Engineered regulatory elements

Abstract

The present disclosure provides for, among other things, methods and compositions comprising engineered nucleic acids, such as engineered regulatory elements, that allow for improved transcriptional activity of an operably linked polynucleotide in cells.

Description

ENGINEERED REGULATORY ELEMENTSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 590,220, filed on October 13, 2023, the contents of which are hereby incorporated in their entirety by reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which is hereby incorporated by reference in its entirety. The electronic copy, created on October 9, 2024, is named 70012_SeqListing.xml, and is 272,758 bytes in size.BACKGROUND

[0003] Gene regulatory elements, such as promoters and enhancers, can possess specific activities and can function differently in different contexts, such as in different cell types. Such regulatory elements are often adapted for use in therapies, such as cell and gene therapies, to treat diseases that benefit from expression of a particular gene and / or therapeutic pay loads. Thus, regulatory elements for regulating the expression of therapeutic payloads can ultimately impact overall functionality of engineered cells or vectors for gene therapy. However, problems with transcriptional strength persist in known regulatory elements commonly used to drive payload expression. By way of example only, engineered cell therapy applications may require low integrated transgene copy number for regulatory and safety reasons; however, known promoters can be ineffective in driving sufficient therapeutic payload expression in engineered cell populations at the desired copy number, particularly in cases where payloads are large and / or multicistronic. Accordingly, there is a need to identify and develop regulatory elements that can effectively drive pay load expression, particularly for use in therapy.SUMMARY

[0004] Provide herein are engineered regulatory elements 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 a nucleotide sequence selected from: SEQ ID NOs: 1-108. In some aspects, the nucleotide sequence is at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs: 1-108. In some aspects, the nucleotide sequence is 100% identical to a nucleotide sequence selected from: SEQ ID NOs: 1-108.

[0005] Also provided herein are engineered regulatory elements comprising one or more TP63 transcription factor binding sites (TFBSs). In some aspects, the one or more TP63 TFBSscomprise a 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: SEQ ID NOs 217-220, and reverse complements thereof. In some aspects, the one or more TP63 TFBSs comprise a sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs 217-220, and reverse complements thereof. In some aspects, the one or more TP63 TFBSs are selected from SEQ ID NOs 217-220, and reverse complements thereof. In some aspects, any of the one or more TP63 TFBSs comprises a TP63 TFBS half- site motif, wherein the TP63 TFBS half-site motif comprises a 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: SEQ ID NOs 221-233, and reverse complements thereof. In some aspects, the TP63 TFBS half-site motif comprises a sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs 221-233, and reverse complements thereof. In some aspects, the TP63 TFBS half-site motif is selected from SEQ ID NOs 221-233 and reverse complements thereof. In some aspects, the TP63 TFBS comprises two TP63 TFBS half-site motifs. In some aspects, the two TP63 TFBS half-site motifs are operatively linked by a nucleic acid linker, optionally wherein the linker is between 1-10 basepairs. In some aspects, the two TP63 TFBS half-site motifs comprise (a) a first half-site motif 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: SEQ ID NOs 221-233; and (b) a second half-site motif 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 reverse complement of a nucleotide sequence selected from: SEQ ID NOs 221-233.

[0006] In some aspects, the engineered regulatory element comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 TP63 TFBSs. In some aspects, the engineered regulatory element comprises at least two TP63 TFBSs.

[0007] The engineered regulatory element of any one of claims 4-13, comprising 1-500, 1-100, 1-50, 2-20, or 2-10 TP63 TFBSs. In some aspects, the engineered regulatory element further comprises at least one additional non-TP63 TFBS. In some aspects, the at least one additional non-TP63 TFBS is selected from: a BARX2 TFBS, a NHEH1 TFBS, a TP73 TFBS, a HOXC10 TFBS, a NFE2 TFBS, a ATF4 TFBS, a HES1 TFBS, a FOS TFBS, a JUN TFBS, and a JUNB TFBS. In some aspects, the BARX2 TFBS comprises a 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, and / or the NHEH1 TFBS comprises a 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 SEQ ID NO: 235, and / or the TP73 TFBS comprises a 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: 236, and / or the HOXC10 TFBS comprises a 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: 237, and / or the NFE2 TFBS comprises a 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: 238, and / or the ATF4 TFBS comprises a 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: 239, and / or the HES1 TFBS comprises a 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: 240, and / or the FOS TFBS comprises a 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: 241, and / or the JUN TFBS comprises a 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: 242, and / or the JUNB TFBS comprises a 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: 243.

[0008] In some aspects, the at least one additional non-TP63 TFBS comprises a BARX2 TFBS, a NHEH1 TFBS, or both a BARX2 TFBS and a NHEH1 TFBS. In some aspects, the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5, or more than 5 BARX2 TFBSs. In some aspects, the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5, or more than 5 NHEH1 TFBSs.

[0009] In some aspects, the engineered regulatory element is operably linked to a core promoter. In some aspects, the core promoter comprises a sequence of a promoter selected from: minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB- SCP3 minimal promoter, a DPR containing SCP3 promoter, a minP promoter, a NFkB response element, a CREB response element, a NF AT response element, a SRF response element 1, a SRF response element 2, an API response element, a TCF-EEF response element promoter fusion, a Hypoxia responsive element, a SMAD binding element, a STAT3 binding site, 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 aspects, the core promoter is selected froma minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB-SCP3 minimal promoter, and a DPR containing SCP3 promoter.

[0010] Also provided herein are engineered regulatory elements 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 a nucleotide sequence selected from: SEQ ID NOs: 109-216. In some aspects, the engineered regulatory element comprises a nucleotide sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs: 109-216. In some aspects, the engineered regulatory element comprises a nucleotide sequence that is 100% identical to a nucleotide sequence selected from: SEQ ID NOs: 109-216.

[0011] Also provided herein are heterologous constructs comprising: any one of the engineered regulatory elements provided herein; and a heterologous payload, wherein the engineered regulatory element is operably linked to the heterologous payload.

[0012] In some aspects, the heterologous pay load comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding one or more polypeptides. In some aspects, the one or more polypeptides comprise at least one effector molecule. In some aspects, the one or more polypeptides comprise two or more separate polypeptides comprising a first effector molecule, a second effector molecule, optionally a third effector molecule, optionally a fourth effector molecule. In some aspects, the polynucleotide comprises E1-L1-E2, optionally wherein the polynucleotide comprises E1-L1-E2-L2-E3-L3-E4, wherein El is a nucleotide sequence encoding the first effector molecule, LI is a first linker molecule, E2 is a nucleotide sequence encoding the second effector molecule, L2 is a second linker molecule, E3 is a nucleotide sequence encoding the third effector molecule, L3 is a third linker molecule, and E4 is a nucleotide sequence encoding the fourth effector molecule. In some aspects, LI, L2, L3, and L4 are independently selected from: an internal ribosome entry site (IRES) and one or more nucleotide sequences encoding one or more 2A ribosome skipping elements. In some aspects, the linker nucleotide sequence encodes one or more 2A ribosome skipping elements. In some aspects, the one or more 2A ribosome skipping elements comprise elements that are each selected from: P2A, T2A, E2A, and F2A.

[0013] In some aspects, the at least one effector molecule or each effector molecule is selected from one or more therapeutic classes, wherein the one or more therapeutic classes are selected from: a chimeric receptor, 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 aspects, the chimeric receptor is a chimeric antigen receptor (CAR).

[0014] In some aspects, the at least one effector molecule or each effector molecule is a human- derived effector molecule.

[0015] In some aspects, the one or more polypeptides comprise the first effector molecule and the second effector molecule, and wherein: the first effector molecule and the second effector molecule are independently selected from a first CAR and a second CAR, or the first effector molecule and the second effector molecule are independently selected from a first CAR and a cytokine, or the first effector molecule and the second effector molecule are independently selected from the first cytokine and a second cytokine, and optionally wherein the first CAR is an activating CAR (aCAR) and the second CAR is an inhibitory CAR (iCAR), optionally wherein the first CAR and / or the second CAR is a bivalent CAR.

[0016] In some aspects, the one or more polypeptides comprise the first effector molecule, the second effector molecule, and the third effector molecule, and wherein the first effector molecule, the second effector molecule, and the third effector molecule are independently selected from: a first CAR, a second CAR, and a cytokine, or a first CAR, a first cytokine, and a second cytokine.

[0017] In some aspects, the one or more polypeptides comprise the first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule, and wherein the first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule are independently selected from: a first CAR, a second CAR, a first cytokine, and a second cytokine.

[0018] Also provided herein are vectors comprising any one of the heterologous constructs provided herein. Also provided herein are dual expression vectors comprising any one of the heterologous constructs provided herein and a second construct comprising an additional payload.

[0019] In some aspects, the vector or dual expression vector is a viral vector, optionally wherein the viral vector is a retroviral vector.

[0020] Also provided herein are immunoresponsive cells comprising any one of the heterologous constructs, the vectors, or the dual expression vectors provided herein. In some aspects, the immunoresponsive cell is selected from: a Natural Killer (NK) cell, 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 B cell, a macrophage, a tumorinfiltrating 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. In some aspects,the immunoresponsive cell is a NK cell or a T cell. In some aspects, the immunoresponsive cell expresses an activating immune receptor. In some aspects, the activating immune receptor comprises an antigen recognizing receptor. In some aspects, the immunoresponsive cell is autologous. In some aspects, the immunoresponsive cell is allogeneic.

[0021] Also provided herein are pharmaceutical compositions comprising any one of the vectors, the dual expression vectors, or the immunoresponsive cells provided herein, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

[0022] Also provided herein are methods of increasing expression of a target gene or a heterologous payload, the method comprising use of any one of the engineered regulatory elements, the vectors, or the dual expression vectors provided herein to increase expression of the target gene. In some aspects, the target gene is an immunomodulatory gene.

[0023] Also provided herein are methods of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of any one of the vectors, the dual expression vectors, the immunoresponsive cells, or the pharmaceutical compositions provided herein.

[0024] Also provided herein is a kit for treating and / or preventing a disease or disorder, comprising any one of the immunoresponsive cells or the pharmaceutical compositions provided herein. In some aspects, the disease or disorder comprises a tumor. In some aspects, 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.BRIEF DESCRIPTION OF THE DRAWING

[0025] FIG. 1A shows a schematic of exemplary engineered regulatory element designs that include engineered enhancer sequences operably linked to a core promoter to generate engineered promoter candidates, as well as the multicistronic pay load constructs.

[0026] FIG. IB shows a schematic of a construct used for assessing engineered regulatory element designs that include engineered enhancer sequences operably linked to a core promoter.

[0027] FIG. 2 shows a graph of performance metrics of selected 1stgeneration engineered promoter candidates based on transduction efficiency.

[0028] FIG. 3 shows flow cytometry scatterplots from exemplary selected engineered promoter constructs SB10698 (TP63), 10977 (NHLH1), 10961 (BARX2), 10947 (ERF039), and 10944 (bHLHl).

[0029] FIG. 4A shows a graph of performance metrics of selected 1stgeneration engineered promoter candidates based on surface CAR protein expression measured on flow cytometer and quantified as fluorescent intensity values.

[0030] FIG. 4B shows a graph of performance metrics of selected 1stgeneration engineered promoter candidates based on surface CAR protein expression measured on flow cytometer and quantified as fluorescent intensity values.

[0031] FIG. 5 shows a graph of performance metrics of selected 1stgeneration engineered promoter candidates based on surface CAR protein expression measured on flow cytometer and quantified as fluorescent intensity values.

[0032] FIGS. 6A-6C show graphs of the percentage of new promoter constructs pairing enhancers with the various core promoters outperforming the SV40 promoter as benchmark.

[0033] FIG. 7A shows a graph of the performance of candidate promoters that include TP63 TFBSs.

[0034] FIG. 7B shows a graph of the performance of candidate promoters that include BARX2, NHLH1 and TP63 TFBSs.

[0035] FIGS. 8A-8B show flow cytometry scatterplots of performance comparison between 1st generation promoters and 2nd generation promoters.

[0036] FIG. 8C shows graphs of the performance comparisons between the 1stgeneration promoters and 2ndgeneration promoters for aCAR (middle) and iCAR (right).

[0037] FIG. 9 shows schematics of a libraries / constructs used for assessing engineered enhancers and engineered promoters.

[0038] FIG. 10 shows graphs of performance evaluation of MPRA library screening for library 1 (top) and library 2 (bottom).

[0039] FIG. 11 shows a graph of Day 7 and Day 14 iCAR MFI (of iCAR+ cells) for NK cells transduced with various constructs.

[0040] FIG. 12 shows exemplary flow plots from the NV (left), SB 12515 (middle), andSB 12896 (right) transduced cells at 25 pl volume on Day 7, as well as normalized viral copy number (VCN) of the transduced cells.

[0041] FIG. 13A shows graphs of transduction efficiency (left) and both aCAR and iCAR pay load expression in transduced NK cells (middle and right).

[0042] FIG. 13B shows graphs of transduction efficiency and both aCAR (top) and iCAR payload (bottom) expression in transduced NK cells.

[0043] FIGS. 14A-14B show graphs of surface expression of IL15 (FIG. 14A; left and right), as well as secreted IL15 (FIG. 14B; left) and IL21 (FIG. 14B; right) in SB 12515 vs SB 12896 transduced cells.

