tracrRNA. A tracrRNA can interact with and thereby promote recruitment of a nuclease (e.g., Cas9) to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is illustrated here, other CRISPR systems can be used, such as the Cpfl system. Nucleases can include derivatives thereof, such as Cas9 functional mutants, e.g., a Cas9 “nickase” mutant that in general mediates cleavage of only a single strand of a defined nucleotide sequence as opposed to a complete double-stranded break typically produced by Cas9 enzymes.

[0267] In general, the components of a CRISPR system interact with each other to form a Ribonucleoprotein (RNP) complex to mediate sequence specific cleavage. In some CRISPRsystems, each component can be separately produced and used to form the RNP complex. In some CRISPR systems, each component can be separately produced in vitro and contacted (i.e., “complexed”) with each other in vitro to form the RNP complex. The in vitro produced RNP can then be introduced (i.e., “delivered”) into a cell’s cytosol and / or nucleus, e.g., a T cell’s cytosol and / or nucleus. The in vitro produced RNP complexes can be delivered to a cell by a variety of means including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNP), virus like particles (VLP), and sonication. In a particular example, in vitro produced RNP complexes can be delivered to a cell using a Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation systems, Miltenyi CliniMACS electroporation systems, Neon electroporation systems, and BTX electroporation systems. CRISPR nucleases, e.g., Cas9, can be produced in vitro (i.e., synthesized and purified) using a variety of protein production techniques known to those skilled in the art CRISPR system RNAs, e.g., an sgRNA, can be produced in vitro (i.e., synthesized and purified) using a variety of RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0268] An in vitro produced RNP complex can be complexed at different ratios of nuclease to gRNA. An in vitro produced RNP complex can be also be used at different amounts in a CRISPR-mediated editing system. For example, depending on the number of cells desired to be edited, the total RNP amount added can be adjusted, such as a reduction in the amount of RNP complex added when editing a large number of cells in a reaction.

[0269] In some CRISPR systems, each component (e.g., Cas9 and an sgRNA) can be separately encoded by a polynucleotide with each polynucleotide introduced into a cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multicistronic vector, see description of exemplary multicistronic systems below) and introduced into a cell. Following expression of each polynucleotide encoded CRISPR component within a cell (e.g., translation of a nuclease and transcription of CRISPR RNAs), an RNP complex can form within the cell and can then direct site-specific cleavage.

[0270] Some RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS)domain such that if a Cas9 RNP complex is delivered into a cell’s cytosol or following translation of Cas9 and subsequent RNP formation, the NLS can promote further trafficking of a Cas9 RNP into the nucleus.

[0271] The engineered cells described herein can be engineered using non-viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using non-viral methods. The engineered cells described herein can be engineered using viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using viral methods such as adenoviral, retroviral, lentiviral, or any of the other viral-based delivery methods described herein.

[0272] In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target the same gene or general genomic locus at more than target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other. In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus. For example, two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.

[0273] In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. TALEN is a engineered site-specific nuclease, which is composed of the DNA- binding domain of TALE (transcription activator-like effectors) and the catalytic domain of restriction endonuclease Fokl. By changing the amino acids present in the highly variable residue region of the monomers of the DNA binding domain, different artificial TALENs can be created to target various nucleotides sequences. The DNA binding domain subsequently directs the nuclease to the target sequences and creates a doublestranded break. TALEN-based systems are described in more detail in U.S. Ser. No. 12 / 965,590; U.S. Pat No. 8,450,471; U.S. Pat No. 8,440,431; U.S. Pat No. 8,440,432; U.S. Pat No.10,172,880; and U.S. Ser. No. 13 / 738,381, all of which are incorporated by reference herein in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474;2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties for all purposes.Other Engineering Delivery Systems

[0274] Also provided herein are various additional means to introduce engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0275] Electroporation can used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing a cargo / payload into a target cell or entity’s interior compartment through applying an electrical field to transiently permeabilize the outer membrane or shell of the target cell or entity. In general, the method involves placing cells or target entities between two electrodes in a solution containing a cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cells is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of cells, at least some, if not a majority, of the cells remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., number of cells, concentration of cargo, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors including, but not limited to, the type of cell or other recipient entity, the cargo to be delivered, the efficiency of internalization desired, and the viability desired. Optimization of such criteria are within the scope of those skilled in the art A variety devices and protocols can be used for electroporation. Examples include, but are not limited to, Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ systems, and Bio-Rad® electroporation systems.

[0276] Other means for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity include, but are not limited to, sonication, gene gun, hydrodynamic injection, and cell membrane deformation by physical means.

[0277] Compositions and methods for delivering engineered mRNAs in vivo, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Then 2019 Apr 10; 27(4):710-728) and Kaczmarek et al. (Genome Med. 2017; 9: 60.), each herein incorporated by reference for all purposes.Methods of Use

[0278] Methods for treatment of diseases are also encompassed by this disclosure. Said methods include administering a therapeutically effective amount of a engineered nucleic acid, engineered cell, or isolated cell as described above. In some aspects, provided herein are methods of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0279] In some aspects, provided herein is a method of increasing expression of a target gene, e.g. a tumor suppressor gene.

[0280] In some aspects, provided herein a methods of increasing expression of a target gene, e.g. an immunomodulatory gene. Exemplary immunomodulatory genes include, for example and without limitation, cytokines, chemokines, receptors thereof, and derivatives thereof.

[0281] In some aspects, provided herein are methods of stimulating a cell-mediated immune response to a tumor cell in a subject, the method comprising administering to a subject having a tumor a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0282] In some aspects, provided herein are methods of providing an anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0283] In some aspects, provided herein are methods of treating a subject having cancer, the method comprising administering a therapeutically effective dose of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0284] In some aspects, provided herein are methods of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the engineered cells, isolated cells, or compositions disclosed herein.

[0285] In some embodiments, the administering comprises systemic administration. In some embodiments, the administering comprises intratumoral administration. In some embodiments, the isolated cell is derived from the subject In some embodiments, the isolated cell is allogeneic with reference to the subject.

[0286] In some embodiments, the method further comprises administering a checkpoint inhibitor, the checkpoint inhibitor is selected from: an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti- TIM-3 antibody, an anti-TIGIT antibody, an anti- VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti- CD27 antibody, an anti-TNFa antibody, an anti-TREMl antibody, and an anti-TREM2 antibody. In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0287] In some embodiments, the tumor is selected from: an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colorectal tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0288] Some methods comprise selecting a subject (or patient population) having a tumor (or cancer) and treating that subject with engineered cells or delivery vehicles that modulate tumor- mediated immunosuppressive mechanisms.

[0289] The methods provided herein also include delivering a preparation of engineered cells or delivery vehicles. A preparation, in some embodiments, is a substantially pure preparation, containing, for example, less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other tha engineered cells. A preparation may comprise 1x105cells / kg to lxl 07cells / kg cells.In vivo Expression

[0290] The methods provided herein also include delivering a composition in vivo capable of producing the engineered cells described herein, e.g., capable of delivering any of the engineered nucleic acids described herein to a cell in vivo. Such compositions include any of the viral-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genomic editing systems, or any of the other engineering delivery systems described herein capable of engineering a cell in vivo.

[0291] The methods provided herein also include delivering a composition in vivo capable of producing any of the effector molecules described herein. The methods provided herein also include delivering a composition in vivo capable of producing two or more of theeffector molecules described herein. Compositions capable of in vivo production of effector molecules include, but are not limited to, any of the engineered nucleic acids described herein. Compositions capable of in vivo production of effector molecules can be a naked mRNA or a naked plasmid.Pharmaceutical Compositions

[0292] The engineered nucleic acid or engineered cell can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the engineered nucleic acids or engineered cells, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient The precise nature of the carrier or other material can depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0293] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or engineered oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol can be included.

[0294] For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included, as required.

[0295] Whether it is a polypeptide, nucleic acid, small molecule or other pharmaceutically useful compound according to the present disclosure that is to be given to an individual, administration is preferably in a “therapeutically effective amount” or “prophylactically effective amount”(as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and timecourse of administration, will depend on the nature and severity of protein aggregation diseasebeing treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0296] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.Kits

[0297] Certain aspects of the present disclosure relate to kits for the treatment and / or prevention of disease or disorder. In certain embodiments, the disease or disorder is a cancer (e.g., solid tumors). In certain embodiments, the kit includes a therapeutic or prophylactic composition comprising an effective amount of one or more chimeric receptors of the present disclosure, isolated nucleic acids of the present disclosure, vectors of the present disclosure, and / or cells of the present disclosure (e.g., immunoresponsive cells). In some embodiments, the kit comprises a sterile container. In some embodiments, such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. The container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0298] In some embodiments, therapeutic or prophylactic composition is provided together with instructions for administering the therapeutic or prophylactic composition to a subject having or at risk of developing cancer (e.g., a solid tumor). In some embodiments, the instructions may include information about the use of the composition for the treatment and / or prevention of the disorder. In some embodiments, the instructions include, without limitation, a description of the therapeutic or prophylactic composition, a dosage schedule, an administration schedule for treatment or prevention of the disorder or a symptom thereof, precautions, warnings, indications, counter-indications, over-dosage information, adverse reactions, animal pharmacology, clinical studies, and / or references. In some embodiments, the instructions can be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.Enumerated Embodiments:Embodiment 1 An engineered enhancer comprising: a. a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NOs: 2-16, and 192-194, wherein the nucleotide sequence does not comprise SEQ ID NO: 1; or b. a nucleotide sequence at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to any one or more of SEQ ID NOs: 2-16, and 192-194; wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or M0 macrophage.Embodiment 2 The engineered enhancer of embodiment 1, wherein the nucleotide sequence is at least 96% identical to any one of: SEQ ID NOs: 2-16, and 192-194.Embodiment 3 The engineered enhancer of embodiment 1, wherein the nucleotide sequence is 100% identical to any one of: SEQ ID NOs: 2-16, and 192-194.Embodiment 4 The engineered enhancer of embodiment 1, wherein the engineered enhancer comprises: a. a nucleotide sequence at least 91.5% identical to SEQ ID NO: 2, wherein the engineered enhancer does not comprise the sequence TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168); b. a nucleotide sequence at least 96.4% identical to SEQ ID NO: 3, wherein the engineered enhancer does not comprise the sequence TGTGACTCACA (SEQ ID NO: 169); c. a nucleotide sequence at least 94.8% identical to SEQ ID NO: 4, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCC (SEQ ID NO: 170);d. a nucleotide sequence at least 98.4% identical to SEQ ID NO: 5, wherein the engineered enhancer does not comprise the sequence ATAAAT (SEQ ID NO: 171); e. a nucleotide sequence at least 89.8% identical to SEQ ID NO: 6, wherein the engineered enhancer does not comprise the sequence TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172); f. a nucleotide sequence at least 92.8% identical to SEQ ID NO: 7, wherein the engineered enhancer does not comprise the sequence TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173); g- a nucleotide sequence at least 90.4% identical to SEQ ID NO: 8, wherein the engineered enhancer does not comprise the sequence AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174); h. a nucleotide sequence at least 93.5% identical to SEQ ID NO: 9, wherein the engineered enhancer does not comprise the sequence CAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175); i. a nucleotide sequence at least 91.6% identical to SEQ ID NO: 10, wherein the engineered enhancer does not comprise the sequence CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176);J- a nucleotide sequence at least 90% identical to SEQ ID NO: 11, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177); k. a nucleotide sequence at least 91.2% identical to SEQ ID NO: 12, wherein the engineered enhancer does not comprise the sequence TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178); or1. a nucleotide sequence at least 91.6% identical to SEQ ID NO: 13, wherein the engineered enhancer does not comprise the sequence AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179).Embodiment 5 An engineered enhancer comprising a variant of SEQ ID NO: 1, wherein the variant of SEQ ID NO: 1 comprises an ablation of any one or more regions selected from: (a) positions 76-110 of SEQ ID NO: 1; (b) positions 122-132 of SEQ ID NO: 1;(c) positions 157-172 of SEQ ID NO: 1; (d) positions 1-6 of SEQ ID NO: 1; (e) positions 7-36 of SEQ ID NO: 1; (f) positions 37-66 of SEQ ID NO: 1; (g) positions 67-96 of SEQ ID NO: 1; (h) positions 97-126 of SEQ ID NO: 1; (i) positions 127-156 of SEQ ID NO: 1; (j) positions 157-186 of SEQ ID NO: 1; (k) positions 187-216 of SEQ ID NO: 1; and(1) positions 217-246 of SEQ ID NO: 1; wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or MO macrophage.Embodiment 6 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 76-110 of SEQ ID NO: 1, wherein the sequence corresponding to position 76-110 of SEQ ID NO: 1 is: TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168).Embodiment 7 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 122-132 of SEQ ID NO: 1, wherein the sequence corresponding to position 122-132 of SEQ ID NO: 1 is: TGTGACTCACA (SEQ ID NO: 169).Embodiment 8 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 157-172 of SEQ ID NO: 1, wherein the sequence corresponding to position 157-172 of SEQ ID NO: 1 is: GGGGACACAATGTTCC (SEQ ID NO: 170).Embodiment 9 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 1-6 of SEQ ID NO: 1, wherein the sequence corresponding to position 1-6 of SEQ ID NO: 1 is: ATAAAT (SEQ ID NO: 171).Embodiment 10 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 7-36 of SEQ ID NO: 1, wherein the sequence corresponding to position 7-36 of SEQ ID NO: 1 is: TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172).Embodiment 11 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 37-66 of SEQ ID NO: 1,wherein the sequence corresponding to position 37-66 of SEQ ID NO: 1 is: TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173).Embodiment 12 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 67-96 of SEQ ID NO: 1, wherein the sequence corresponding to position 67-96 of SEQ ID NO: 1 is: AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174).Embodiment 13 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 97-126 of SEQ ID NO: 1, wherein the sequence corresponding to position 97-126 of SEQ ID NO: 1 is: CAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175).Embodiment 14 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 127-156 of SEQ ID NO: 1, wherein the sequence corresponding to position 127-156 of SEQ ID NO: 1 is: CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176).Embodiment 15 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 157-186 of SEQ ID NO: 1, wherein the sequence corresponding to position 157-186 of SEQ ID NO: 1 is: GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177).Embodiment 16 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 187-216 of SEQ ID NO: 1, wherein the sequence corresponding to position 187-216 of SEQ ID NO: 1 is: TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178).Embodiment 17 The engineered enhancer of embodiment 5, wherein the engineered enhancer comprises a sequence corresponding to position 217-246 of SEQ ID NO: 1, wherein the sequence corresponding to position 217-246 of SEQ ID NO: 1 is: AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179).Embodiment 18 An engineered macrophage specific promoter comprising an engineered enhancer of any one of the preceding embodiments operably linked to a minimal promoter.Embodiment 19 The engineered macrophage specific promoter of embodiment 18, wherein the minimal promoter is selected from: a hybrid YBTATA-SCP3 (“YB-SCP3”), SCP3, SCP3 containing DPR, minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB TATA, minTK, inducer molecule responsive promoters, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb, and tandem repeats thereof.Embodiment 20 The engineered macrophage specific promoter of embodiment 19, wherein the minimal promoter is YB-SCP3.Embodiment 21 The engineered macrophage specific promoter of embodiment 20, wherein the minimal promoter comprises the sequenceEmbodiment 22 The engineered macrophage specific promoter of embodiment 18, wherein the minimal promoter comprises the sequenceEmbodiment 23 A heterologous construct comprising: a. the engineered enhancer of any one of embodiments 1-17 or the engineered macrophage-specific promoter of any one of embodiments 18-22; and b. a heterologous payload,wherein the engineered enhancer or engineered promoter is operably linked to the heterologous payload.Embodiment 24 The heterologous construct of embodiment 23, wherein the heterologous payload comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding a polypeptide.Embodiment 25 The heterologous construct of embodiment 24, wherein the polypeptide comprises at least one effector molecule.Embodiment 26 The heterologous construct of any one of embodiments 24-25, wherein the polypeptide comprises a first effector molecule and a second effector molecule.Embodiment 27 The heterologous construct of embodiment 26, wherein the polynucleotide comprises a nucleotide sequence encoding the first effector molecule, a linker nucleotide sequence, and a nucleotide sequence encoding the second effector.Embodiment 28 The heterologous construct of embodiment 27, wherein the linker nucleotide sequence encodes one or more 2A ribosome skipping elements.Embodiment 29 The heterologous construct of embodiment 28, wherein the one or more 2A ribosome skipping elements comprise elements that are each selected from the group consisting of: P2A, T2A, E2A, and F2A.Embodiment 30 The heterologous construct of any one of embodiments 25-29, wherein the at least one effector molecule or each effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a polynucleotide molecule, a coactivation molecule, a tumor microenvironment modifier, a receptor, a ligand, a transcription factor, an antibody, a peptide, and an enzyme.Embodiment 31 The heterologous construct of any one of embodiments 25-30, wherein the at least one effector molecule or each effector molecule is a human-derived effector molecule.Embodiment 32 A heterologous construct for inducing a macrophage to transition from an M2 state to an Ml state, comprising: a. the engineered enhancer of any one of embodiments 1-17 or the engineered macrophage-specific promoter of any one of embodiments 18-22; and b. a heterologous pay load encoding a master regulator of polarization to an Ml macrophage, wherein the engineered enhancer or the engineered macrophage-specific promoter of a) is operably linked to the heterologous pay load and configured to induce expression of the heterologous payload.Embodiment 33 The heterologous construct of embodiment 32, wherein the master regulator of polarization to an Ml macrophage is a cytokine.Embodiment 34 The heterologous construct of embodiment 33, wherein the cytokine is IFNgamma, IFNalpha, TNF alpha, GM-CSF, IL-12, IL-12p70, IL-12p40, IL-12p35, IL-6, IL-23, IL-lalpha, IL- 1 beta, or a derivative thereof.Embodiment 35 The heterologous construct of embodiment 33 or 34, wherein the cytokine is modified to comprise a membrane tethering domain, optionally wherein the membrane tethering domain is or comprises a transmembrane-intracellular domain and / or transmembrane domain of a protein selected from: PDGFR-beta, CDS, CD28, CD3zeta- chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA, or a functional portion thereof, optionally wherein the membrane tethering domain is or comprises a transmembrane domain of