[0044] FIG. 14C shows graphs of copy number of cells with secreted IL 15 (top) and IL21 (bottom) in SB 12515 vs SB 12896 transduced cells.

[0045] FIG. 15A shows exemplary flow plots of different donor NK cells for SB 12896 and SB12515.

[0046] FIGS. 15B-15D show graphs of promoter strength metrics for SB 12896 and SB 12515 across NK cells from different donors.

[0047] FIG. 16A shows a schematic of engineered NK cells expressing Payload 5 and the DLD- 1 target cell lines.

[0048] FIGS. 16B-16D show the results of a killing assay comparing the engineered promoter of SB 12896 to SV40 for killing of TA+PA- cells.

[0049] FIG. 16E-16F show the results of a killing assay comparing the engineered promoter of SB 12896 to SV40 for killing of TA+PA+ cells.

[0050] FIG. 16G shows a graph of the results of a killing assay comparing the engineered promoter of SB 12896 to SV40.

[0051] FIG. 17A shows graphs of performance of engineered NK promoter candidates (top, middle, and bottom) compared to SV40 (SB 12515) in payload expression.

[0052] FIG. 17B shows graphs of performance of engineered NK promoter candidates compared to SV40 (SB 12515) in killing of TA+PA- target cells and TA+PA+ “healthy” cells (left and right).

[0053] FIG. 18A shows graphs of performance of candidate promoters compared to an SV40 promoter (SB 12515) in NK cells (left and right).

[0054] FIG. 18B shows graphs of performance of candidate promoters compared to an SV40 promoter (SB 12515) in T cells (left and right).

[0055] FIG. 18C shows graphs of the correlation of performance of candidate promoters compared to an SV40 promoter (SB 12515) in NK cells compared to T cells (top and bottom).

[0056] FIG. 18D depicts graphs of performance of candidate promoters in NK and T cell in a single graph.

[0057] FIG. 18E shows graphs of flow cytometry results for the SFFV control vs. SB 13498 (top and bottom).DETAILED DESCRIPTIONI. Definitions

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the disclosed subjectmatter belongs. Generally, nomenclatures utilized in connection with, and techniques of,immunology, oncology, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject-matter claimed or otherwise provided herein. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject-matter described.

[0059] As used herein, singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0060] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90% to 100%, includes 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%, etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth.

[0061] 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.

[0062] Agent: The term “agent” as used herein may refer to a compound, molecule, or entity of any chemical class including, for example, polypeptides, nucleic acids (e.g., engineered nucleic acids as described herein), saccharides, lipids, small molecules, metals, or combinations thereof. In some embodiments, an agent is or comprises a natural product in that it is found in and / or is obtained from nature. In some embodiments, an agent is or comprises one or more entities that is man-made in that it is designed, engineered, modified, and / or produced through action of the hand of man and / or is not found in nature. In many aspects, the present disclosure provides for engineered nucleic acids comprising particular engineered transcriptional regulatory elements as described herein (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein). In some aspects, an agent may be utilized in isolated or pure form (e.g., an isolated polynucleotide); in some embodiments, an agent may be utilized in crude form. In some aspects, potential agents are provided as collections or libraries, for example that may be screened to identify or characterize active agents within them. Some particular embodiments of agents that may be utilized in accordance with the present disclosureinclude small molecules, antibodies, antibody fragments, aptamers, nucleic acids (e.g., siRNAs, shRNAs, DNA / RNA hybrids, antisense oligonucleotides, ribozymes), peptides, peptide mimetics. In some aspects, an agent as described herein is encoded on a coding nucleotide sequence that is operably linked to an engineered regulatory element as provided herein (e.g., an engineered enhancer sequence and / or an engineered promoter including an engineered enhancer sequence). In some aspects, an agent is an effector molecule as described herein.

[0063] Approximately: The term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0064] Biologically active: The phrase “biologically active” refers to an agent or substance that has activity in a biological system (e.g., an isolated cell, a cell in a particular tissue, a cell in culture, or a cell in an organism, etc.). For instance, an agent or substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active, or have biological activity (e.g., a biologically active agent, bioactive agent, bioactive molecule, etc.). It will be appreciated by those skilled in the art that often only a portion or fragment of a biologically active substance is required (e.g., is necessary and sufficient) for an activity to be present; in such circumstances, that portion or fragment is considered to be a “biologically active” portion or fragment.

[0065] Expression cassette: An “expression cassette” is a polynucleotide construct, generated recombinantly or synthetically, that includes a regulatory element (e.g., any one of the engineered regulatory elements described herein, such as an engineered enhancer sequence and / or an engineered promoter including an engineered enhancer sequence described herein) operably linked to a selected polynucleotide (e.g., a coding sequence, such as a gene) to facilitate expression of said selected polynucleotide in a host cell, or in a cell-free environment. Heterologous constructs as described herein may include one or more expression cassettes.

[0066] Heterologous: The term “heterologous” as used herein with respect to a nucleotide sequence, amino acid sequence, or polypeptide, refers to a compound or agent which is either foreign (e.g., exogenous, such as it is not found in nature) to a given host cell, or which is naturally found in a given host cell (e.g., is endogenous), however, said compound or agent is in the context of a heterologous construct, e.g., employing heterologous nucleic acid, as described herein. A heterologous nucleotide sequence as found endogenously may also be produced in anunnatural, e.g., greater than expected or greater than naturally found, amount in a cell. A heterologous nucleotide sequence, or a nucleic acid comprising a heterologous nucleotide sequence, possibly differs in sequence from an endogenous nucleotide sequence but encodes the same protein as found endogenously. Specifically, heterologous nucleotide sequences are those not found in the same relationship to a host cell in nature. Any recombinant or artificial nucleotide sequence is understood to be heterologous. A non-limiting example of a heterologous polynucleotide, e.g., an engineered regulatory element as described herein, is a nucleotide sequence such as an engineered enhancer sequence operably linked to a promoter with which it is not natively associated. Heterologous polynucleotides comprising such engineered regulatory elements may further be used to control expression of coding sequences (e.g., genes) in place of the native, or wild-type, promoter of said coding sequence.

[0067] Identity: The term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Accordingly, reference to “identity” or “homology” in the context of a particular sequence (e.g., nucleotide sequence, amino acid sequence, etc.) have the same meaning unless the context indicates otherwise. Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain aspects, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CAB IOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM 120 weight residue table, a length penalty of 12, and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix. Optimal alignment of sequences forcomparison can also 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, BESTFIT, 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.). Another 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 / ).

[0068] Isolated: The term “isolated” refers to a substance and / or entity that has been: (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and / or in an experimental setting), and / or (2) produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and / or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0069] Core promoter: A “core promoter” comprises a sequence of a promoter that maintains the ability to initiate, or induce, transcription of a target coding sequence, e.g., a gene. Accordingly, a core promoter can be a heterologous, engineered promoter.

[0070] Nucleic acid: The term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. In some aspects, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some aspects, “nucleic acid” refers to an oligonucleotide chain comprising individual nucleic acid residues. The terms “oligonucleotide” and “polynucleotide” can be used interchangeably. In some aspects, “nucleic acid” encompasses RNA as well as single and / or double- stranded DNA and / or cDNA.Furthermore, the terms “nucleic acid,” “DNA.” “RNA.” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so- called “peptide nucleic acids,” which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present disclosure. The terms “nucleotide sequence encoding an amino acid sequence” or “coding sequence” includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences that encode proteins and / or RNA may include introns. A coding sequence may also refer to a nucleotide sequence that produces a bioactive nucleic acids (e.g., a mRNA, miRNA, siRNA, shRNA, etc.). Nucleic acids can be isolated or purified from natural sources, produced using recombinant expression systems and optionally isolated or purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. The term “nucleic acid segment” is used herein to refer to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In many embodiments, a nucleic acid segment comprises at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, at least 40, or more residues. In some aspects, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5- methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8- oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some aspects, the present disclosure is directed to “unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and / or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery.

[0071] Operably linked: The term “operably linked” refers to polynucleotide sequences or amino acid sequences placed into a functional relationship with one another. For instance, an regulatory element (e.g., as an engineered enhancer sequence and / or an engineered promoter including an engineered enhancer sequence) is operably linked to a coding sequence if it regulates, or contributes to modulation of, the transcription of the coding sequence. Operably linked DNA sequences encoding regulatory sequences are typically contiguous to a coding sequence. However, certain regulatory elements (e.g., enhancers) can function when separated from a promoter by up to several kilobases or more. Additionally, multi-cistronic constructs can include multiple coding sequences which use only one regulatory element by including a 2A self-cleaving peptide, an IRES element, etc. as described herein. Accordingly, some polynucleotide elements (e.g., engineered regulatory elements provided herein) may be operably linked to one or more coding sequences, but not contiguous with said one or more coding sequences.

[0072] Protein: The term “protein” refers to a polypeptide (z.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and / or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a biologically active portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some aspects, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids.

[0073] Substantially: The term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0074] Subject: The term “subject” means a mammal (e.g., a human, in some embodiments including prenatal human forms, a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, asheep, cattle, a primate, and / or a pig). In some aspects, a subject is suffering from a relevant disease, disorder or condition (e.g., cancer). In some aspects, a subject is susceptible to a disease, disorder, or condition. In some aspects, a subject displays one or more symptoms or characteristics of a disease, disorder or condition. In some aspects, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some aspects, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition. A subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease. In some aspects, a subject is an individual to whom therapy is administered, e.g., a recipient. In many aspects of the present disclosure, a subject has, or is susceptible to, a disease or disorder, such as cancer.

[0075] Therapeutic agent: The phrase “therapeutic agent” in general refers to any agent that elicits a desired pharmacological effect when administered to an organism (e.g., a subject). In some aspects, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some aspects, the appropriate population may be a population of model organisms. In some aspects, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some aspects, a therapeutic agent is a substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some aspects, a “therapeutic agent” is an agent that has been or is required to be approved by a government agency before it can be marketed for administration to humans. In some aspects, a “therapeutic agent” is an agent for which a medical prescription is required for administration to humans.

[0076] Therapeutically effective amount: The term “therapeutically effective amount” refers to an amount of a therapeutic protein (e.g., an effector molecule) which confers a therapeutic effect on the treated subject, at a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In particular, the “therapeutically effective amount” refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) may vary,for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0077] Treatment: The term “treatment” (also “treat” or “treating”) refers to any administration of a substance that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and / or reduces incidence of one or more symptoms, features, and / or causes of a particular disease, disorder, and / or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and / or condition and / or of a subject who exhibits only early signs of the disease, disorder, and / or condition. Alternatively, or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and / or condition. In some aspects, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and / or condition. In some aspects, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and / or condition.

[0078] Vector: “Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. The terms “vector” and “plasmid” may be used interchangeably. In some aspects, vectors are capable of extra-chromosomal replication and / or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and / or prokaryotic cell. Vectors capable of directing the expression of operatively linked coding sequences (e.g., genes) are referred to herein as “expression vectors.” An expression vector typically comprises an expression cassette. Vectors and plasmids include, but are not limited to, replication vectors, probe generation vectors, sequencing vectors, integrating vectors, phagemids, prokaryotic plasmids, eukaryotic plasmids, plant synthetic chromosomes, episomes, viral vectors (e.g., animal virus vectors), cosmids, and artificial chromosomes.II. Engineered Regulatory Elements

[0079] The present disclosure provides methods and compositions comprising engineered nucleic acids that comprise engineered regulatory elements (e.g., engineered enhancer sequences and / or engineered promoters that include engineered enhancer sequences). As used herein, a“regulatory element” refers to polynucleotide sequences that modulates (e.g., initiate, induce, activate, increase, or otherwise regulate) transcription of downstream genes operably linked to the regulatory element. Such engineered regulatory elements are useful for improving protein expression in a cell. By way of example, the engineered regulatory elements described herein can be useful for driving high expression of multiple proteins encoded in complex, multicistronic systems. By way of other example, an engineered regulatory element described herein can allow for improved transcription of an operably linked polynucleotide (e.g., a gene) in a cell, such as improved transcription in an NK cell and / or a T cell. In some instances, an engineered regulatory element described herein can allow for improved transcription of an operably linked polynucleotide in a cell (e.g., an NK cell and / or T cell), as compared to a known regulatory element (e.g., SV40, SFFV) operably linked to the same polynucleotide. In some instances, vectors comprising an engineered regulatory element described herein can drive higher expression of one or more proteins at the same or lower integrated copy number, as compared to a vector comprising a known regulatory element (e.g., SV40, SFFV) used to drive expression of the same one or more proteins. In some aspects, an engineered regulatory element described herein improves selective transcription in an NK cell and / or a T cell as compared to non-NK cell and / or a non-T cell, respectively.

[0080] In many aspects, an engineered nucleic acid provided by the present disclosure is or comprises an engineered regulatory element, as described herein (e.g., any one of SEQ ID NOs: 1-216 and / or any one of the transcription factor binding sites (TFBSs) described herein, e.g., any one of the TP63 TFBSs described herein). In some aspects, an engineered regulatory element is provided in an expression cassette, a heterologous construct, a vector, or other polynucleotide sequence.