B7-1 protein, or a functional portion thereof.Embodiment 36 The heterologous construct of embodiment 32, wherein the master regulator of polarization to an Ml macrophage is a transcription factor selected from IRF7 or a derivative thereof, or p65 / RelA or a derivative thereof.Embodiment 37 A heterologous construct for stabilizing a macrophage in an M2 polarization state, comprising:a. the engineered enhancer of any one of embodiments 1-17 or the engineered macrophage-specific promoter of any one of embodiments 18-22; and b. a heterologous pay load encoding a master regulator of polarization to an M2 macrophage, wherein the engineered enhancer or the engineered macrophage-specific promoter of a) is operably linked to the heterologous pay load and configured to induce expression of the heterologous payload.Embodiment 38 The heterologous construct of embodiment 37, wherein the master regulator of polarization to an M2 macrophage is a cytokine selected from the group consisting of IL-10, IL-4, IL-13, IL-21, TGF-beta, M-CSF, or a derivative thereof.Embodiment 39 The heterologous construct of embodiment 37 or 38, wherein the cytokine is modified to comprise a membrane tethering domain, optionally wherein the membrane tethering domain is or comprises a transmembrane-intracellular domain and / or transmembrane domain of a protein selected from: PDGFR-beta, CDS, CD28, CD3zeta- chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA, or a functional portion thereof, optionally wherein the membrane tethering domain is or comprises a transmembrane domain of B7-1 protein, or a functional portion thereof.Embodiment 40 The heterologous construct of embodiment 37, wherein master regulator of polarization to an M2 macrophage is a transcription factor.Embodiment 41 The heterologous construct of embodiment 40 wherein the transcription factor is selected from the group consisting of EGR2, STAT3, PPAR-gamma, PPAR- alpha, and Myc.Embodiment 42 The heterologous construct of any one of embodiments 32-41, wherein the M2 state is an M2c state, an M2a state, or an M2b state.Embodiment 43 A vector comprising the heterologous construct of any one of embodiments 23-42.Embodiment 44 A dual expression vector comprising the heterologous construct of any one of embodiments 23-42 and a second construct comprising a nucleotide sequence encoding an activating immune receptor.Embodiment 45 An immunoresponsive cell comprising the heterologous construct of any one of embodiments 23-42, the vector of embodiment 43, or the dual expression vector of embodiment 44.Embodiment 46 The immunoresponsive cell of embodiment 45, wherein the immunoresponsive cell is selected from the group consisting of: a macrophage, a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (HL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.Embodiment 47 The immunoresponsive cell of embodiment 46, wherein the immunoresponsive cell is a macrophage.Embodiment 48 The immunoresponsive cell of embodiment 47, wherein the macrophage is a tumor-resident macrophage.Embodiment 49 The immunoresponsive cell of any one of embodiments 45-48, wherein the immunoresponsive cell expresses an activating immune receptor.Embodiment 50 The immunoresponsive cell of embodiment 45-49, wherein the activating immune receptor comprises an antigen recognizing receptor.Embodiment 51 The immunoresponsive cell of any one of embodiments 45-50, wherein the immunoresponsive cell is autologous.Embodiment 52 The immunoresponsive cell of any one of embodiments 45-51, wherein the immunoresponsive cell is allogeneic.Embodiment 53 A pharmaceutical composition comprising the vector of embodiment 43, the dual expression vector of embodiment 44, or the immunoresponsive cell of any one of embodiments 45-52, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.Embodiment 54 A method of increasing expression of a target gene, the method comprising use of the engineered enhancer or the engineered macrophage-specific promoter of any one of embodiments 1-22, the vector of embodiment 43, or the dual expression vector of embodiment 44 to increase expression of the target gene.Embodiment 55 The method of embodiment 54, wherein the target gene is an immunomodulatory gene.Embodiment 56 A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the vector of embodiment 43, the dual expression vector of embodiment 44, the immunoresponsive cell of any one of embodiments 45-52, or the pharmaceutical composition of embodiment 53.Embodiment 57 A kit for treating and / or preventing a disease or disorder, comprising the immunoresponsive cell of any one of embodiments 45-52 or the pharmaceutical composition of embodiment 53.Embodiment 58 The kit of embodiment 57, wherein the disease or disorder comprises a tumor.Embodiment 59 The kit of embodiment 57 or 58, wherein the kit further comprises written instructions for using the immunoresponsive cell or the pharmaceutical composition for treating and / or preventing the disease or disorder in a subjectEmbodiment 60 An engineered enhancer comprising a nucleotide sequence having at least 75%, 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% sequence identity to any one or more of SEQ ID NOs:17 - 31, wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or MO macrophage.Embodiment 61 The engineered enhancer of embodiment 60, wherein the nucleotide sequence is at least 96% identical to any one of: SEQ ID NOs: 17 - 31.Embodiment 62 The engineered enhancer of embodiment 60, wherein the nucleotide sequence is 100% identical to any one of: SEQ ID NOs: 17 - 31.Embodiment 63 An engineered macrophage specific promoter comprising an engineered enhancer of any one of the preceding embodiments operably linked to a minimal promoter.Embodiment 64 The engineered macrophage specific promoter of embodiment 63, wherein the macrophage specific promoter further comprises a spacer sequence, optionally wherein the spacer sequence is between the engineered enhancer and the minimal promoter.Embodiment 65 The engineered macrophage specific promoter of embodiment 63 or 64, wherein the minimal promoter is selected from: a hybrid YBTATA-SCP3 (“YB-SCP3”), SCP3, SCP3 containing DPR, minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB TATA, minTK, inducer molecule responsive promoters, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb, and tandem repeats thereof.Embodiment 66 The engineered macrophage specific promoter of embodiment 65, wherein the minimal promoter is YB-SCP3.Embodiment 67 The engineered macrophage specific promoter of embodiment 66, wherein the minimal promoter comprises the sequenceEmbodiment 68 The engineered macrophage specific promoter of any one of embodiments 60 - 67, wherein the engineered macrophage specific promoter is selected from the group consisting of SEQ ID NOs: 225-239.Embodiment 69 A heterologous construct comprising a. the engineered enhancer of any one of embodiments 30-62 or the engineered macrophage-specific promoter of any one of embodiments 63-68; and b. a heterologous payload, wherein the engineered enhancer or engineered promoter is operably linked to the heterologous payload.Embodiment 70 The heterologous construct of embodiment 69, wherein the heterologous payload comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding a polypeptide.Embodiment 71 The heterologous construct of embodiment 70, wherein the polypeptide comprises at least one effector molecule.Embodiment 72 The heterologous construct of any one of embodiments 69-71, wherein the polypeptide comprises a first effector molecule and a second effector molecule.Embodiment 73 The heterologous construct of embodiment 72, wherein the polynucleotide comprises a nucleotide sequence encoding the first effector molecule, a linker nucleotide sequence, and a nucleotide sequence encoding the second effector.Embodiment 74 The heterologous construct of embodiment 73, wherein the linker nucleotide sequence encodes one or more 2A ribosome skipping elements.Embodiment 75 The heterologous construct of embodiment 74, wherein the one or more 2A ribosome skipping elements comprise elements that are each selected from the group consisting of: P2A, T2A, E2A, and F2A.Embodiment 76 The heterologous construct of any one of embodiments 71-75, wherein the at least one effector molecule or each effector molecule is selected from a therapeuticclass, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a polynucleotide molecule, a coactivation molecule, a tumor microenvironment modifier, a receptor, a ligand, a transcription factor, an antibody, a peptide, and an enzyme.Embodiment 77 The heterologous construct of any one of embodiments 71-76, wherein the at least one effector molecule or each effector molecule is a human-derived effector molecule.Embodiment 78 A heterologous construct for inducing a macrophage to transition from an M2 state to an Ml state, comprising: a. the engineered enhancer of any one of embodiments 60-62 or the engineered macrophage-specific promoter of any one of embodiments 63-68; and b. a heterologous pay load encoding a master regulator of polarization to an Ml macrophage, wherein the engineered enhancer or the engineered macrophage-specific promoter of a) is operably linked to the heterologous pay load and configured to induce expression of the heterologous payload.Embodiment 79 The heterologous construct of embodiment 79, wherein the master regulator of polarization to an Ml macrophage is a cytokine.Embodiment 80 The heterologous construct of embodiment 80, wherein the cytokine is IFNgamma, IFNalpha, TNF alpha, GM-CSF, IL-12, IL-12p70, IL-12p40, IL-12p35, IL-6, IL-23, IL-lalpha, IL- 1 beta, or a derivative thereof.Embodiment 81 The heterologous construct of embodiment 79 or 80, wherein the cytokine is modified to comprise a membrane tethering domain, optionally wherein the membrane tethering domain is or comprises a transmembrane-intracellular domain and / or transmembrane domain of a protein selected from: PDGFR-beta, CDS, CD28, CD3zeta- chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA, or a functional portion thereof, optionally wherein themembrane tethering domain is or comprises a transmembrane domain of B7-1 protein, or a functional portion thereof.Embodiment 82 The heterologous construct of embodiment 79, wherein the master regulator of polarization to an Ml macrophage is a transcription factor selected from IRF7 or a derivative thereof, or p65 / RelA or a derivative thereof.Embodiment 83 A heterologous construct for stabilizing a macrophage in an M2 polarization state, comprising: a. the engineered enhancer of any one of embodiments 60-62 or the engineered macrophage-specific promoter of any one of embodiments 63-68; and b. a heterologous pay load encoding a master regulator of polarization to an M2 macrophage, wherein the engineered enhancer or the engineered macrophage-specific promoter of a) is operably linked to the heterologous pay load and configured to induce expression of the heterologous payload.Embodiment 84 The heterologous construct of embodiment 83, wherein the master regulator of polarization to an M2 macrophage is IL-10, IL-4, IL-13, IL-21, TGF-beta, M-CSF, or a derivative thereof.Embodiment 85 The heterologous construct of embodiment 83 or 84, wherein the cytokine is modified to comprise a membrane tethering domain, optionally wherein the membrane tethering domain is or comprises a transmembrane-intracellular domain and / or transmembrane domain of a protein selected from: PDGFR-beta, CDS, CD28, CD3zeta- chain, CD4, 4-1BB, 0X40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, LNGFR, NKG2D, EpoR, TNFR2, B7-1, and BTLA, or a functional portion thereof, optionally wherein the membrane tethering domain is or comprises a transmembrane domain of B7-1 protein, or a functional portion thereof.Embodiment 86 The heterologous construct of any one of embodiments 78-85, wherein the M2 state is an M2c state, an M2a state, or an M2b state.Embodiment 87 A vector comprising the heterologous construct of any one of embodiments 69-86.Embodiment 88 A dual expression vector comprising the heterologous construct of any one of embodiments 69-86 and a second construct comprising a nucleotide sequence encoding an activating immune receptor.Embodiment 89 An immunoresponsive cell comprising the heterologous construct of any one of embodiments 69-86, the vector of embodiment 87, or the dual expression vector of embodiment 88.Embodiment 90 The immunoresponsive cell of embodiment 89, wherein the immunoresponsive cell is selected from the group consisting of: a macrophage, a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.Embodiment 91 The immunoresponsive cell of embodiment 90, wherein the immunoresponsive cell is a macrophage.Embodiment 92 The immunoresponsive cell of embodiment 91, wherein the macrophage is a tumor-resident macrophage.Embodiment 93 The immunoresponsive cell of any one of embodiments 89-92, wherein the immunoresponsive cell expresses an activating immune receptor.Embodiment 94 The immunoresponsive cell of embodiment 89-93, wherein the activating immune receptor comprises an antigen recognizing receptor.Embodiment 95 The immunoresponsive cell of any one of embodiments 89-94, wherein the immunoresponsive cell is autologous.Embodiment 96 The immunoresponsive cell of any one of embodiments 89-95, wherein the immunoresponsive cell is allogeneic.Embodiment 97 A pharmaceutical composition comprising the vector of embodiment 87, the dual expression vector of embodiment 88, or the immunoresponsive cell of any one of embodiments 89-96, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.Embodiment 98 A method of increasing expression of a target gene, the method comprising use of the engineered enhancer or the engineered macrophage-specific promoter of any one of embodiments 60-68, the vector of embodiment 87, or the dual expression vector of embodiment 88 to increase expression of the target gene.Embodiment 99 The method of embodiment 98, wherein the target gene is an immunomodulatory gene.Embodiment 100 A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the vector of embodiment 87, the dual expression vector of embodiment 88, the immunoresponsive cell of any one of embodiments 89-96, or the pharmaceutical composition of embodiment 97.Embodiment 101 A kit for treating and / or preventing a disease or disorder, comprising the immunoresponsive cell of any one of embodiments 89-96 or the pharmaceutical composition of embodiment 97.Embodiment 102 The kit of embodiment 101, wherein the disease or disorder comprises a tumor.Embodiment 103 The kit of embodiment 101 or 102, wherein the kit further comprises written instructions for using the immunoresponsive cell or the pharmaceutical composition for treating and / or preventing the disease or disorder in a subject.Embodiment 104 An engineered enhancer comprising a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to any one of: (a)ttgcgagcttctaagtccaattAAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO:194); (b)(SEQ ID NO: 24); (c)Embodiment 105 An engineered macrophage specific promoter comprising a nucleotide sequence at least 75%, 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 one of: (a)Embodiment 106 A heterologous construct comprising an engineered macrophage specific promoter of embodiment 105 and a heterologous payload, optionally, wherein the heterologous payload encodes a master regulator of polarization, wherein the master regulator of polarization polarizes to an M2 phenotype.Embodiment 107 The heterologous construct of embodiment 106, wherein the heterologous payload comprises IL4 and / or IL10, optionally wherein the heterologous payload comprises: (a) IL4; (b) IL10; (c) tethered IL4; (d) tethered IL10; (e) IL4 and IL10; or (f) a tethered polypeptide comprising IL4 and IL10.Embodiment 108 The heterologous construct of embodiment 107, wherein: A) the IL4 and / or IL 10 comprises IL4, optionally, (a) having a secretion signal, optionally encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to (i)C (SEQ ID NO: 272), (b) encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identical to (i)or (c) comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to (i)HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS (SEQ ID NO: 264), or (ii)MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTIMREKYSKCSS (SEQ ID NO: 73);B) the IL4 and / or IL 10 comprises IL10, optionally (a) having a secretion signal, optionally encoded by a nucleotide sequence at least 75%, 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 (i)C (SEQ ID NO: 272), (b) encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identical to (i)(SEQ ID NO: 70), or (c) comprises an animo acid sequence at least 75%, 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 (i)(SEQ ID NO: 266), or (ii)(SEQ ID NO: 71); or C) the IL4 and / or IL 10 comprises IL4 and IL10, optionally (a) wherein the nucleotide encoding the IL4 and / or the nucleotide encoding the IL10 comprise a nucleotide sequence encoding a secretion signal, optionally encoded by a nucleotide sequence at least 75%, 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 (i)C (SEQ ID NO: 272); and / or (b) wherein the IL4 encoding nucleotide sequence and the IL10 encoding nucleotide sequence are separated by a 2A peptide encoding sequence, optionally at least 75%, 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 toEmbodiment 109 The heterologous construct of embodiment 108, wherein the IL4 encoding sequence is 5’ relative to the IL 10 encoding sequence, optionally, wherein the nucleotide sequence encoding the IL4 and IL 10 is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical toEmbodiment 110 The heterologous construct of embodiment 108, wherein the IL 10 encoding sequence is 5’ relative to the IL4 encoding sequence, optionally, wherein the nucleotide sequence encoding the IL4 and IL10 is at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical toEmbodiment 111 The heterologous construct of embodiment 106, wherein the heterologous pay load comprises a tethered IL4 comprising IL4 and a transmembrane domain linked to the IL4 via a linker, optionally further comprising a secretion signal, optionally, wherein the (a) the secretion signal is encoded by a nucleotide sequence at least 75%, at least80%, 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 (i)C (SEQ ID NO: 272); (b) the IL4 is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to (i)(c) the transmembrane domain is encoded by a nucleotide sequence at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical toG (SEQ ID NO: 94); and / or (d) the linker is encoded by a nucleotide sequence at least 75%, 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 toEmbodiment 112 The heterologous construct of embodiment 111, wherein the tethered IL4 is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical toEmbodiment 113 The heterologous construct of embodiment 106, wherein the heterologous pay load comprises a tethered IL10 comprising IL 10 and a transmembrane domain linked to the IL4 via a linker, optionally further comprising a secretion signal, optionally, wherein the (a) the secretion signal is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to (i)C (SEQ ID NO: 272); (b) the IL10 is encoded by a nucleotide sequence at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to (i)(SEQ ID NO: 70); (c) the transmembrane domain is encoded by anucleotide sequence at least 75%, 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(SEQ ID NO: 94); and / or (d) the linker is encoded by a nucleotide sequence at least 75%, 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 toEmbodiment 114 The heterologous construct of embodiment 113, wherein the tethered IL 10 is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical toEmbodiment 115 The heterologous construct of embodiment 107, wherein the heterologous payload comprises a tethered polypeptide comprising IL4 and IL10, optionally, wherein the tethered polypeptide comprising IL4 and IL10 further comprises a transmembrane domain linked to the IL4 or IL10 via a linker, optionally further comprising a secretion signal.Embodiment 116 The heterologous construct of embodiments 115, wherein: (a) the heterologous pay load comprises from 5 ’-3’, nucleotide sequences encoding a secretion signal, IL4, a first linker, IL10, a second linker, and a transmembrane domain; or (b) theheterologous pay load comprises from 5 ’-3’, nucleotide sequences encoding a secretion signal, IL10, a first linker, IL4, a second linker, and a transmembrane domain.Embodiment 117 The heterologous construct of embodiment 116, wherein: (a) the first linker is encoded by a nucleotide sequence at least 75%, 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 Gggggtggaggttcagggggtggaggttcaggtggtggcggtagt (SEQ ID NO: 268); (b) the second linker is encoded by a nucleotide sequence at least 75%, 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 toTCAGGCGGCGGTGGTAGTGGAGGCGGAGGCTCAGGCGGCGGAGGTTCCGGAGGTGGCGGTTCCGGCGGAGGATCTCTTCAA (SEQ ID NO: 269); or (c) the transmembrane domain is encoded by a nucleotide sequence at least 75%, 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 toTTGCTGCCTAGCTGGGCCATCACACTGATCTCCGTGAACGGCATCTTCGTGATCTGCTGCCTGACCTACTGCTTCGCCCCTAGATGCAGAGAGCGGAGAAGAAACGAGCGGCTGAGAAGAGAAAGCGTGCGGCCTGTG (SEQ ID NO: 270).Embodiment 118 The heterologous construct of embodiment 116, wherein: (a) the transmembrane domain is encoded by a secretion signal at least 75%, 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 toATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCATGTGCCGGCAACTTTGTCCACGGA (SEQ ID NO: 271); (b) the transmembrane domain is encoded by a secretion signal at least 75%, 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 ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC (SEQ ID NO: 272).Embodiment 119 The heterologous construct of embodiment 116, wherein: (a) the heterologous payload is at least 75%, 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(SEQ ID NO:217); or (b) the heterologous payload is at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical toEmbodiment 120 The heterologous construct of embodiment 113 or 114, wherein the engineered promoter comprises a nucleotide sequence at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical to a sequence selected from a sequence listed in Table A, and wherein the master regulator of polarization that polarizes to M2 phenotype comprises a nucleotide sequence at least 75%, 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 a sequence selected from a sequence listed in Table 4.Embodiment 121 A vector comprising the heterologous construct of any one of embodiments 106-120.Embodiment 122 An immunoresponsive cell comprising the heterologous construct of any one of embodiments 106-120, or the vector of embodiment 121, optionally wherein theimmunoresponsive cell is selected from the group consisting of: a macrophage, a T cell, aCD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer(NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell, optionally wherein the immunoresponsive cell is a macrophage, optionally wherein the macrophage is a tumor-resident macrophage, optionally wherein the immunoresponsive cell expresses an activating immune receptor, optionally wherein the activating immune receptor comprises an antigen recognizing receptor, optionally wherein the immunoresponsive cell is autologous or allogeneic.Embodiment 123 A pharmaceutical composition comprising the vector of embodiment 121, or the immunoresponsive cell of any one of embodiment 122, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.Sequence TablesTable 1: Exemplary Engineered Polarization-Specific EnhancersTable 2: Ablation variants of Enhancer 7 (SB11785). SEO ID NO: 1)Table 3: Exemplary minimal promotersTable 4: Sequences Of Exemplary Effector Molecules, Reporter Molecules, and Payload ComponentsTable 5. Exemplary Signal Secretion PeptidesTable 6: Exemplary hinge or spacer domainsTable 10 : Exemplary Engineered Polarization-Specific EnhancersTable 11: Exemplary engineered macrophage-specific promotersTable 13; Exemplary Macrophage-Specific Promoters (Annotated)Table 14: Exemplary Effector Pavload Components (Annotated)EXAMPLES