[0081] In some aspects, an engineered regulatory element comprises an engineered enhancer sequence. Engineered enhancer sequences provided herein include nucleotide sequences 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% identity to a nucleotide sequence selected from: SEQ ID NOs: 1-108. SEQ ID NOs: 1-108 are shown in Table 1. In some aspects, an engineered enhancer sequence includes a nucleotide sequence that is at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs: 1-108. In some aspects, an engineered enhancer sequence includes a nucleotide sequence that is 100% identical to a nucleotide sequence selected from: SEQ ID NOs: 1-108.

[0082] Engineered enhancer sequences provided herein include engineered enhancer sequences that include one or more TP63 transcription factor binding sites (TFBSs). In some aspects, TP63 TFBSs include a sequence at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% identity to a nucleotide sequence selected from: SEQ ID NOs 217-220, and reverse complements thereof. SEQ ID NOs: 217-220 are shown in Table 3. In some aspects, TP63 TFBSs include a sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs 217-220, and reverse complements thereof. In some embodiments, TP63 TFBSs are selected from SEQ ID NOs 217-220, and reverse complements thereof.

[0083] In some aspects, an engineered enhancer sequence includes at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten TP63 TFBSs. In some aspects, an engineered enhancer sequence includes at least two TP63 TFBSs. In some aspects, an engineered enhancer sequence includes at least three TP63 TFBSs. In some aspects, an engineered enhancer sequence includes at least five TP63 TFBSs. In some aspects, an engineered enhancer sequence includes at least ten TP63 TFBSs. In some aspects, an engineered enhancer sequence includes two TP63 TFBSs. In some aspects, an engineered enhancer sequence includes three TP63 TFBSs. In some aspects, an engineered enhancer sequence includes five TP63 TFBSs. In some aspects, an engineered enhancer sequence includes ten TP63 TFBSs. In some aspects, an engineered enhancer sequence includes 1-500, 1-100, 1- 50, 2-20, or 2-10 TP63 TFBSs.

[0084] In some aspects, TP63 TFBSs include a TP63 TFBS half-site motif. In some aspects, a TP63 TFBS includes two TP63 TFBS half-site motifs. Without wishing to be bound by theory, TP63 generally prefers to bind to DNA as a dimer, and its consensus binding motif is approximately 20 bp (a “full” response element) that are typically made up of two "half-sites" of approximately 10 basepairs each. TP63 dimers can bind to a half-site well, and a total of four TP63 (i.e. tetramer) can bind to a full response element. Two TP63 TFBS half-site motifs are generally operatively linked to each other. Two half-sites can be concatenated directly with each other (e.g., not separated a linker). Two half-sites can be concatenated with each other via a nucleic acid linker and still have TP63 binding. Half-site linkers can be of different lengths, such as a single nucleotide or a linker between 2 and 10 nucleotides in length. Two half-sites can also be on the same or different DNA strands (e.g. half-site in the forward orientation and the other half in the reverse orientation), accordingly a TP63 TFBSs can include a first TP63 TFBS halfsite motif in one orientation operatively linked (e.g., concatenated) to a second TP63 TFBS halfsite motif in the opposite orientation (a reverse complement).

[0085] In some aspects, a TP63 TFBS half-site motif includes a 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: SEQ ID NOs 221-233, and reverse complements thereof. SEQ ID NOs: 221-233 are shown in Table 3. In some aspects, aTP63 TFBS half-site motif includes a sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs 221-233, and reverse complements thereof. In some aspects, a TP63 TFBS half-site motif is selected from SEQ ID NOs 221-233 and reverse complements thereof.

[0086] A TP63 TFBS with two TP63 TFBS half-site motifs can include two of the same TP63 TFBS half-site motifs. A TP63 TFBS with two TP63 TFBS half-site motifs can include two different TP63 TFBS half-site motifs. A TP63 TFBS with two TP63 TFBS half-site motifs can include a TP63 TFBS half-site motif and a reverse complement of the same TP63 TFBS half-site motif. In some aspects, two TP63 TFBS half-site motifs include (a) a first half-site motif 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: SEQ ID NOs 221- 233 ; and (b) a second half-site motif 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 reverse complement of a nucleotide sequence selected from: SEQ ID NOs 221-233.

[0087] In some aspects, an engineered enhancer sequence includes at least one additional non- TP63 TFBS in addition to a TP63 TFBS. In some aspects, an additional non-TP63 TFBS is selected from: a BARX2 TFBS, a NHEH1 TFBS, a TP73 TFBS, a HOXC10 TFBS, a NFE2 TFBS, a ATF4 TFBS, a HES1 TFBS, a FOS TFBS, a JUN TFBS, and a JUNB TFBS. n some aspects, an additional non-TP63 TFBS is selected from: a BARX2 TFBS, a NHEH1 TFBS, or both a BARX2 TFBS and a NHEH1 TFBS.

[0088] In some aspects, an additional non-TP63 TFBS is a BARX2 TFBS. In some aspects, an additional non-TP63 TFBS is a BARX2 TFBS including a 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. In some aspects, an engineered enhancer sequence includes 2, 3, 4, 5, or more than 5 BARX2 TFBSs.

[0089] In some aspects, an additional non-TP63 TFBS is a NHEH1 TFBS. In some aspects, an additional non-TP63 TFBS is a NHEH1 TFBS including a 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: 235. In some aspects, an engineered enhancer sequence includes 2, 3, 4, 5, or more than 5 NHEH1 TFBSs.

[0090] In some aspects, an additional non-TP63 TFBS is a TP73 TFBS. In some aspects, an additional non-TP63 TFBS is a TP73 TFBS including a 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: 236.

[0091] In some aspects, an additional non-TP63 TFBS is a HOXC10 TFBS. In some aspects, an additional non-TP63 TFBS is a HOXC10 TFBS including a 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: 237.

[0092] In some aspects, an additional non-TP63 TFBS is a NFE2 TFBS. In some aspects, an additional non-TP63 TFBS is a NFE2 TFBS including a 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: 238.

[0093] In some aspects, an additional non-TP63 TFBS is a ATF4 TFBS. In some aspects, an additional non-TP63 TFBS is a ATF4 TFBS including a 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: 239.

[0094] In some aspects, an additional non-TP63 TFBS is a HES1 TFBS. In some aspects, an additional non-TP63 TFBS is a HES1 TFBS including a 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: 240.

[0095] In some aspects, an additional non-TP63 TFBS is a FOS TFBS. In some aspects, an additional non-TP63 TFBS is a FOS TFBS including a 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: 241.

[0096] In some aspects, an additional non-TP63 TFBS is a JUN TFBS. In some aspects, an additional non-TP63 TFBS is a JUN TFBS including a 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: 242.

[0097] In some aspects, an additional non-TP63 TFBS is a JUNB TFBS. In some aspects, an additional non-TP63 TFBS is a JUNB TFBS including a 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: 243.

[0098] In some aspects, TFBSs can be operatively linked. In some aspects, TFBSs can be concatenated directly with each other (e.g., not separated a linker). In some aspects, TFBSs can be concatenated with each other via a nucleic acid linker

[0099] In some aspects, an engineered regulatory element is or includes an engineered promoter. Engineered promoters provided herein include an engineered enhancer sequence described herein (e.g., SEQ ID NOs: 1-108) operatively linked to a core promoter sequence. Core promoter sequences include, but are not limited to, a promoter selected from: minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB- SCP3 minimal promoter, a DPR containing SCP3 promoter, a minP promoter, a NFkB responseelement, a CREB response element, a NF AT response element, a SRF response element 1, a SRF response element 2, an API response element, a TCF-LEF response element promoter fusion, a Hypoxia responsive element, a SMAD binding element, a STAT3 binding site, 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. Core promoter sequences include, but are not limited to, a promoter selected from: a minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB-SCP3 minimal promoter, and a DPR containing SCP3 promoter.

[0100] A core promoter sequence can be a minCMV minimal promoter. A core promoter sequence can be an SV40 promoter. A core promoter sequence can be a B2M promoter. A core promoter sequence can be an SCP3 minimal promoter. A core promoter sequence can be a YB- SCP3 minimal promoter. A core promoter sequence can be a DPR containing SCP3 promoter. Exemplary core promoter sequences are provided in Table 4.

[0101] In some aspects, an engineered promoter includes 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% identity to a nucleotide sequence selected from: SEQ ID NOs: 109- 216. SEQ ID NOs: 109-216 are shown in Table 1. In some aspects, an engineered promoter includes a nucleotide sequence that is at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs: 109-216. In some aspects, an engineered promoter includes a nucleotide sequence that is 100% identical to a nucleotide sequence selected from: SEQ ID NOs: 109-216.

[0102] In some aspects, an engineered enhancer sequence is or comprises one or more enhancer segments (e.g., separate TP63 TFBSs). In some aspects, one or more enhancer segments of an engineered enhancer sequence are contiguous. In some aspects, one or more enhancer segments of an engineered enhancer sequence are non-contiguous. In some aspects, one or more enhancer segments of an engineered enhancer sequence are contiguous, and one or more engineered enhancer sequences of the same engineered enhancer sequence are noncontiguous. In some aspects, a nucleotide sequence of about 1, about 5, about 10, about 15, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, or about 200 nucleotides is located between the first enhancer segment and the second enhancer segment. In some aspects, a nucleotide sequence of about 1 to about 200, about 1 to about 100, about 1 to about 50, about 1 to about 25, about 10 to about 200, about 10 to about 100, about 10 to about 50, about 10 to about 25, about 25 to about 200, about 25 to about 100, about 25 to about 50, about 50 to about 200, about 50 to about 150, about 50 to about 100, about 50 to about 75, about 100 to about 200, about 100 to about 150,about 25 to about 100, about 30 to about 100, about 40 to about 100, about 25 to about 75, or about 25 to about 50 nucleotides is located between the first enhancer segment and the second enhancer segment.

[0103] In some aspects, an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) induces expression of an operably linked coding sequence (e.g., a gene) at a comparable level, or strength, to an alternative regulatory element, e.g., as measured by percentage of transcriptional activity as compared to said alternative regulatory element (e.g., a promoter, such as an SFFV promoter, an SV40 promoter, or a viral LTR). In some aspects, an engineered regulatory element induces expression of an operably linked coding sequence at a strength of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more as compared to an alternative regulatory element (e.g., an SFFV promoter, an SV40 promoter, or a viral LTR).

[0104] In some aspects, an engineered regulatory element (e.g., engineered enhancer sequences, engineered promoters that include engineered enhancer sequences, and / or enhancer segment) may comprise about 5 base pairs in length, about 10 base pairs in length , about 20 base pairs in length, about 30 base pairs in length, about 40 base pairs in length, about 50 base pairs in length, about 60 base pairs in length, about 70 base pairs in length, about 80 base pairs in length, about 90 base pairs in length, about 100 base pairs in length, about 200 base pairs in length, about 300 base pairs in length, about 400 base pairs in length, about 500 base pairs in length, about 600 base pairs in length, about 700 base pairs in length, about 800 base pairs in length, about 900 base pairs in length, about 1000 base pairs in length, about 1,100 base pairs in length, about 1,500 base pairs in length, about 2,000 base pairs in length, about 5,000 base pairs in length, or about 10,000 base pairs in length.

[0105] In some aspects, an engineered regulatory element (e.g., engineered enhancer sequences, engineered promoters that include engineered enhancer sequences, and / or enhancer segment) is between 5-10,000 base pairs in length. In some aspects, an engineered regulatory element is between 5-5,000 base pairs in length. In some aspects, an engineered regulatory element is between 5-2,000 base pairs in length. In some aspects, an engineered regulatory element is between 5-1,100 base pairs in length. In some aspects, an engineered regulatory element is between 5-1,000 base pairs in length. In some aspects, an engineered regulatory element is between 5-900 base pairs in length. In some aspects, an engineered regulatory element is between 5-800 base pairs in length. In some aspects, an engineered regulatory element is between 5-700 base pairs in length. In some aspects, an engineered regulatory element is between 5-600 base pairs in length. In some aspects, an engineered regulatoryelement is between 5-500 base pairs in length. In some aspects, an engineered regulatory element is between 5-400 base pairs in length. In some aspects, an engineered regulatory element is between 5-300 base pairs in length. In some aspects, an engineered regulatory element is between 5-200 base pairs in length. In some aspects, an engineered regulatory element is between 5-100 base pairs in length.

[0106] In some aspects, an engineered promoter that includes engineered enhancer sequences is between 500-10,000 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 500-5,000 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 500- 2,000 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 500-1,100 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 500-1,000 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 500-900 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 500-800 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 500-700 base pairs in length.In some aspects, an engineered promoter that include engineered enhancer sequences is between 500-600 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 600-1,100 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 700-1,100 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 600-1,500 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 700-1,200 base pairs in length. In some aspects, an engineered promoter that include engineered enhancer sequences is between 700-1,500 base pairs in length.

[0107] In some aspects, an engineered enhancer sequence is between 5-500 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-400 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-300 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-200 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-150 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-100 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-90 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-80 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-70 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-60 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-50 base pairs in length. In some aspects, anengineered enhancer sequence is between 5-40 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-30 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-20 base pairs in length. In some aspects, an engineered enhancer sequence is between 5-10 base pairs in length.