[0246] Below are examples of specific embodiments for carrying out the present disclosure.The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0247] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art Such techniques are explained fully in the literature.See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman andCompany, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition);Sambrook, et al, Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods InEnzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington’sPharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B(1992).Example 1: M2 promoters engineered from ATAC-Seq nominated enhancers

[0248] ATAC-Seq was performed on MO, Ml , and M2c polarized macrophages to identify regions of differentially open chromatin in each polarization state. Regions of putative enhancers specific for each polarization state were bioinformatically selected from the ATAC-Seq data. 40 putative enhancers were then combined with a minimal promoter to create candidate M2-state selective promoter. See FIG. 4A. A constitutive promoter control (EPS) was screened in parallel to determine strong, non-selective promoter activity.

[0249] The SB constructs described in this experiment had the following insert structure: Enhancer sequence - minimal promoter (YBTATA) with flanking spacers and barcode sequence - mCherry payload. The enhancer sequence of exemplary SB constructs are shown in Table 1, Table 10, and Table 11. The mCherry payload sequence is shown in Table 4. The YBTATA minimal promoter sequence with flanking spacers is shown in Table 3.

[0250] Results are shown in FIG. 4B. Data below the dotted lines represent the cutoff for non state-selective promoter activity. Promoter strength was normalized to constitutive promoter (% of EPS). As shown, 8 promising M2-state selective promoters were identified (SB09959, SB09965, SB09955, SB09963, SB09964, SB09956, SB09966, and SB09935). These promoters exhibited high selectivity for the M2 state (>10-100-fold) with no measurable activity in the M0 or Ml states. These promoters exhibited <15% of the strength of the EPS promoter.Example 2: Re-engineering of selected M2 promoters from ATAC-Seq nominated enhancers

[0251] Eight M2-state selective promoter constructs identified in Example 1 (SB09959,SB09965, SB09955, SB09963, SB09964, SB09956, SB09966, and SB09935) were found to be highly selective but had lower promoter strength as compared to EPS. Accordingly, these promoters selected for re-engineering (see FIG. 5, left panel, pink circles) by combining the enhancer regions from these eight constructs with a variety of minimal or core promoter sequences. The-re-engineering yielded promoters with improved strength and selectivity over the M0 and / or Ml state, as compared to the original promoters (see FIG. 5, right panel, green oval).

[0252] FIG. 6 depicts the promoter constructs tested (top panel) and promoter activity across different macrophage polarization conditions.

[0253] The 8 putative enhancers selected from earlier screening were paired with 5 minimal or core promoters (6 total including the original, minPro 1). In the bottom panel of FIG. 6, each group of 3 of the same colored-bars represent a single enhancer and minimal promoter pairing that was tested in the MO, Ml, and M2c polarization states, respectively (while the x-axis of FIG. 6 shows only MO labeling, promoter activity of each construct is depicted as three lines of the same color, representing activity in the MO, Ml, and M2c polarization states, from left to right). Candidate promoter activity was assessed via reporter expression normalized to expression of a strong, constitutive promoter (EFS). minPro 1, 2 and 3 correspond to a consensus YBTATA promoter, a miniaturized CMV promoter (minCMV), and a minimal IK promoter (minTK), respectively. MinPro4 is the SCP3 promoter sequence, and minPro 5 is a hybrid sequence between the YBTATA sequence and the SCP3 sequence. MinProti is a fully synthetic, compact promoter hit that was derived from SPECS library screening (SB09846). MinPro 6 is an array of five of the same ELF1 transcription factor binding sites repeated in tandem linked to a minCMV promoter. Exemplary minimal promoter sequences are described in Table 3. Blue dashed lines divide up the enhancer-based promoter candidates by each minimal / core promoter type. Promoter strength and macrophage state-selective activity was assessed according to methods described in Example 1. Tested core promoters improved the dynamic range of promoter strength over the original minimal promoter used, YBTATA (minProl). For many promoter candidates, M2c-state selective promoter strength was increased to >100% of EFS for many promoter candidates.

[0254] FIG. 7 depicts M2 state selective activity over the Ml and M0 states, respectively. Promoter strength was normalized to a strong, constitutive EFS promoter control (SB07683) that has no state-selective activity. The top 8 strongest and most M2c selective promoters were identified to be SB11754 (Enhancer 8 - minPro2), SB11755 (Enhancer 1 - minPro3), SB11760 (Enhancer 6 - minPro3), SB11771 (Enhancer 1 - minPro4), SB11776 (Enhancer 6 - minPro4), SB11779 (Enhancer 1 - minProS), SB11784 (Enhancer 6 - minProS), and SB11785 (Enhancer 7 - minProS). See FIG. 7, Left panel. These top 8 promoters exhibited at least 10-fold selective activity in the M2c state over either the M0 or Ml states (left panel). When grouping selective activity by the same enhancer elements (right panel), enhancer elements 1 (red dots) and 6(brown dots) were the most robust enhancers that were selective across nearly all minimal and core promoters. See FIG. 7, right panel. As indicated in FIG. 7, constructs clustered more closely by their enhancer than by their core promoters suggesting that enhancers were the main drivers of selectivity.

[0255] FIG. 8 plots the “original” promoter design (with YBTATA as the minimal promoter) and best next generation promoter design in a pair- wise fashion.

[0256] The color, size, and text of each data point represents the enhancer, the promoter strength, and the minimal / core promoter, respectively. Five of the eight enhancers tested (enhancer 1, 6, 7 and 8) had improved M2c-selective activity and promoter strength with tested core promoters. The pairing of the enhancer elements with different minimal or core promoters improved M2c selectivity over the Ml and / or M0 states, and boosted overall promoter strength in unpredictable ways.Example 3: Engineering and Evaluation of Fourth Generation Enhancers and Promoters

[0257] Table 10 provides exemplary enhancer sequences that were tested in SB constructs for this study. Table 11 provides exemplary engineered macrophage-specific promoter sequences that were tested in SB constructs for this study. Table 12 provides sequences to native enhancers used as controls for this study.

[0258] Fifteen 4th generation enhancer sequences listed in Table 10 were generated based on combinations of regulatory elements derived from five enhancer sequences. Generally, active elements from each of the enhancers were retained while repressive elements were ablated.Methods

[0259] All 4th generation enhancer sequences were paired with minProS (YB-SCP3). All promoters were engineered to express a fluorescent reporter protein (mCherry) to evaluate promoter activity via flow cytometry.