[0108] In some aspects, an engineered regulatory element (e.g., an engineered promoters that include engineered enhancer sequences) comprises at least one spacer sequence. In some aspects, an engineered regulatory element comprises at least one spacer located between an engineered enhancer sequence (e.g., comprising concatenated TFBSs) and a core promoter (e.g., a minimal promoter described herein).

[0109] In some aspects, engineered nucleic acids (e.g., heterologous constructs, as described herein) are configured to produce multiple agents (e.g., one or more effector molecules) that can be encoded in one or more coding sequences that are operably linked to an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein), as provided herein. For example, engineered nucleic acids may be configured to produce 2-20 different agents. In some aspects, engineered 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 agents. In some aspects, 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 agents. Provided agents are, in many cases, therapeutic agents, such as those described herein.

[0110] In some aspects, engineered nucleic acids (e.g., heterologous constructs, as described herein) may comprise multicistronic regions, i.e., more than one separate polypeptide (e.g., therapeutic agents, effector molecules, and the like) can be produced from a single mRNA transcript transcribed from said multicistronic region. For example, a heterologous payload can include a polynucleotide that encodes two or more polypeptides, such as a first effector molecule and a second effector molecule, or a first, second, and third effector molecule, or a first, second,third, and fourth effector molecule, where each polypeptide and / or effector molecule is a separately expressed peptide. Multicistronic regions may be created through the use of various linkers, e.g., a first coding sequence can be linked to a second coding sequence with a linker, for example creating a construct comprising, from 5’ to 3’, a first coding sequence, a linker, and a second coding sequence. 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 aspects, a multicistronic region comprises up to two, about to three, up to four, up to five, up to six, up to seven, up to eight, up to nine, up to ten, up to fifteen, up to twenty, or more coding sequences each linked by a linker (e.g., a first linker, a second linker, a third linker, a fourth linker, and so on).

[0111] In some aspects, a heterologous pay load includes a polynucleotide of the form El-El- E2, El-El -E2-E2-E3, and / or E1-L1-E2-L2-E3-L3-E4 where El is a nucleotide sequence encoding a first effector molecule, LI is a first linker molecule, E2 is a nucleotide sequence encoding a second effector molecule, L2 is a second linker molecule, E3 is a nucleotide sequence encoding a third effector molecule, L3 is a third linker molecule, and E4 is a nucleotide sequence encoding a fourth effector molecule. In some aspects, LI, L2, L3, and L4 are independently selected from: an internal ribosome entry site (IRES) and one or more nucleotide sequences encoding one or more 2A ribosome skipping elements.

[0112] In some aspects, engineered nucleic acids of the present disclosure (e.g., heterologous constructs, as described herein) comprise 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 aspects, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some aspects, the WPRE comprises the alpha, beta, and gamma components of the WPRE element. In some aspects, the WPRE comprises the alpha component of the WPRE element.III. Heterologous Constructs

[0113] Certain aspects of the present disclosure relate to polynucleotides (e.g., isolated polynucleotides) comprising one or more engineered regulatory elements (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) to produce a heterologous construct. In some aspects, a provided heterologous construct further comprises one or more expression cassettes.

[0114] In some aspects, a heterologous construct comprises an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) that is operably linked to a coding polynucleotide sequence (e.g., a gene, or coding sequence for a bioactive molecule). In some aspects, a heterologous construct comprises an engineered regulatory element that is operably linked to a polynucleotide sequence encoding at least one effector molecule (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth). In some aspects, an effector molecule comprises a bioactive molecule. In some aspects, an effector molecule comprises a polypeptide. In some aspects, an effector molecule comprises a polynucleotide (e.g., a mRNA, miRNA, siRNA, shRNA, etc.). In some aspects, an effector molecule is a human-derived effector molecule.

[0115] In some aspects, a heterologous construct comprises a nucleotide sequence encoding two or more effector molecules under the transcriptional control of an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) of the present disclosure. In some aspects, a heterologous construct comprises a nucleotide sequence encoding two or more effector molecules each under the transcriptional control of separate engineered regulatory elements.

[0116] In some aspects, a heterologous construct comprises a nucleotide sequence encoding two or more effector molecules that are in the same reading frame and are expressed as a single polypeptide chain. In some aspects, two or more effector molecules that are expressed as a single polypeptide chain may comprise one or more peptide cleavage sites (e.g., auto-cleavage sites, or cleavage sites for an intracellular protease) which when cleaved separate the two or more effector molecules. 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.

[0117] In some aspects, two or more effector molecules that are expressed as a single polypeptide chain comprise a T2A peptide cleavage site. In some aspects, two or more effector molecules that are expressed as a single polypeptide chain comprise an E2A peptide cleavage site. In some aspects, two or more effector molecules that are expressed as a single polypeptide chain comprise a T2A and an E2A peptide cleavage site.

[0118] In some aspects, a polynucleotide sequence encoding a first effector molecule is linked to a polynucleotide sequence encoding a second effector molecule by a linker polynucleotide sequence. In some aspects, a linker polynucleotide sequence comprises a polynucleotide sequence encoding at least one 2A ribosome skipping element. In some aspects, a 2A ribosome skipping element is a T2A, a P2A, a E2A, or a F2A element.

[0119] Any suitable effector molecule known in the art can be encoded in or expressed by a polynucleotide within a provided heterologous construct. In some aspects, an effector molecule (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth) is a therapeutic molecule. 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, receptors, ligands, antibodies, polynucleotides, peptides, shRNAs, miRNAs, and enzymes. Accordingly, in some aspects, an effector molecule (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth) belongs to a therapeutic class 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 chimeric receptor (e.g., chimeric antigen receptor [CAR]), a ligand, an antibody, a peptide, a polynucleotide molecule, a co-activation molecule, a tumor microenvironment modifier, an RNA molecule (e.g., mRNA, miRNA, siRNA, shRNA, etc.), and an enzyme.

[0120] In some aspects, an effector molecule is a receptor is a chimeric antigen receptor (CAR). In some aspects, CARs are engineered receptors that graft or confer a specificity of interest onto an immune effector cell. In certain aspects, CARs can be used to graft the specificity of an antibody onto an immunoresponsive cell, such as a T cell or NK cell. In some aspects, CARs of the present disclosure comprise an extracellular antigen-binding domain (e.g., an scFv) fused to a transmembrane domain, fused to one or more intracellular signaling domains. In some aspects, the extracellular antigen -binding domain of a CAR of the present disclosure specifically binds to one or more antigens expressed on a tumor cell. Antigen-binding domains of the present disclosure can include any domain that binds to the antigen including, without limitation, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a bispecific antibody, a conjugated antibody, a human antibody, a humanized antibody, and a functional fragment thereof, including but not limited to a single-domain antibody (sdAb) such as a heavy chain variable domain (VH), a light chain variable domain (VL) and a variable domain (VHH) of camelid derived nanobody, and to an alternative scaffold known in the art to function as antigen-binding domain, such as a recombinant fibronectin domain, a T cell receptor(TCR), a recombinant TCR with enhanced affinity, or a fragment thereof, e.g., single chain TCR, and the like. In some instances, it is beneficial for the antigen-binding domain to be derived from the same species in which the CAR will ultimately be used in. For example, for use in humans, it may be beneficial for the antigen-binding domain of the CAR to comprise human or humanized residues for the antigen-binding domain of an antibody or antibody fragment.

[0121] In some aspects, the transmembrane domain of a CAR of the present disclosure comprises a hydrophobic alpha helix that spans at least a portion of a cell membrane. It has been shown that different transmembrane domains can result in different receptor stability. After antigen recognition, receptors cluster and a signal is transmitted to the cell. In some aspects, the transmembrane domain of a CAR of the present disclosure can comprise the transmembrane domain of a CD8 polypeptide, a CD28 polypeptide, a CD25 polypeptide, a CD7 polypeptide, a CD3-zeta polypeptide, a CD4 polypeptide, a 4- IBB polypeptide, an 0X40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a LAX polypeptide, a LAT polypeptide, a PD- 1 polypeptide, a LAG-3 polypeptide, a TIM3 polypeptide, a KIR3DS1 polypeptide, a KIR3DL1 polypeptide, an NKG2D polypeptide, an NKG2A polypeptide, a TIGIT polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a LIR-1 (LILRB1) polypeptide, a SIRPa polypeptide, or can be a synthetic peptide, or any combination thereof.

[0122] In some aspects, a CAR of the present disclosure can also comprise a spacer region that links the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to orient in different directions to facilitate antigen recognition. In some aspects, the spacer region may be a hinge from a human protein. For example, the spacer (also referred to herein as “hinge”) may be a human Ig (immunoglobulin) hinge, including without limitation an IgG4 hinge, an IgG2 hinge, a CD8a hinge, or an IgD hinge. In some aspects, the spacer region may comprise an IgG4 hinge, an IgG2 hinge, an IgD hinge, a CD28 hinge, a KIR2DS2 hinge, an LNGFR hinge, or a PDGFR-beta extracellular linker. In some aspects, the spacer region is localized between the antigen-binding domain and the transmembrane domain.

[0123] In some aspects, a CAR of the present disclosure comprises one or more cytoplasmic domains or regions. The cytoplasmic domain or region of the CAR may include an intracellular signaling domain. An intracellular signaling domain is typically responsible for activation of one or more effector functions of an immune cell (e.g., a T cell or an NK cell) that has been engineered to express a CAR of the present disclosure. For example, an effector function of a T cell may be cytolytic activity or helper activity, such as the secretion of cytokines. Accordingly, in some aspects the term "intracellular signaling domain" refers to the portion of a protein which transduces an effector function signal and directs the cell to perform a specialized function.While the entire intracellular signaling domain may be employed, in many instances it is not necessary to use the entire chain. In aspects where a truncated portion of the intracellular signaling domain is used, such a truncated portion may be used in place of the corresponding intact chain as long as the truncated portion transduces the effector function signal. Certain aspects of the present disclosure relate to chimeric inhibitory receptors. Chimeric inhibitory receptors are useful, for example, as NOT logic gates for controlling cell activity, such as immune cell activity. In some aspects, chimeric inhibitory receptors of the present disclosure specifically bind to one or more antigens that are expressed on normal cells but not on tumor cells. In some aspects, the chimeric inhibitory receptor comprises an antigen-binding domain, a transmembrane domain of the present disclosure (e.g., any suitable transmembrane domain used in conjunction with a chimeric receptor of the present disclosure), and an intracellular domain. In some aspects, the chimeric inhibitory receptor may inhibit one or more activities of a cell, such as an immunoresponsive cell.

[0124] In some aspects, a CAR of the present disclosure comprises one or more components of a natural killer (NK) cell, thereby forming an NK CAR. The NK component may be a transmembrane domain, a hinge domain, or a cytoplasmic domain from any suitable natural killer cell receptor, including without limitation, a killer cell immunoglobulin-like receptor (KIR), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS DPI; a natural cytotoxicity receptor (NCR), such as NKp30, NKp44, NKp46; a signaling lymphocyte activation molecule (SLAM) family of immune cell receptor, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; an Fc receptor (FcR), such as CD16, and CD64; and an Ey49 receptor, such as EY49A and EY49C. In some aspects, the NK-CAR may interact with an adaptor molecule or intracellular signaling domain, such as DAP12. The structural components as described above of a CAR are also applicable to the structure of an NK CAR. Exemplary configurations and sequences of CARs comprising NK receptor components are described in International Patent Publication WO2014 / 145252, published September 18, 2014.

[0125] Exemplary CARs and CAR architectures are further described in WO2021035093, WO2020223445, WO2021168317, WO2021168298, WO2022115565, WO 2022 / 236142, and PCT / US2023 / 069829, which are hereby incorporated by reference in their entirety.

[0126] In some aspects, an effector molecule is a human-derived effector molecule. In some aspects, at least one effector molecule is a human-derived effector molecule. In some aspects, each effector molecule is a human-derived effector molecule.

[0127] In some aspects, a heterologous pay load includes a first effector molecule and a second effector molecule. In some aspects, a heterologous payload includes a first effector molecule and a second effector molecule, where the first effector molecule and the second effector molecule are independently selected from a first CAR and a second CAR. In some aspects, the first CAR is an activating CAR (aCAR) and the second CAR is an inhibitory CAR (iCAR), optionally wherein the first CAR and / or the second CAR is a bivalent CAR.

[0128] In some aspects, a heterologous pay load includes a first effector molecule and a second effector molecule, where the first effector molecule and the second effector molecule are independently selected from a first CAR and a cytokine. In some aspects, a heterologous payload includes a first effector molecule and a second effector molecule, where the first effector molecule and the second effector molecule are independently selected from the first cytokine and a second cytokine.

[0129] In some aspects, a heterologous pay load includes a first effector molecule, a second effector molecule, and a third effector molecule. In some aspects, a heterologous payload includes a first effector molecule, a second effector molecule, and a third effector molecule, where the first effector molecule, the second effector molecule, and the third effector molecule are independently selected from a first CAR, a second CAR, and a cytokine. In some aspects, a heterologous payload includes a first effector molecule, a second effector molecule, and a third effector molecule, where the first effector molecule, the second effector molecule, and the third effector molecule are independently selected from a first CAR, a first cytokine, and a second cytokine.