[0260] These 4th generation enhancer sequences were benchmarked against a shortened human EFla (EFS) promoter for promoter strength. The EFS promoter also serves as a constitutive control. Additionally, previously designed 3rd generation M2 state-specific promoters were used as positive controls (SB13749 or SB14005) for state specific activity. Allnative enhancer-based promoter constructs were also included as controls (SB11779, SB11754, SB11758, SB 11760, and SB11785 corresponding to Enhancers 1, 4, 6, 7, and 8, respectively).

[0261] Macrophage preparation: On Day 0, monocytes were isolated from human PBMCs using a CD 14+ selection kit and subsequently differentiated to macrophages with M-CSF.

[0262] Macrophage transduction: On Day 4, macrophages were plated at 100,000 cells / well in a 96 well ULA plate and transduced with vpx lentivirus for each construct Transductions were performed in triplicate. On Day 6, macrophages were polarized to M0, Ml , or M2c states such that one well of each transduction was polarized to each state. On Day 8, flow cytometry was used to evaluate reporter expression of each construct in each polarization state.Results

[0263] FIG. 9 demonstrates all 4th generation promoters have >100% the strength of EFS in the M2c state, and all 4th generation promoters have <10% the strength of EFS in the M0 or Ml states, suggesting low basal activity in the off-target states.

[0264] Many 4th generation enhancers, including SB 14084, SB 14087, SB 14088, SB 14090, SB14091, SB14092, SB14093, SB14095, SB14096, and SB14097 constructs, are stronger than the previously identified 3rd generation promoter, SB 13749, in the on-target M2c state.

[0265] FIG. 10A and FIG. 10B depict the same data. In both figures, M2c-state selective activity over the M0 state is plotted on the x-axis, whereas M2c-state selective activity over the Ml state is plotted on the y-axis. For both figures, the size of the data point represents the strength of the promoter in the M2c state as a percentage of EFS.

[0266] In FIG. 10A, the dots are colored by generation of enhancer engineering. The black dot is the constitutive EFS control used as a benchmark for promoter strength. The blue dots represent the activity of the native enhancer (Generation 1) with no engineering or modifications to the enhancer sequence. The red dots represent the activity from previously designed “best” 3rd-generation enhancer-based M2c-state specific promoters. The magenta data points represent the 4th generation enhancer-based M2c-state specific promoters tested herein.

[0267] In FIG. 10B, the dots are colored by the native enhancer or combinations of native enhancers from which they are derived.

[0268] 4thgeneration enhancer-based M2c-state specific promoters outperform the best 3rdgeneration promoters (SB13749 and SB14005). Specifically, SB14090, SB14091, and SB14092all have >~1 OO-fold M2c-state specificity over both MO and Ml states. They’re promoter strength also range from >250-650% of EFS.

[0269] The top three 4th-generation promoters contain regions derived from enhancers 1 (SB11779), 6 (SB11758), and 7 (SB11760).Example 4: Evaluation of Fourth Generation Promoters for Driving Cvtokine Expression

[0270] In this example, state-selective promoters derived from enhancers, SB 14090- SB14092, are used to drive the expression of cytokines IL-4 and IL-10. Constructs with the best ability to repress expression of Ml surface and cytokine markers as measured by flow cytometry and Luminex are identified. Monocistronic IL-4, monocistronic IL- 10, and bicistronic IL-4 / IL-10 (in two different orientations) are evaluated.Method

[0271] On day 0, monocytes are isolated from PBMCs using CD14+ selection and are differentiated to macrophages for 4 days using M-CSF (50 ng / mL).

[0272] On day 4, macrophages are transduced with a construct comprising a sequence presented in Table 15. Each transduction is performed in 4x replicate. Later that day, cells are polarized to M2c (50 ng / mL M-CSF + 20 ng / mL TGFbeta + 20 ng / mL IL-10).

[0273] On day 6, two replicates are transpolarized to Ml (washed out the M2c media and changed to media containing 50 ng / mL M-CSF and 50 ng / mL IFNgamma). The rest of the replicates are fed with M2c media (50 ng / mL M-CSF + 20 ng / mL TGFbeta + 20 ng / mL IL-10).

[0274] On day 8, supernatants are harvested for Luminex and cells are harvested for flow cytometry.

[0275] An 11-plex Luminex is performed according to manufacturer protocol to quantify levels of IFNgamma, TNFalpha, IL-6, IL-12p70, IL-18, IL-23, MIPIbeta, GROalpha, IFNalpha, IL-10, and IL-4. In addition, the following flow markers are evaluated: CD80, CD40, CD163, CD206, C5aR, and ITGB5.

[0276] Table 15 shows construct sequences that are evaluated for the ability to repress expression of Ml -associated markers in the present example.Table 15: Nucleotide Sequences of Pa^oad: State-Selective Promoter CombinationsExample 5: Furflier Engineering of candidate M2 promoter

[0277] 12 ablation mutants were generated based on SB 11785 (enhancer 7 - minProS), one of the top M2c selective promoters described in Example 2. To generate the ablation mutants, regions of the enhancer 7 sequence were ablated and replaced with inert sequences. Each of the ablated enhancers were paired with minProS.

[0278] Table 1 provides the enhancer sequences of exemplary tested SB constructs for thisExample and Example 6. Table 2 describes each construct and its ablated regions.

[0279] On Day 0, monocytes were isolated from primary human PBMCs using a CD14+ selection kit and subsequently differentiated to macrophages with M-CSF. On Day 4, macrophages were plated at 100,000 cells / well in a 96 well ULA plate and transduced with vpx lentivirus for each construct tested at an MOI of 2 infectious units per cell. Transductions wereperformed in triplicate. On Day 6, macrophages were polarized to M0, Ml, or M2c states such that one well of each transduction was polarized to each state. On Day 8, flow cytometry was used to evaluate reporter expression of each construct in each polarization state.

[0280] Results are shown in FIG. 11 and FIG. 12. As depicted in FIG. 11, M2c selectivity was improved for a number of ablation mutants including SB13123, SB13124, andSB13129 as compared to SB11785, the native promoter. As depicted in FIG. 12, repressive elements, non-specific activator elements, and state-specific active elements were identified. For example, SB13119, SB13120, SB13121, SB13126, SB13127, and SB13128 each comprise ablations of active elements. SB13125 comprises an ablation of a non-specific activator element.SB13129 and SB13130 each comprise ablations of repressive elements. Table 7 shows the results of strength and selectivity studies for the ablation mutants.Table 7: Selectivity of Ablation VariantsExample 6: Evaluation of Next-generation State-Selective Promoters

[0281] In Example 5, active elements were identified within SB11785 that are important for promoter strength as well as elements that repressed promoter activity. In this example, nextgeneration promoters were generated to identify engineered promoters having increased selectivity and strength. Next generation promoters were designed to combine active elements while removing or ablating multiple repressive elements. The results of strength and selectivity studies using these next-generation promoters are shown in Table 8. FIG. 13 shows the results of the performance of the next-generation promoters.Table 8: Results of Next Generation EnhancersExample 7: Evaluation of Cvtokine Expression by Next-generation State- Selective Promoters

[0282] In this example, state-selective promoter, SB14005 1 (SEQ ID NO: 194), is used to drive the expression of cytokines IL-4 and IL- 10. Constructs with the best ability to repress expression of Ml surface and cytokine markers as measured by flow cytometry and Luminex are identified. Monocistronic IL-4, monocistronic IL- 10, and bicistronic IL-4 / IL-10 (in two different orientations) are evaluated.Method

[0283] On day 0, monocytes are isolated from PBMCs using CD 14+ selection and are differentiated to macrophages for 4 days using M-CSF (50 ng / mL).

[0284] On day 4, macrophages are transduced with a construct comprising a sequence presented in Table 9. Each transduction is performed in 4x replicate. Later that day, cells are polarized to M2c (50 ng / mL M-CSF + 20 ng / mL TGFbeta + 20 ng / mL IL-10).

[0285] On day 6, two replicates are transpolarized to Ml (washed out the M2c media and changed to media containing 50 ng / mL M-CSF and 50 ng / mL IFNgamma). The rest of the replicates are fed with M2c media (50 ng / mL M-CSF + 20 ng / mL TGFbeta + 20 ng / mL IL-10).

[0286] On day 8, supernatants are harvested for Luminex and cells are harvested for flow cytometry.

[0287] An 11-plex Luminex is performed according to manufacturer protocol to quantify levels of IFNgamma, TNFalpha, IL-6, IL-12p70, IL-18, IL-23, MIPIbeta, GROalpha, IFNalpha,IL-10, and IL-4. In addition. The following flow markers are evaluated: CD80, CD40, CD163,CD206, C5aR, and ITGB5.

[0288] Table 9 shows construct sequences that are evaluated for the ability to repress expression of Ml -associated markers in the present example.Table 9: Nucleotide Sequences of State-Selective Promoter : Payload Combinations

Claims

CLAIMSWhat is claimed is:

1. An engineered enhancer comprising: a. a nucleotide sequence at least 75%, 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 one or more of: SEQ ID NOs: 2-16, and 192-194, wherein the nucleotide sequence does not comprise SEQ ID NO: 1; or b. a nucleotide sequence at least 89.5%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to any one or more of SEQ ID NOs: 2-16, and 192-194; wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or M0 macrophage.

2. The engineered enhancer of claim 1, wherein the engineered enhancer comprises: a. a nucleotide sequence at least 91.5% identical to SEQ ID NO: 2, wherein the engineered enhancer does not comprise the sequence TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168); b. a nucleotide sequence at least 96.4% identical to SEQ ID NO: 3, wherein the engineered enhancer does not comprise the sequence TGTGACTCACA (SEQ ID NO: 169); c. a nucleotide sequence at least 94.8% identical to SEQ ID NO: 4, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCC (SEQ ID NO: 170); d. a nucleotide sequence at least 98.4% identical to SEQ ID NO: 5, wherein the engineered enhancer does not comprise the sequence ATAAAT (SEQ ID NO: 171);e. a nucleotide sequence at least 89.8% identical to SEQ ID NO: 6, wherein the engineered enhancer does not comprise the sequence TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172); f. a nucleotide sequence at least 92.8% identical to SEQ ID NO: 7, wherein the engineered enhancer does not comprise the sequence TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173); g- a nucleotide sequence at least 90.4% identical to SEQ ID NO: 8, wherein the engineered enhancer does not comprise the sequence AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174); h. a nucleotide sequence at least 93.5% identical to SEQ ID NO: 9, wherein the engineered enhancer does not comprise the sequence CAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175); i. a nucleotide sequence at least 91.6% identical to SEQ ID NO: 10, wherein the engineered enhancer does not comprise the sequence CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176); j- a nucleotide sequence at least 90% identical to SEQ ID NO: 11, wherein the engineered enhancer does not comprise the sequence GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177); k. a nucleotide sequence at least 91.2% identical to SEQ ID NO: 12, wherein the engineered enhancer does not comprise the sequence TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178); or1. a nucleotide sequence at least 91.6% identical to SEQ ID NO: 13, wherein the engineered enhancer does not comprise the sequence AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179).

3. An engineered enhancer comprising a variant of SEQ ID NO: 1, wherein the variant of SEQ ID NO: 1 comprises an ablation of any one or more regions selected from:(a) positions 76-110 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 76-110 of SEQ ID NO: 1 is: TGAAACAGGAAGTCAGCTTCACAGCAGGAAGCAGA (SEQ ID NO: 168);(b) positions 122-132 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 122-132 of SEQ ID NO: 1 is: TGTGACTCACA (SEQ ID NO: 169);(c) positions 157-172 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 157-172 of SEQ ID NO: 1 is: GGGGACACAATGTTCC (SEQ ID NO: 170);(d) positions 1-6 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 1-6 of SEQ ID NO: 1 is: ATAAAT (SEQ ID NO: 171);(e) positions 7-36 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 7-36 of SEQ ID NO: 1 is: TTCTTGATAGAATTTAAATGTTAAGTGTCC (SEQ ID NO: 172)(f) positions 37-66 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 37-66 of SEQ ID NO: 1 is: TGAAATGTGTTTACTTCTGGATCAGAAATG (SEQ ID NO: 173);(g) positions 67-96 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 67-96 of SEQ ID NO: 1 is: AGAATGTACTGAAACAGGAAGTCAGCTTCA (SEQ ID NO: 174);(h) positions 97-126 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 97-126 of SEQ ID NO: 1 is: CAGCAGGAAGCAGACCTCAAAGAAATGTGA (SEQ ID NO: 175);(i) positions 127-156 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 127-156 of SEQ ID NO: 1 is: CTCACATAGTCTTTTGAATGTGCTCCACTT (SEQ ID NO: 176);(j) positions 157-186 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 157-186 of SEQ ID NO: 1 is: GGGGACACAATGTTCCCCACAGCTTGCCCA (SEQ ID NO: 177);(k) positions 187-216 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 187-216 of SEQ ID NO: 1 is: TCTCATCCATTCTAACTTTCCCATGGGACA (SEQ ID NO: 178); and(1) positions 217-246 of SEQ ID NO: 1, optionally wherein the sequence corresponding to position 217-246 of SEQ ID NO: 1 is: AAAAGCATCATAACAAAGAATTAGAGGAGA (SEQ ID NO: 179); wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or MO macrophage.

4. An engineered enhancer comprising a nucleotide sequence having at least 75%, 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% sequence identity to any one or more of SEQ ID NOs: 17-31, wherein the engineered enhancer induces greater transcriptional activity in an M2 macrophage as compared to an Ml or MO macrophage, optionally wherein the engineered enhancer comprises a nucleotide sequence having at least at least 75%, 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% sequence identity to any one of SEQ ID NOs 24-26.

5. An engineered macrophage specific promoter comprising an engineered enhancer of any one of the preceding claims operably linked to a minimal promoter, optionally wherein the macrophage specific promoter further comprises a spacer sequence, optionally wherein the spacer sequence is between the engineered enhancer and the minimal promoter, optionally wherein the minimal promoter is selected from: a hybrid YBTATA- SCP3 (“YB-SCP3”), SCP3, SCP3 containing DPR, minP, NFkB response element, CREB response element, NF AT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB TATA, minTK, inducer molecule responsive promoters, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb, and tandem repeats thereof, optionally wherein the minimal promoter is YB-SCP3, optional wherein the minimal promoter comprises anucleotide sequence at least 75%, 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 TCTAGAGGGTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGTCCGCCTGGAGACCTCGAGCCGAGTGGTCGTGCCTCCATAGAA (SEQ ID NO: 50), optionally wherein the engineered macrophage specific promoter further comprises a spacer between the engineered enhancer and the minimal promoter, optionally wherein the spacer comprises a sequence CGGATCAACT.

6. The engineered macrophage specific promoter of claim 5, wherein the engineered macrophage specific promoter comprises: a. a nucleotide sequence at least 75%, 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 a nucleotide sequence selected from the group consisting of SEQ ID NOs: 225- 239, and SEQ ID NO: 262 , or b. a nucleotide sequence at least 75%, 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 SEQ ID NO: 232, or c. a nucleotide sequence at least 75%, 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 SEQ ID NO: 233, or d. a nucleotide sequence at least 75%, 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 SEQ ID NO: 234, or e. a nucleotide sequence at least 75%, 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 SEQ ID NO: 262.

7. A heterologous construct comprising:a. the engineered enhancer of any one of claims 1-4 or the engineered macrophagespecific promoter of claim 5 or 6; and b. a heterologous payload, wherein the engineered enhancer or engineered promoter is operably linked to the heterologous payload, optionally, wherein the heterologous payload encodes a master regulator of polarization, wherein the master regulator of polarization polarizes to an M2 phenotype.

8. The heterologous construct of claim 7, wherein the heterologous payload comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding a polypeptide, optionally wherein the polypeptide comprises at least one effector molecule, optionally wherein the polypeptide comprises a first effector molecule and a second effector molecule.

9. The heterologous construct of claim 8, wherein the polynucleotide comprises a nucleotide sequence encoding the first effector molecule, a linker nucleotide sequence, and a nucleotide sequence encoding the second effector, optionally wherein the linker nucleotide sequence encodes one or more 2A ribosome skipping elements, optionally wherein the one or more 2A ribosome skipping elements comprise elements that are each selected from the group consisting of: P2A, T2A, E2A, and F2A.

10. The heterologous construct of claim 8 or claim 9, wherein the at least one effector molecule or each effector molecule is selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of: a cytokine, a chemokine, a homing molecule, a growth factor, a polynucleotide molecule, a co-activation molecule, a tumor microenvironment modifier, a receptor, a ligand, a transcription factor, an antibody, a peptide, and an enzyme11. The heterologous construct of any one of claims 7-10, wherein the heterologous pay load comprises IL4 and / or IL10, optionally wherein the heterologous payload comprises:(a) IL4;(b) IL10;(c) tethered IL4;(d) tethered IL 10;(e) IL4 and IL10; or(f) a tethered polypeptide comprising IL4 and IL 10.

12. A vector comprising the heterologous construct of any one of claims 7-11.

13. A dual expression vector comprising the heterologous construct of any one of claims 7- 11 and a second construct comprising a nucleotide sequence encoding an activating immune receptor.

14. An immunoresponsive cell comprising the heterologous construct of any one of claims 7- 11, the vector of claim 12, or the dual expression vector of claim 13, optionally wherein the immunoresponsive cell is selected from the group consisting of: a macrophage, a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (HL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell, optionally wherein the immunoresponsive cell is a macrophage, optionally wherein the macrophage is a tumorresident macrophage, optionally wherein the immunoresponsive cell expresses an activating immune receptor, optionally wherein the activating immune receptor comprises an antigen recognizing receptor, optionally wherein the immunoresponsive cell is autologous or allogeneic.

15. A pharmaceutical composition comprising the vector of claim 12, the dual expression vector of claim 13, or the immunoresponsive cell of claim 14, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

16. A method of increasing expression of a target gene, the method comprising use of the engineered enhancer of any one of claims 1-4, the engineered macrophage-specific promoter of claim 5 or claim 6, the vector of claim 12, or the dual expression vector of claim 13 to increase expression of the target gene, optionally wherein the target gene is an immunomodulatory gene.