[0130] In some aspects, a heterologous pay load includes a first effector molecule, a second effector molecule, a third effector molecule, and a fourth effector molecule. In some aspects, a heterologous payload includes a first effector molecule, a second effector molecule, a third effector molecule, and a fourth effector molecule, where the first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule are independently selected from: a first CAR, a second CAR, a first cytokine, and a second cytokine.

[0131] In general, a heterologous payload (including multicistronic payloads described herein) can include any length polynucleotide capable of being expressed using an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) provided herein. In some aspects, a heterologous payload includes a polynucleotide at least 1000 nucleotides (nt) in length. In some aspects, a heterologous payload includes a polynucleotide at least 1500 nt in length. In some aspects, a heterologous payload includes a polynucleotide at least 2000 nt in length. In someaspects, a heterologous payload includes a polynucleotide at least 2500 nt in length. In some aspects, a heterologous payload includes a polynucleotide at least 3000 nt in length. In some aspects, a heterologous payload includes a polynucleotide at least 3500 nt in length. In some aspects, a heterologous payload includes a polynucleotide at least 4000 nt in length. In some aspects, a heterologous payload includes a polynucleotide at least 4500 nt in length. In some aspects, a heterologous payload includes a polynucleotide 5000 nt or less in length. In some aspects, a heterologous payload includes a polynucleotide 4800 nt or less in length. In some aspects, a heterologous payload includes a polynucleotide 4700 nt or less in length. In some aspects, a heterologous payload includes a polynucleotide 4500 nt or less in length.

[0132] In some aspects, a heterologous payload includes a polynucleotide between 2000-4500 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 2000- 4700 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 2000-4800 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 2000-5000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 2200-4500 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 2200-4700 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 2200-4800 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 2200-5000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 1000-4500 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 1000-4700 nt in length. In some aspects, a heterologous pay load includes a polynucleotide between 1000-4800 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 1000-5000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 100-4500 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 100- 4700 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 100-4800 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 100-5000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-4500 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-4700 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-4800 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-5000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-6000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-7000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-8000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-9000 nt in length. In some aspects, a heterologous payload includes apolynucleotide between 10-10,000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-25,000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-50,000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-100,000 nt in length. In some aspects, a heterologous payload includes a polynucleotide between 10-500,000 nt in length. In some aspects, a heterologous pay load includes a polynucleotide between 10-1,000,000 nt in length.

[0133] In some aspects, 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 polynucleotides in a heterologous construct of the present disclosure include: CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9 / CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CC125, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5 CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, CX3CL1, or combinations thereof.

[0134] In some aspects, an effector molecule is a cytokine. Non-limiting examples of cytokines that may be encoded by polynucleotides in a heterologous construct of the present disclosure include: IL-l-alpha, IL-l-beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL- 13, IL-15, IL-16, IL-17, 11-17A, IL-17B / C / D, I1-17E (IL-25), IL-17F, IL-18, IL-19, IL-20, IL- 21, IL-22, IL-23, IL-24, IL-25, IL-26, 11-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL- 35, IL-36-alpha, IL-36-beta, IL-36-gamma, IL-37, IL-38, IFN-alpha, IFN-beta, IFN-gamma, TGF-beta, GM-CSF, CSF-l / M-CSF, G-CSF, SCF, TNF-alpha, TNF-beta, growth hormone (GH), prolactin (PRL), erythropoietin (EPO), leptin, FLT3 ligand, or combinations thereof.

[0135] In some aspects, an effector molecule is a tumor microenvironment modifier. Suitable tumor microenvironment modifiers for use as an effector molecule include, but are not limited to, adenosine deaminase, TGF-beta inhibitors, immune checkpoint inhibitors, and HPGE2, or any combination thereof.

[0136] In some aspects, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one TGF-beta inhibitor. Suitable TGF-beta inhibitors for use as an effector molecule include, but are not limited to, an anti-TGF-beta peptide, an anti-TGF-beta antibody, a TGF-beta-TRAP, or combinations thereof.

[0137] In some aspects, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising 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-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-TNF-alpha antibodies, anti-TREMl antibodies, and anti- TREM2 antibodies, or combinations and / or functional fragments thereof.

[0138] 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).

[0139] In some aspects, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one therapy that treats a disease or disorder. In some aspects, a disease or disorder includes cancer.

[0140] In some aspects, a heterologous construct of the present disclosure is configured to produce an effector molecule comprising at least one gene replacement therapy. A gene therapy is a therapy that replaces a non-functional, partially functional, or missing disease-related gene with an engineered version of the same disease-related gene, thereby providing a therapeutically relevant amount of the disease-related gene product. In some aspects, a gene replacement therapy is a therapy that treats a disease or disorder, such as cancer.

[0141] In some aspects, an effector molecule as described may comprise a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the effector molecule’s N- terminus. Without wishing to be bound by theory, a secretion signal peptide or signal sequence is understood to direct newly synthesized proteins destined for secretion or membrane insertion to the proper protein processing pathways. In aspects with two or more effector molecules, each effector molecule can comprise a secretion signal.

[0142] In some aspects, a secretion signal peptide operably associated with an effector molecule can be a native secretion signal peptide (e.g., a secretion signal peptide that is natively associated with the given effector molecule). In some aspects, a secretion signal peptideoperably associated with an effector molecule can be a non-native secretion signal peptide native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments of interest, e.g., those related to diseases or disorders, such as cancer.IV. Vectors / Plasmids

[0143] Another aspect of the disclosure relates to a vector comprising a nucleotide sequence encoding an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) ,e.g., a heterologous construct), as described herein. In some aspects, a vector is an expression vector. Such an expression vector comprises a nucleotide sequence encoding any engineered regulatory element disclosed herein (e.g., any of the NK cell and / or T cell specific regulatory elements herein) operably linked a coding nucleotide sequence (e.g., for an effector molecule, e.g., as described herein) to allow for expression of said coding nucleotide sequence in a cell or cell-free extract. A wide variety of expression vectors can be employed for expressing a nucleic acid molecule encoding engineered regulatory elements of the present disclosure including, without limitation, viral expression vectors, prokaryotic expression vectors, eukaryotic expression vectors (e.g., yeast expression vectors, insect expression vectors, mammalian expression vectors, etc.), and cell-free extract expression vectors.

[0144] It is further understood that expression vectors useful to practice aspects of methods described herein may include additional promoters (e.g., inducible, constitutive, cell- specific), enhancer elements, or both. Expression vectors may include polynucleotides encoding protein tags, or epitope tags, to aid in isolation, purification or selection (e.g., poly-His tags, FLAG-tags, hemagglutinin tags, fluorescent protein tags, bioluminescent tags, and nuclear localization tags). As described herein, coding sequences for such protein tags, or epitope tags, can be fused to a coding sequence or can be included in a separate expression cassette. Non-limiting examples of expression vectors, along with well-established reagents and conditions for making and using an expression construct from such expression vectors are readily available from commercial vendors that include, without limitation, BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences- Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection, making and use of an appropriate expression vector are routine procedures well within the scope of one skilled in the art and from the teachings herein.

[0145] In some aspects, a vector comprises a transposon / transposase system to incorporate nucleotides of the present disclosure into a host cell genome. In some aspects, a transposonsystem used in accordance with the present disclosure is a Sleeping Beauty transposon / transposase or the piggyBac transposon / transposase.

[0146] In some aspects, 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 aspects, 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 aspects, a vector of the present disclosure is an adenoviral vector (A5 / 35).

[0147] In some aspects, 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., WO 01 / 96584; WO 01 / 29058; and US 6,326,193). A number of viral based systems have been developed for gene transfer into mammalian cells. A selected gene (e.g., alone, or within an expression cassette, or heterologous construct) can be inserted into a vector and packaged in retroviral particles using techniques known in the art. A 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.

[0148] In some aspects, vectors of the present disclosure include regulatory elements, including engineered and / or native regulatory elements, such as engineered enhancer sequences and / or native enhancers, that regulate the frequency of transcriptional initiation. In general, the spacing between regulatory elements may be flexible, so that transcriptional function is preserved when regulatory elements are inverted or moved relative to one another. Depending on the promoter (e.g., an engineered promoter described herein), individual elements may function either cooperatively or independently to activate transcription.

[0149] In some aspects, 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.

[0150] In some aspects, a vector of the present disclosure can further comprise a selectable marker gene and / or a reporter gene to facilitate identification and selection of certain cells (e.g., those comprising an engineered regulatory element, such an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) from a population of cells that have been transduced with said vector. In some aspects, a selectable marker may be encoded by a polynucleotide that is separate from a vector and used in a co-transfection procedure. A selectable marker or a reporter gene may be flanked with appropriate regulatory sequences to allow expression in a host cell. In some aspects, a selectable marker comprises an antibiotic resistance gene. Examples of antibiotic resistance genes include, without limitation, kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, and combinations thereof.

[0151] In some aspects, a reporter gene 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 that has an easily detectable property, such as enzymatic activity or fluorescence. Expression of a reporter gene can be assayed at a suitable time after a polynucleotide comprising said reporter gene has been introduced into a recipient or host cell that is capable of expressing said reporter gene. Examples of reporter genes include, without limitation, genes encoding for luciferase (or variants or fragments of luciferase, e.g., nanoluciferase), genes encoding for betagalactosidase, genes encoding for chloramphenicol acetyl transferase, genes encoding for secreted alkaline phosphatase, and genes encoding for green fluorescent protein (GFP) (or common variants or fragments of GFP, e.g., RFP, YFP, etc.). Suitable expression systems are well known in the art and may be prepared using known techniques or obtained commercially.

[0152] In some aspects, a vector can include a dual expression vector system, such as a vector encoding multiple cassettes, where each cassette encodes a separate heterologous construct that can drive expression of a heterologous payload. For example, a dual expression vector system can include vectors where separate cassettes each include separate engineered regulatory elements (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) each driving expression of separate heterologous pay loads. Separate engineered regulatory elements can include one of the engineered promoters described herein. In some aspects, each of the engineered regulatory elements in a dual expression vector include one of the engineered promoters described herein.V. Engineering Cells

[0153] The present disclosure further provides for methods and compositions for preparing and using engineered cells (e.g., immunoresponsive cells such as, e.g., NK cells or T cells) comprising a heterologous construct comprising an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) as provided herein.

[0154] In some aspects, engineered cells comprise an engineered regulatory element (e.g., engineered enhancer sequence, and / or an engineered promoter that includes engineered enhancer sequences provided herein).

[0155] Also provided herein are compositions and methods for engineering cells that are capable of producing one or more effectors molecules (e.g., a first effector molecule, a second effector molecule, a third effector molecule, and so forth). Effector molecules of the present disclosure may be encoded in one or more heterologous constructs or expression cassettes as described herein or otherwise known in the art.

[0156] In some aspects, cells of the present disclosure are engineered to produce effector molecules through introduction (i.e., delivery) of one or more polynucleotides that encode for one or more effector molecules (e.g., those provided herein). For example, polynucleotide heterologous constructs or expression cassettes encoding one or more effector molecules can be any of the engineered nucleic acids described herein. 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 (e.g., including all methods described herein). One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

[0157] Engineered cells can include an immunoresponsive cell having any of the heterologous constructs described herein. In some aspects, an immunoresponsive cell is selected from: a Natural Killer (NK) cell, 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 B cell, a macrophage, 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. In some aspects, an immunoresponsive cell is a NK cell. In some aspects, an immunoresponsive cell is a T cell.

[0158] In some aspects, an immunoresponsive cell expresses an activating immune receptor. In some aspects, an immunoresponsive cell expresses an activating immune receptor that is an antigen recognizing receptor, e.g., a CAR.

[0159] In some aspects, an immunoresponsive cell is autologous. In some aspects, an immunoresponsive cell is allogeneic.A. Viral-Mediated Delivery

[0160] Viral vector-based delivery platforms can be engineered to deliver certain nucleic acids of interest into a host cell in order to create an engineered cell as described herein. In general, a viral vector-based delivery platform can be used to create an engineered cell by introducing (z.e., delivering, or transducing) a heterologous nucleic acid, e.g., a transgene, expression cassette, or a heterologous construct as described herein, into a particular host cell. In many aspects of the present disclosure, a viral vector-based delivery platform delivers a nucleic acid comprising an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) as described herein, or a heterologous construct as described herein. In some aspects, a transduced nucleic acid is integrated into a host cell genome. Viruses created for use in viral vector-based delivery platforms can be referred to as recombinant viruses or engineered viruses. It is understood that a recombinant virus may encode one or more viral genes needed for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), in some cases referred to as cis-acting elements or cis-acting genes, in addition to nucleic acids to be delivered to a host cell.

[0161] In some aspects, recombinant viruses may be used to deliver nucleic acids encoding one or more genes (e.g., transgenes), expression cassettes, or heterologous constructs. In many aspects, delivered nucleic acids comprise one or more nucleotide sequences comprising an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) as described herein. In some aspects, delivered nucleic acids (e.g., heterologous constructs) are configured to express one or more effector molecules.

[0162] A viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding genes (e.g., transgenes), expression cassettes, or heterologous constructs described herein, and referred to as trans-acting elements or trans-acting 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 a vector encoding a transgene expression cassette, or heterologous construct. The number of viral vectors used can depend on the packaging capacity of the above-mentioned viral vector-based platforms, and one skilled in the art can select the appropriate number of viral vectors.

[0163] In general, any of viral vector-based system can be used for the in vitro expression of engineered nucleic acids (e.g., heterologous constructs), e.g., for the production of molecules, such as effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivodelivery of the engineered nucleic acids comprising nucleotide sequences encoding one or more effector molecules under transcriptional control of an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein). 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.

[0164] Viral vector-based delivery platforms can utilize 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 et al., 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 et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873- 9880).

[0165] Provided engineered nucleic acid sequences may be preceded with one or more nucleic acid sequences that by themselves, or via their encoded polypeptide sequence, target a subcellular compartment. Upon introduction (i.e., delivery) of engineered nucleic acids into a host cell, infected cells (i.e., engineered cells) can express polypeptides encoded by said engineered nucleic acids (e.g., one or more effector molecules), and in some case secrete, said polypeptides. Vaccinia vectors and 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 vectorsuseful for the introduction (z.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.

[0166] The viral vector-based delivery platforms described herein can utilize 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 more engineered nucleic acids (e.g., genes, expression cassettes, heterologous constructs, etc.).

[0167] In some aspects, a recombinant virus is created from a virus selected from: a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a viruslike particle (VLP).

[0168] A viral vector-based delivery platform as used in accordance with the present disclosure 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 said vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., genes, expression cassettes, heterologous constructs, etc.) into a target cell to provide permanent integration and / or expression of said engineered nucleic acids. 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 et al., J. Virol. 66:2731-2739 (1992); Johann et al, J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al, 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.

[0169] A viral vector-based delivery platform as used in accordance with the present disclosure 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-baseddelivery platforms can be HIV-based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs). Lentiviral-based delivery platforms can be SIV, or FIV-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.

[0170] A viral vector-based delivery platform as used in accordance with the present disclosure 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 et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995;Sakamoto et al., 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 WO 96 / 13597, each herein incorporated by reference for all purposes.

[0171] A viral vector-based delivery platform as used in accordance with the present disclosure 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 an engineered nucleic acids, e.g., those encoding one or more effector molecules (see, e.g., West et al., 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, et al., 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 of which are 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 al., Mol. Cell. Biol. 5:3251- 3260 (1985); Tratschin et al., Mol. Cell, Biol. 4:2072-2081 (1984); Muzyczka, PNAS 81:64666470 (1984); and Samuiski et al., J. Virol. 63:03822-3828 (1989), each of which areherein 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, AAV.RhlO, AAV11 and variants thereof.

[0172] A viral vector-based delivery platform as used in accordance with the present disclosure 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 Ther. 2009 May; 17(5): 767-777), herein incorporated by reference for all purposes.

[0173] A viral vector-based delivery platform as used in accordance with the present disclosure can be engineered to target (z.e., infect or transduce) a range of cells, target a narrow subset of cells, or target a specific cell. In general, an envelope protein chosen for a viral vector-based delivery platform will determine viral tropism. A virus used in a viral vector-based delivery platform can be pseudotyped to target a specific cell of interest. A 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. A 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.B. Lipid Structure Delivery Systems

[0174] Engineered nucleic acids of the present disclosure (e.g., any of engineered nucleic acid as 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.

[0175] 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., an engineered nucleic acid, such as any engineered nucleic acid as 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 aspects, 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 of which are herein incorporated by reference for all purposes

[0176] 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 structures between 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 engineered nucleic acid as 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 a lipid bilayer.

[0177] A liposome used in accordance with the present disclosure 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 / 01161and 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 of which are hereby incorporated by reference for all purposes.

[0178] 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 WO 03 / 015757A1, WO 04029213A2, and WO 02 / 100435A1, each of which are herein incorporated by reference in their entirety

[0179] 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 of which are herein incorporated by reference for all purposes.

[0180] 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 engineered nucleic acid as 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. 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.

[0181] 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 nm, 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 engineered nucleic acid as 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 withalkaline 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.

[0182] 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 said producer 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 engineered nucleic acid as described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. A nano vesicle, 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.

[0183] 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 / pay loads, such as any engineered nucleic acid or viral system described herein, by absorbing into a membrane of a target cell 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 compositionscan 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, which will be appreciated by those skilled in the art.

[0184] 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 a membrane and the single-chain lipid’ s hydrophobic tails form the micelle center. Micelles typically refer to lipid structures only containing a lipid monolayer. 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.

[0185] Nucleic-acid vectors, such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of a nucleic acid by serum nucleases or off-target stimulation of an immune system by free nucleic acids. Similarly, viral delivery systems exposed directly to serum can trigger an undesired immune response and / or neutralization of a viral delivery system. Therefore, encapsulation of an engineered nucleic acid and / or viral delivery system can be used to avoid degradation, while also avoiding potential off- target affects. In certain examples, an engineered nucleic acid and / or viral delivery system is fully encapsulated within a delivery vehicle, such as within an aqueous interior of an LNP, or other vesicle or lipid system as described herein. Encapsulation of an engineered nucleic acid and / or viral delivery system within an LNP or other lipid system 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, a desired lipid formulation, such as MC3 or MC3-like containing compositions, is provided to a droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agents, such that a 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. Lor 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. Pollowing droplet generation, delivery vehicles encapsulating a cargo / payload (e.g., an engineered nucleic acid and / or viral delivery system) can be further treated or engineered to prepare them for administration.C. Nanoparticle Delivery

[0186] 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 of non-immunogenic materials and generally avoid eliciting immunity to a 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), each of which are herein incorporated by reference for all purposes.D. Genomic Editing Systems

[0187] A genomic editing system can be used to engineer a host genome to encode a engineered nucleic acid, such as any engineered nucleic acid described herein. 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 (e.g., those described herein).

[0188] A transposon system can be used to integrate an engineered nucleic acid, e.g., an engineered nucleic acid as described herein, 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 TIR- 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., an 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.

[0189] A nuclease genomic editing system can be used to engineer a host genome to encode a engineered nucleic acid, such as an engineered nucleic acid of the present disclosure. Without wishing to be bound by theory, in general, nuclease-mediated gene editing systems used to introduce an exogenous gene, or nucleic acid, 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 byusing another DNA 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 a 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 a lesion that is included between the 5’ and 3’ complimentary ends within an HRT (e.g. , a gene or a portion of a gene, or engineered nucleic acid as described herein) can be incorporated (z.e., “integrated”) into a 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 an endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any engineered nucleic acid as described herein, e.g., those encoding one or more effector molecules).

[0190] In some examples, an 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, an HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.

[0191] The identical, or substantially identical, sequences found at the 5’ and 3’ ends of an HR template, with respect to an exogenous sequence to be introduced, are generally referred to as arms (HR arms). HR arms can be identical to regions of an endogenous genomic target locus (i.e., 100% identical). HR arms in some examples can be substantially identical to regions of an 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.

[0192] Each HR arm, i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can 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 immediately adjacent 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.

[0193] 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 zinc-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.

[0194] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid as described herein, e.g., those encoding one or more 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 purposes. In general, a CRISPR- mediated gene editing system comprises a CRIS PR-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 Cpf 1 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.

[0195] 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 CRISPR systems, 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 cellusing 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.

[0196] 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 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.

[0197] 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.

[0198] 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.

[0199] Engineered cells described herein can be engineered using non- viral methods, e.g., 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., 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 other viral-based delivery methods described herein.

[0200] 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 directcleavage 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.

[0201] In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. TALEN is an 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.E. Other Engineering Delivery Systems

[0202] Various additional means to introduce engineered nucleic acids (e.g., any engineered nucleic acid as described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0203] Electroporation can be 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 a 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 engineered nucleic acid as 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 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, recoveryconditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors including, but not limited to, type of cell or other recipient entity, cargo to be delivered, efficiency of internalization desired, and viability desired. Optimization of such criteria is 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.

[0204] Other means for introducing engineered nucleic acids (e.g., any of engineered nucleic acid as 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.

[0205] Compositions and methods for delivering engineered mRNAs in vivo, such as naked plasmids or mRNA, are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10; 27(4): 710-728) and Kaczmarek et al. (Genome Med. 2017; 9: 60.), each of which are herein incorporated by reference for all purposes.VI. Methods of Use

[0206] Methods and compositions for treating a subject with a disease or disorder are encompassed by the present disclosure. Provided methods include administering to a subject with a disease or disorder a therapeutically effective amount of any engineered nucleic acid (e.g., a heterologous construct, vector, dual expression vector), engineered cell (e.g., an isolated engineered cell), and / or pharmaceutical composition described herein that include an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) described herein.

[0207] In some aspects, methods and compositions provided herein may be used to treat a disease or disorder. In some aspects, a disease or disorder includes cancer.

[0208] Also described herein are methods of increasing expression of a target gene or a heterologous payload that include use of an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) described herein, such as any of the vectors or dual expression vectors that include an engineered regulatory element described herein. In some aspects, a target gene is an immunomodulatory gene.VII. Pharmaceutical Compositions

[0209] Provided engineered nucleic acids or engineered cells 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 non-toxic 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.

[0210] 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.

[0211] 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.

[0212] Composition provided herein can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.VIII. Kits

[0213] Certain aspects of the present disclosure relate to kits for the treatment and / or prevention of disease or disorder. In certain aspects, a disease or disorder is a cancer (e.g., a tumor, such as solid tumors). In certain aspects, a kit includes a therapeutic or prophylactic composition comprising an effective amount of one or more engineered nucleic acids (e.g., isolate engineered nucleic acids) of the present disclosure, vectors of the present disclosure, and / or engineered cells of the present disclosure that include an engineered regulatory element (e.g., an engineered enhancer sequence and / or engineered promoter including engineered enhancer sequences described herein) described herein. In some aspects, a kit comprises a sterile container. In some aspects, such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. A container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0214] In some aspects, a therapeutic or prophylactic composition (e.g., an engineered cell, such as an engineered immunoresponsive cell described herein) is provided together with instructions for administering said therapeutic or prophylactic composition to a subject having or at risk of developing a particular disease or disorder (e.g., cancer). In some aspects, the instructions may include information about use of a composition for the treatment and / or prevention of a disease or disorder. In some aspects, the instructions include, without limitation, a description of a therapeutic or prophylactic composition, a dosage schedule, an administration schedule for treatment or prevention of a disease or disorder or a symptom thereof, precautions, warnings, indications, counter-indications, over-dosage information, adverse reactions, animal pharmacology, clinical studies, and / or references. In some aspects, the instructions can be printed directly on a container (when present), or as a label applied to a container, or as a separate sheet, pamphlet, card, or folder supplied in or with a container.

[0215] Throughout the description, where agents, compounds, entities, and so forth (e.g., provided engineered nucleic acids) are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are agents, compounds, entities, and so forth of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