17. A method of treating a subject in need thereof, the method comprising administering to the subject a therapeutically effective dose of the vector of claim 12, the dual expression vector of claim 13, the immimoresponsive cell of claim 14, or the pharmaceutical composition of claim 15.

18. A kit for treating and / or preventing a disease or disorder, comprising the immimoresponsive cell of claim 14 or the pharmaceutical composition of claim 15, optionally wherein the disease or disorder comprises a tumor, optionally wherein the kit further comprises written instructions for using the immimoresponsive cell or the pharmaceutical composition for treating and / or preventing the disease or disorder in a subject19. An engineered enhancer comprising a nucleotide sequence at least 75%, 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 one of:(a)(SEQ ID NO: 24);(c)(SEQ ID NO: 25); and(d)20. An engineered macrophage specific promoter comprising a nucleotide sequence at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% identical to any one of:(a)ACCGTCAGTCCGCCTGGAGACCTCGAGCCGAGTGGTCGTGCCTCCATAGAA (SEQ IDNO: 262);(b)(SEQ ID NO: 232);(c)(SEQ ID NO: 233); and(d)(SEQ ID NO: 234).

21. A heterologous construct comprising an engineered macrophage specific promoter of claim 20 and a heterologous payload, optionally, wherein the heterologous payload encodes a master regulator of polarization, wherein the master regulator of polarization polarizes to an M2 phenotype.

22. The heterologous construct of claim 21, wherein the heterologous payload comprises IL4 and / or IL10, optionally wherein the heterologous payload comprises:(a) IL4;(b) IL10;(c) tethered IL4;(d) tethered IL10;(e) IL4 and IL10; or(f) a tethered polypeptide comprising IL4 and IL10.

23. The heterologous construct of claim 22, wherein:A) the IL4 and / or IL10 comprises IL4, optionally(a) having a secretion signal, optionally encoded by a nucleotide sequence at least 75%, 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(i)GTGCCGGCAACTTTGTCCACGGA (SEQ ID NO: 271), or(ii) G(b) encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to(i)(SEQ ID NO: 263), or(ii)(SEQ ID NO: 72), or(c) comprises an amino acid sequence at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to(i)HKCDITLQEIIKTLNSLTEQKTLCTELTVTDIFAASKNTTEKETFCRAATVL RQFYSHHEKDTRCLGATAQQFHRHKQLIRFLKRLDRNLWGLAGLNSCPV KEANQSTLENFLERLKTIMREKYSKCSS (SEQ ID NO: 264), or(ii)MGLTSQLLPPLFFLLACAGNFVHGHKCDITLQEIIKTLNSLTEQKTLCTELT VTDIFAASKNTTEKETFCRAATVLRQFYSHHEKDTRCLGATAQQFHRHK QLIRFLKRLDRNLWGLAGLNSCPVKEANQSTLENFLERLKTTMREKYSKC SS (SEQ ID NO: 73);B) the IL4 and / or IL10 comprises IL10, optionally(a) having a secretion signal, optionally encoded by a nucleotide sequence at least 75%, 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(i)ATGGGTCTCACCTCCCAACTGCTTCCCCCTCTGTTCTTCCTGCTAGCAT GTGCCGGCAACTTTGTCCACGGA (SEQ ID NO: 271), or(ii) ATGCACAGCTCAGCACTGCTCTGTTGCCTGGTCCTCCTGACTGGGGTGAGGGCC (SEQ ID NO: 272),(b) encoded by a nucleotide sequence at least 75%, 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(i)GACATCTTCATCAACTACATAGAAGCCTACATGACAATGAAGATACGAAAC (SEQ ID NO: 265), or(ii)(SEQ ID NO: 70), or(c) comprises an animo acid sequence at least 75%, 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(i)(SEQ ID NO: 266), or(ii)(SEQ ID NO: 71); orC) the IL4 and / or IL 10 comprises IL4 and IL10, optionally(a) wherein the nucleotide encoding the IL4 and / or the nucleotide encoding the IL10 comprise a nucleotide sequence encoding a secretion signal, optionally encoded by anucleotide sequence at least 75%, 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(i)(SEQ ID NO: 271), or(ii)(b) wherein the IL4 encoding nucleotide sequence and the IL 10 encoding nucleotide sequence are separated by a 2A peptide encoding sequence, optionally at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% at least 99% or 100% identical to(SEQ ID NO: 267).

24. The heterologous construct of claim 23, wherein the IL4 encoding sequence is 5’ relative to the IL 10 encoding sequence, optionally, wherein the nucleotide sequence encoding the IL4 and IL10 is at least 75%, 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(SEQ ID NO: 221).

25. The heterologous construct of claim 23, wherein the IL 10 encoding sequence is 5’ relative to the IL4 encoding sequence, optionally, wherein the nucleotide sequence encoding theIL4 and IL10 is at least 75%, 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(SEQ ID NO: 223).

26. The heterologous construct of claim 21, wherein the heterologous payload comprises a tethered IL4 comprising IL4 and a transmembrane domain linked to the IL4 via a linker, optionally further comprising a secretion signal, optionally, wherein the(a) the secretion signal is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least99% or 100% identical to(i)(SEQ ID NO: 271), or(ii)(SEQ ID NO: 272);(b) the IL4 is encoded by a nucleotide sequence at least 75%, 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(i)(ii)(SEQ ID NO: 72);(c) the transmembrane domain is encoded by a nucleotide sequence at least 75%, at least80%, 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(SEQ ID NO: 94); and / or(d) the linker is encoded by a nucleotide sequence at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical to(SEQ ID NO: 92) or (SEQ ID NO: 268).

27. The heterologous construct of claim 26, wherein the tethered IL4 is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% identical to(SEQ ID NO: 215).

28. The heterologous construct of claim 21, wherein the heterologous payload comprises a tethered IL10 comprising IL 10 and a transmembrane domain linked to the IL4 via a linker, optionally further comprising a secretion signal, optionally, wherein the(a) the secretion signal is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical to(i)(ii)(b) the IL10 is encoded by a nucleotide sequence at least 75%, 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(i)(SEQ ID NO: 265), or(ii)G G G G C C C C C C G GCC C G C G(SEQ ID NO: 70);(c) the transmembrane domain is encoded by a nucleotide sequence at least 75%, at least80%, 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(SEQ ID NO: 94); and / or(d) the linker is encoded by a nucleotide sequence at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical to(SEQ ID NO: 92) ort (SEQ ID NO: 268).

29. The heterologous construct of claim 28, wherein the tethered IL10 is encoded by a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% identical to(SEQ ID NO: 80).

30. The heterologous construct of claim 22, wherein the heterologous payload comprises a tethered polypeptide comprising IL4 and IL10, optionally, wherein the tethered polypeptidecomprising IL4 and IL 10 further comprises a transmembrane domain linked to the IL4 or IL 10 via a linker, optionally further comprising a secretion signal.

31. The heterologous construct of claim 30, wherein:(a) the heterologous pay load comprises from 5 ’-3’, nucleotide sequences encoding a secretion signal, IL4, a first linker, IL10, a second linker, and a transmembrane domain; or(b) the heterologous pay load comprises from 5 ’-3’, nucleotide sequences encoding a secretion signal, IL10, a first linker, IL4, a second linker, and a transmembrane domain.

32. The heterologous construct of claim 31, wherein:(a) the first linker is encoded by a nucleotide sequence at least 75%, 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 toGggggtggaggttcagggggtggaggttcaggtggtggcggtagt (SEQ ID NO: 268);(b) the second linker is encoded by a nucleotide sequence at least 75%, 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(SEQ ID NO: 269); or(c) the transmembrane domain is encoded by a nucleotide sequence at least 75%, 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(SEQ ID NO: 270).

33. The heterologous construct of claim 31, wherein:(a) the transmembrane domain is encoded by a secretion signal at least 75%, 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(SEQ ID NO: 271);(b) the transmembrane domain is encoded by a secretion signal at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or100% identical to(SEQ ID NO: 272).

34. The heterologous construct of claim 31, wherein:(a) the heterologous payload is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to(SEQ ID NO: 217); or(b) the heterologous payload is at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical toa (SEQ ID NO: 219).

35. The heterologous construct of claim 28 or 29, wherein the engineered promoter comprises a nucleotide sequence at least 75%, at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to a sequence selected from a sequence listed in Table 13, and wherein the master regulator of polarization that polarizes to M2 phenotype comprises a nucleotide sequence at least 75%, 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 a sequence selected from a sequence listed in Table 14.

36. A vector comprising the heterologous construct of any one of claims 21-35.

37. An immunoresponsive cell comprising the heterologous construct of any one of claims 21-35, or the vector of claim 36, optionally wherein the immunoresponsive cell is selected from the group consisting of: a macrophage, a T cell, a CD8+ T cell, a CD4+ T cell, a gamma-delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a viral-specific T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell, optionally wherein the immunoresponsive cell is a macrophage, optionally wherein the macrophage is a tumor-resident macrophage, optionally wherein the immunoresponsive cell expresses an activating immune receptor, optionally wherein the activating immune receptor comprises an antigen recognizing receptor, optionally wherein the immunoresponsive cell is autologous or allogeneic.

38. A pharmaceutical composition comprising the vector of claim 36, or the immunoresponsive cell of any one of claim 37, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

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