[0216] The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0217] Practice of the invention will be more fully understood from the foregoing examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way.Enumerated Embodiments:Embodiment 1 An engineered regulatory element 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 a nucleotide sequence selected from: SEQ ID NOs: 1-108.Embodiment 2 The engineered regulatory element of embodiment 1, wherein the nucleotide sequence is at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs: 1-108.Embodiment 3 The engineered regulatory element of embodiment 2, wherein the nucleotide sequence is 100% identical to a nucleotide sequence selected from: SEQ ID NOs: 1-108.Embodiment 4 An engineered regulatory element comprising one or more TP63 transcription factor binding sites (TFBSs).Embodiment 5 The engineered regulatory element of embodiment 4, wherein the one or more TP63 TFBSs comprise a 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: SEQ ID NOs 217-220, and reverse complements thereof.Embodiment 6 The engineered regulatory element of embodiment 5, wherein the one or more TP63 TFBSs comprise a sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs 217-220, and reverse complements thereof.Embodiment 7 The engineered regulatory element of embodiment 6, wherein the one or more TP63 TFBSs are selected from SEQ ID NOs 217-220, and reverse complements thereof.Embodiment 8 The engineered regulatory element of embodiment 4, wherein any of the one or more TP63 TFBSs comprises a TP63 TFBS half-site motif, wherein the TP63 TFBS half-site motif comprises a 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: SEQ ID NOs 221-233, and reverse complements thereof.Embodiment 9 The engineered regulatory element of embodiment 8, wherein the TP63 TFBS half-site motif comprises a sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs 221-233, and reverse complements thereof.Embodiment 10 The engineered regulatory element of embodiment 9, wherein the TP63 TFBS half-site motif is selected from SEQ ID NOs 221-233 and reverse complements thereof.Embodiment 11 The engineered regulatory element of any one of embodiments 8-10, wherein the TP63 TFBS comprises two TP63 TFBS half-site motifs.Embodiment 12 The engineered regulatory element of embodiment 11, wherein the two TP63 TFBS half-site motifs are operatively linked by a nucleic acid linker, optionally wherein the linker is between 1-10 base pairs.Embodiment 13 The engineered regulatory element of embodiment 11 or 12, wherein the two TP63 TFBS half-site motifs comprise (a) a first half-site motif 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: SEQ ID NOs 221- 233; and (b) a second half-site motif 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 reverse complement of a nucleotide sequence selected from: SEQ ID NOs 221-233.Embodiment 14 The engineered regulatory element of any one of embodiments 4-13, comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least 9, or at least 10 TP63 TFBSs.Embodiment 15 The engineered regulatory element of any one of embodiments 4-13, comprising at least two TP63 TFBSs.Embodiment 16 The engineered regulatory element of any one of claims 4-13, comprising 1-500, 1-100, 1-50, 2-20, or 2-10 TP63 TFBSs.Embodiment 17 The engineered regulatory element of any one of claims 4-16, further comprising at least one additional non-TP63 TFBS.Embodiment 18 The engineered regulatory element of claim 17, wherein the at least one additional non-TP63 TFBS is selected from: a BARX2 TFBS, a NHLH1 TFBS, a TP73 TFBS, a HOXC10 TFBS, a NFE2 TFBS, a ATF4 TFBS, a HES1 TFBS, a FOS TFBS, a JUN TFBS, and a JUNB TFBS.Embodiment 19 The engineered regulatory element of embodiment 18, wherein: the BARX2 TFBS comprises a 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, and / or the NHLH1 TFBS comprises a 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: 235, and / orthe TP73 TFBS comprises a 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: 236, and / or the HOXCIO TFBS comprises a 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: 237, and / or the NFE2 TFBS comprises a 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: 238, and / or the ATF4 TFBS comprises a 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: 239, and / or the HES1 TFBS comprises a 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: 240, and / or the FOS TFBS comprises a 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: 241, and / or the JUN TFBS comprises a 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: 242, and / or the JUNB TFBS comprises a 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: 243.Embodiment 20 The engineered regulatory element of any one of embodiments 18-19, wherein the at least one additional non-TP63 TFBS comprises a BARX2 TFBS, a NHEH1 TFBS, or both a BARX2 TFBS and a NHEH1 TFBS.Embodiment 21 The engineered regulatory element of embodiment 20, wherein the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5, or more than 5 BARX2 TFBSs.Embodiment 22 The engineered regulatory element of embodiment 20, wherein the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5, or more than 5 NHEH1 TFBSs.Embodiment 23 The engineered regulatory element of any one of the preceding embodiments, wherein the engineered regulatory element is operably linked to a core promoter.Embodiment 24 The engineered regulatory element of embodiment 23, wherein the core promoter comprises a sequence of a promoter selected from: minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB-SCP3 minimal promoter, a DPR containing SCP3 promoter, a minP promoter, a NFkB response element, a CREB response element, a NFAT response element, a SRF response element 1, a SRF response element 2, an API response element, a TCF-EEF response element promoter fusion, a Hypoxia responsive element, a SMAD binding element, a STAT3 binding site, 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 25 The engineered regulatory element of embodiment 23 or 24, wherein the core promoter is selected from a minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB-SCP3 minimal promoter, and a DPR containing SCP3 promoter.Embodiment 26 An engineered regulatory element 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 a nucleotide sequence selected from: SEQ ID NOs: 109-216.Embodiment 27 The engineered regulatory element of embodiment 26, comprising a nucleotide sequence at least 95% identical to a nucleotide sequence selected from: SEQ ID NOs: 109-216.Embodiment 28 The engineered regulatory element of embodiment 27, comprising a nucleotide sequence that is 100% identical to a nucleotide sequence selected from: SEQ ID NOs: 109-216.Embodiment 29 A heterologous construct comprising: a. the engineered regulatory element of any one of embodiments 1-28; and b. a heterologous pay load,Embodiment 30 The heterologous construct of embodiment 29, wherein the engineered regulatory element is operably linked to the heterologous payload.Embodiment 31 The heterologous construct of embodiment 29, wherein the heterologous payload comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding one or more polypeptides.Embodiment 32 The heterologous construct of embodiment 30, wherein the one or more polypeptides comprise at least one effector molecule.Embodiment 33 The heterologous construct of any one of embodiments 30-31, wherein the one or more polypeptides comprise two or more separate polypeptides comprising a first effector molecule, a second effector molecule, optionally a third effector molecule, optionally a fourth effector molecule.Embodiment 34 The heterologous construct of embodiment 32, wherein the polynucleotide comprises E1-L1-E2, optionally wherein the polynucleotide comprises E1-L1-E2-L2-E3-L3-E4, wherein El is a nucleotide sequence encoding the first effector molecule, LI is a first linker molecule, E2 is a nucleotide sequence encoding the second effector molecule, L2 is a second linker molecule, E3 is a nucleotide sequence encoding the third effector molecule, L3 is a third linker molecule, and E4 is a nucleotide sequence encoding the fourth effector molecule.Embodiment 35 The heterologous construct of embodiment 33, wherein LI, L2, L3, and L4 are independently selected from: an internal ribosome entry site (IRES) and one or more nucleotide sequences encoding one or more 2A ribosome skipping elements.Embodiment 36 The heterologous construct of embodiment 34, wherein the linker nucleotide sequence encodes one or more 2A ribosome skipping elements.Embodiment 37 The heterologous construct of embodiment 34 or 35, wherein the one or more 2A ribosome skipping elements comprise elements that are each selected from: P2A, T2A, E2A, and F2A.Embodiment 38 The heterologous construct of any one of embodiments 31-36, wherein the at least one effector molecule or each effector molecule is selected from one or more therapeutic classes, wherein the one or more therapeutic classes are selected from: a chimeric receptor, a cytokine, a chemokine, a homing molecule, a growth factor, apolynucleotide molecule, a co-activation molecule, a tumor microenvironment modifier, a receptor, a ligand, a transcription factor, an antibody, a peptide, and an enzyme.Embodiment 39 The heterologous construct of embodiment 37, wherein the chimeric receptor is a chimeric antigen receptor (CAR).Embodiment 40 The heterologous construct of any one of embodiments 31-38, wherein the at least one effector molecule or each effector molecule is a human-derived effector molecule.Embodiment 41 The heterologous construct of any one of embodiments 31-39, wherein the one or more polypeptides comprise the first effector molecule and the second effector molecule, and wherein: i. the first effector molecule and the second effector molecule are independently selected from a first CAR and a second CAR, or ii. the first effector molecule and the second effector molecule are independently selected from a first CAR and a cytokine, or iii. the first effector molecule and the second effector molecule are independently selected from the first cytokine and a second cytokine, and optionally wherein the first CAR is an activating CAR (aCAR) and the second CAR is an inhibitory CAR (iCAR), optionally wherein the first CAR and / or the second CAR is a bivalent CAR.Embodiment 42 The heterologous construct of any one of embodiments 31-40, wherein the one or more polypeptides comprise the first effector molecule, the second effector molecule, and the third effector molecule, and wherein the first effector molecule, the second effector molecule, and the third effector molecule are independently selected from: i. a first CAR, a second CAR, and a cytokine, or ii. a first CAR, a first cytokine, and a second cytokine.Embodiment 43 The heterologous construct of any one of embodiments 31-41, wherein the one or more polypeptides comprise the first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule, and wherein the first effector molecule, the second effector molecule, the third effector molecule, and thefourth effector molecule are independently selected from: a first CAR, a second CAR, a first cytokine, and a second cytokine.Embodiment 44 A vector comprising the heterologous construct of any one of embodiments 29-42.Embodiment 45 A dual expression vector comprising the heterologous construct of any one of embodiments 29-42 and a second construct comprising an additional payload.Embodiment 46 The vector or dual expression vector of embodiment 43 or 44, wherein the vector or dual expression vector is a viral vector, optionally wherein the viral vector is a retroviral vector.Embodiment 47 An immunoresponsive cell comprising the heterologous construct of any one of embodiments 29-42, the vector of embodiment 43 or 45, or the dual expression vector of embodiment 44 or 45.Embodiment 48 The immunoresponsive cell of embodiment 46, wherein the immunoresponsive cell is selected from: a Natural Killer (NK) cell, 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 B cell, a macrophage, 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 49 The immunoresponsive cell of embodiment 47, wherein the immunoresponsive cell is a NK cell or a T cell.Embodiment 50 The immunoresponsive cell of any one of embodiments 46-48, wherein the immunoresponsive cell expresses an activating immune receptor.Embodiment 51 The immunoresponsive cell of embodiment 49, wherein the activating immune receptor comprises an antigen recognizing receptor.Embodiment 52 The immunoresponsive cell of any one of embodiments 46-50, wherein the immunoresponsive cell is autologous.Embodiment 53 The immunoresponsive cell of any one of embodiments 46-51, wherein the immunoresponsive cell is allogeneic.Embodiment 54 A pharmaceutical composition comprising the vector of embodiment 43 or 45, the dual expression vector of embodiment 44 or 45, or the immunoresponsive cell of any one of embodiments 46-52, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.Embodiment 55 A method of increasing expression of a target gene or a heterologous payload, the method comprising use of the engineered regulatory element of any one of embodiments 1-28, the vector of embodiment 43 or 45, or the dual expression vector of embodiment 44 or 45 to increase expression of the target gene.Embodiment 56 The method of embodiment 54, wherein the target gene is an immunomodulatory gene.Embodiment 57 A method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of the vector of embodiment 43 or 45, the dual expression vector of embodiment 44 or 45, the immunoresponsive cell of any one of embodiments 46-52, or the pharmaceutical composition of embodiment 53.Embodiment 58 A kit for treating and / or preventing a disease or disorder, comprising the immunoresponsive cell of any one of embodiments 46-52 or the pharmaceutical composition of embodiment 53.Embodiment 59 The kit of embodiment 57, wherein the disease or disorder comprises a tumor.Embodiment 60 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 subject.EXAMPLES

[0218] The present disclosure will be further illustrated in the following Examples which are given for illustration purposes only and are not intended to limit the disclosure in any way.Example 1: Construction of multicistronic engineered nucleic acids for expressing multiple payloads

[0219] The following multicistronic pay load constructs were designed to express multiple payload components from a single open reading frame. Such payload constructs were used for testing performance of various engineered regulatory element designs that include engineered enhancer sequences operably linked to a core promoter to generate engineered promoter candidates, as described in the following Examples. See FIG. 1A for a schematic of exemplary engineered promoter candidate designs as well as the multicistronic pay load constructs.

[0220] Pay load #, design, and total length (nt and aa) are shown in the Table A below.Table A - Multicistronic payload constructs featuresExample 2: Nomination and screening of first generation strong enhancers in NK cells

[0221] Candidate enhancer sequences were nominated based on bioinformatic analysis of inhouse pooled screening data in NK cells from multiple donors with an MPRA library. Candidate transcription factors upregulated in NK cells were bioinformatically identified, and their corresponding transcription factor binding sites (TFBSs) were arranged in various combinatorial arrangements to create candidate enhancer sequences. Candidate enhancer sequences were operably linked to the minCMV core promoter sequence to create candidate engineered promoters. See FIG. IB.

[0222] Two methods were used to interpret promoter strengths and nominate top engineered promoter candidates: 1) RNA to DNA ratio with top candidates selected based on flat RNA / DNA ratio cut-offs and 2) RNA only methods with top candidates selected based on the highest strength measured as average normalized RNA counts.

[0223] Promoter-payload constructs were designed by linking candidate engineered promoters to Pay load 1. aCAR and iCAR pay loads contain peptide tags for downstream detection purposes. See also FIGS. 1A-1B.

[0224] Promoter activity of the candidate engineered promoters was evaluated in a clonal manner. Briefly, promoter-payload constructs were packaged into self-inactivating (SIN) y-retroviruses (sinvec virus) and transduced into NK cells (D = 0). Briefly, sinvec viruses were generated through co-transfection of test constructs and envelope plasmids into GP2-293 cells, then collected and concentrated. Post-transduction, NK cells were allowed to rest in culture, and surface expression of aCAR and iCAR proteins were detected by flow cytometry at one or more later timepoints, typically at D = 3 to 7, using fluorescent conjugated antibodies that recognized the specific peptide tags. Where applicable, integrated viral copy number (VCN), which is a proxy for copy of integrated transgene, was also assessed from these transduced NK cells. Promoter strength was assessed by measuring pay load expression relative to SFFV promoter as a control promoter.

[0225] Surface expression of aCAR and iCAR was used as an indicator of promoter strength. These expression data were extracted from the raw flow cytometry data gated on live single NK cells. Metrics included 1) total %aCAR+ of live NK cell populations, 2) total %iCAR+ of live NK cell populations, 3) %aCAR+iCAR (%++) of live NK cell populations, 4) mean or median fluorescent intensity (MFI) of aCAR from total %aCAR+ and %++ populations, 5) MFI of iCAR from total %iCAR+ and %++ populations. Promoter strength was primarily quantified as aCAR and iCAR MFI with or without normalization to VCN, and secondarily as extent of %transduced (i.e., %CAR+) populations. VCN normalized MFI values were calculated as follows:CAR MFI x %CAR+population Normalized MFI = -VCN x 100

[0226] FIG. 2 shows performance metrics of selected 1stgeneration engineered promoter candidates based on transduction efficiency. FIG. 3 shows flow cytometry scatterplots from exemplary selected engineered promoter constructs SB10698, 10977, 10961, 109457, and 10944. FIGS. 4A-4B show graphs of performance metrics of selected 1stgeneration engineered promoter candidates based on surface CAR protein expression measured on flow cytometer and quantified as fluorescent intensity values. Selected engineered promoter constructs based on, e.g., higher performance as compared to SFFV in at least one of the metrics above included: SB10968, SB10977, SB10961, SB10941, SB10945, SB10974, SB10978, SB10980, SB10981, SB 10966, SB 10963, SB 10956, SB 10953, SB 10944, and SB 10947. In particular, SB 10968, SB 10977, SB 10961, SB 10947 and SB 10944 exhibited higher %CAR+ population than the SFFV control construct (SB 10984). Enhancer sequences and full promoter sequences from these engineered promoter constructs are shown in Tables 1 and 2, respectively. See Table B below for design details of the first generation promoters assessed. Sequences of exemplary TFBSs are shown in Table 3.Table 1 - Enhancer SequencesTable 2 - Full Promoter SequencesTable B - First generation promoter configurationsExample 3: Design and screening of cell promoters with combinatorial TFBS arrays

[0227] To further engineer stronger engineered promoters, new promoters were designed based on combinatorial arrangements of selective TFBS from Example 1 linked to different core promoter sequences, such as minCMV, SV40, B2M and NKG7 promoter sequences. The new engineered promoters were subsequently linked to a multi-component payload composed of crIL-15, aCAR and iCAR linked by 2A peptides (Pay load 2). aCAR and iCAR pay loads contain peptide tags for downstream detection purposes.

[0228] Engineered promoters were screened in a clonal manner as described in Example 2, through transduction of NK cells with packaged sinvec virus and flow cytometry as downstream readout, but the entire process was handled on an in-house custom-built automated liquid handler (ALH) tailored for high-throughput screening. Flow cytometry assessment on surface aCAR and iCAR expression was performed on D = 6, and SV40 promoter was selected as a control promoter for this payload context.

[0229] Surface expression of aCAR and iCAR quantified as %CAR+ population and CAR MFI with and without VCN normalization were used as proxy for promoter strength. For each expression metric, the test promoters were directly compared with the SV40 control promoter (see below for details), and strong engineered promoter candidates were nominated if they exhibited fold SV40 values of over 1 for both aCAR and iCAR MFI metrics and fold SV40 of at least 0.8 for %CAR+ population metrics.

[0230] Given that expression of the multicomponent pay load (Pay load 2) was driven by a single promoter, and we experimentally observed strong correlations between the MFI expression metrics, iCAR MFIs of iCAR+ and aCAR+iCAR+ (++) NK cell populations were selected as the primary metrics to rank the promoters by strength for the purpose of top promoter candidate nomination.

[0231] Many of the second-generation promoters outperformed SV40 on at least one expression metric (e.g., % CAR+ populations, non-VCN normalized MFI), as seen in FIG. 5 andFIGS. 6A-6C depicting the percentage of new promoter constructs pairing enhancers with the various core promoters outperforming the SV40 promoter as benchmark.

[0232] A number of engineered promoters were selected as the top engineered promoter candidates and are shown in Table C. Among the top engineered promoter candidates, all contained TP63 TFBSs (2, 3, or 5 TP63 TFBSs). Further analysis of the ALH screening results indicated that a majority of the promoter designs having > 5 TP63 TFBSs or 2 TP63 TFBSs performed favorably as compared to the SV40 benchmark, as seen in FIG. 7A. For example, five promoter candidates having ten TP63 TFBSs linked to various core promoters (e.g., SB 12295, SB 12324, SB 12353, SB 12382, SB 12440) improved payload expression as compared to SV40 (data not shown).

[0233] In addition, many of the top engineered promoter candidates (e.g., SB 12305, SB 12310- SB12315,SB12319, SB12372, SB12343, SB12342, SB12344, SB12370, SB12371, SB12300, SB 12373, SB 12304, SB 12309, SB 12496, SB 12377, SB 12368, SB 12339, and SB 12348) were found to contain BARX2, NHLH1, and / or TP63 TFBS, seen in FIG. 7B, suggesting that BARX2, NHLH1 and TP63 TFBS containing enhancers linked to minCMV were among the strongest selected engineered promoter candidates. See Table C below for further details on the design of the top promoters from this Example. Relevant TFBS sequences are shown in Table 3.Table C - Top combinatorial TFBS array promotersTable 3 - TP63 and other non-TP63 sequences

[0234] A performance comparison between the 1st generation promoters described in Example 2 and the 2nd generation promoters selected in this Example are shown in FIGS. 8A-8C (left, middle, and right). The top second-generation promoters exhibited 2-4X pay load expression compared to the SV40 promoter control (SB 10985).Example 4: Design and pooled MPRA screening of cell promoters with combinatorial TFBS arrays

[0235] A separate set of 16,000 synthetic engineered promoters were designed and generated computationally based on combinatorial array of TFBS paired with a core promoter (minCMV). TFBS were selected based on strong promoters from Example 1 and considerations from the literature. These promoters were linked upstream of 12 base pairs (bp) of DNA barcode followed by a multi-component payload as the readout. In total, two libraries were generated with different payloads. SFFV, SV40 and ETR were included in this library as viral control promoters. The length of the control viral promoters was -300-400 bp, while the length of the MPRA library promoters was -200 bp.Library 1 (SB11858): CEA-targeting aCAR, VSIG2-targeting iCAR (Payload 3) Library 2 (SB11859): CEA-targeting aCAR, crIL-15 (Payload 4)

[0236] For each of the libraries, each component of the multi-component pay load was separated by a 2A peptide. See Example 1 for details of the Pay load designs. Primer binding sites were included for downstream next generation sequencing (NGS) applications.

[0237] The synthetic engineered promoters were synthesized as pooled oligos and cloned into plasmids with respective payloads to generate library 1 and 2, and subsequently screened in form of massively parallel reporter assay (MPRA) screens, as seen in the schematic of FIG. 9. Briefly, the pooled plasmids were packaged into sinvec viruses and transduced into NK cells in two biological replicates (D = 0). On D = 3, NK cells were harvested, and RNA and DNA samples were extracted, analyzed by NGS, and assessed for promoter strength.

[0238] Promoter scores were calculated for each MPRA library member separately based on average promoter strength and variations between biological replicate samples.

[0239] Top engineered promoter candidates were selected based on high promoter score from both MPRA libraries for downstream clonal validation. Additional untested engineered promoter candidates with strong predicted score from the proprietary analytical pipelines were also selected for evaluation.

[0240] Results are shown in FIG. 10 (top and bottom). The larger filled circles correspond to the viral promoter controls. As shown, several candidates have promoter strengths comparable to the viral control promoters despite being significantly smaller. Statistical analyses nominated strong TFBS, which was used to inform subsequent machine learning-derived and rationally designed engineered regulatory elements.Example 5: Clonal validation of MPRA derived 2ndgeneration promoters

[0241] Engineered promoter candidates from Example 4 were linked to an internal multicomponent payload composed of IL21, crIL-15, aCAR and iCAR linked by 2A peptides (Payload 5). aCAR and iCAR payloads contained peptide tags for downstream detection purposes. Expression of these payloads were used to evaluate promoter strength.

[0242] Similar to Example 3, these promoter-payload constructs were packaged into sinvec virus and transduced into NK cells utilizing the ALH, with flow cytometry on D = 6 as the downstream readout for promoter strength. SV40 promoter was selected as a control promoter for this pay load context. Promoter strength was measured as surface expression of aCAR and iCAR quantified as %CAR+ population and CAR MFI without VCN normalization.

[0243] Top engineered promoter candidates were selected based on higher aCAR and iCAR MFI of CAR+ population compared to SV40 promoter (i.e., Fold SV40 values of over 1). About half of the MRPA engineered promoter candidates and a majority of machine learning-derived and rationally designed engineered promoter candidates outperformed the SV40 promoter (data not shown). Exemplary engineered regulatory element sequences, including engineered enhancer sequences and engineered promoter sequences of top engineered promoter candidates from the MPRA library screen, are in Table 1 and Table 2.Example 6: Validation and functional characterization of engineered promoter candidates for expressing multiple CARs and armoring cytokines from a quadcistronic construct

[0244] Promoter strength of the top engineered promoter candidates from the previous Examples were re-evaluated in a larger payload system and with functional characterization of their associated CAR-NK cells. Specifically, they were linked to upstream of a quadcistronicconstruct for expressing IL21, crIL-15, a CEA-targeting aCAR, and a VSIG2-targeting iCAR linked by 2A peptides (Pay load 5). Details of Pay load 5 design are provided in Example 1.

[0245] These promoter-payload constructs were screened in a clonal manner similar to example 1. Briefly, the constructs were packaged into sinvec virus and used to transduce NK cells at three different viral doses (by pl volume). Post-transduction the CAR-NK cells were cultured and stained on D = 7, 14 and 21 with fluorescent conjugated antibodies to determine surface expression of aCAR, iCAR and IL 15 by flow cytometry. Supernatant of CAR-NK cells were collected for Luminex assay to determine the levels of secreted IL- 15 and IL-21 payloads. VCN values for the transduced NK cells were quantified by qPCR. SV40 promoter was selected as a control promoter for this payload context.

[0246] Overall promoter strength was determined by surface expression of aCAR, iCAR, and crIL-15 quantified as MEI values of live NK cells, and secreted levels of IL-15 and IL-21. Secreted IL- 15 and IL-21 was quantified as pg / mL normalized by cell density and back- calculated from Luminex standards.

[0247] EIG. 11 depicts a graph of Day 7 and Day 14 iCAR MEI (of iCAR+ cells) for unengineered NK cells (NV control), SB 12515 transduced NK cells (SV40), SB 12894 transduced NK cells (B2M promoter), SB 12895 transduced NK cells (NKG7 promoter), and SB 12896 transduced cells (4xTP63 TEBS-minCMV). As shown, SB 12896 drove higher iCAR expression as compared to SV40, B2M, and NKG7 promoters, at all time points and all viral doses.

[0248] EIG. 12 depicts exemplary flow plots from the NV (left), SB 12515 (middle), and SB 12896 (right) transduced cells at 25 pl volume on Day 7, as well as normalized VCN of the transduced cells. As shown, SB 12896 transduced cells (right) exhibited higher iCAR+, aCAR+, and aCAR iCAR ++ % populations at a lower VCN as compared SB 12515 transduced cells. As shown in EIGS. 13A (middle and right) and 13B (top and bottom), transduction efficiency and both aCAR and iCAR payload expression was enhanced in SB 12896 transduced cells at all viral doses and across NK cell donors, as compared to SB 12515 transduced cells.

[0249] EIG. 14A (left and right), EIG. 14B (left and right) and EIG. 14C (top and bottom) ( depict surface expression of IL15, as well as secreted IL15 and IL21 in SB 12515 vs SB 12896 transduced cells. As shown, SB 12896 drove higher cytokine expression levels as compared to SB 12515, at all viral doses and normalized VCN.

[0250] EIGS. 15A-15D shows promoter strength metrics for SB12896 and SB12515 across NK cells from different donors. As shown, SB 12896 drove higher transduction efficiency (FIG. 15B), and aCAR and iCAR payload expression across all NK donors as compared to SB 12515 (FIGS. 15C-15D).

[0251] In addition, promoters comprising 4X TP63 and 5XTP63 outperformed SV40 across multiple experiments, and with various multicistronic payload constructs. See Table D below. Details of Pay load 2 and Pay load 5 design are provided in Example 1.Table D - TP63-based Promoter Assessment

[0252] Further analysis of the identified TP63 TFBS sites was performed. TP63 generally prefers to bind to DNA as a dimer, and without wishing to be bound by theory, its consensus binding motif is approximately 20 bp (a “full” response element) that are typically made up of two "half-sites" of 10 bp each. TP63 dimers can bind to the half-site well, and a total of four TP63 (i.e., tetramer) can bind to a full response element. Two half sites can be separated by no linkers, or linkers of different lengths (such as 2 and 10 bp linkers) and still have TP63 binding. Two half sites can also be on the same or different DNA strands (e.g., half site in the forward orientation and the other half in the reverse orientation). Accordingly, the “full” TP63 TFBS sites identified were assessed for TP63 “half-site” sequences and motifs. The results of the analysis identifying half-sites and half-site motifs are shown in Table 3.

[0253] Furthermore, killing function of NK cells engineered with these promoter-payload constructs were evaluated in a mixed target serial killing assay. For each round of killing, two engineered DED- 1 (colorectal adenocarcinoma) cell lines were mixed at 1 : 1 ratio and pre -plated in flat-bottom tissue culture plates. The TA+PA- DED-1 target cell line was engineered to express: GFP reporter protein, a CEA target antigen that is recognized by the aCAR (TA+), and an off-target protective antigen Her2 that is not recognized by the iCAR (PA-). The TA+PA+ DED-1 target cell line was engineered to express: mCherry reporter, the same CEA targetantigen recognized by the aCAR (TA+), and on-target protective antigen VSIG2 recognized by the iCAR (PA+).

[0254] Effector cells (unengineered NK cells (NV), SB 12515 engineered NK cells (expressing Payload 5 from the SV40 promoter), or Promoter Candidate - Payload 5 engineered NK cells (e.g., SB 12896 engineered NK cells) were added to pre-plated DLD-1 target cells at E:T ratio of 1:4. (For clarity, E:T ratio refers to the ratio of Effector Cells (NK cells) to the pre-plated DLD-1 target cells.) After approximately 45 hours post co-culture, NK cells in suspension were transferred to new plated target cells for next round of killing, for total of three rounds of killing. Images of the assay wells were automatically taken at four-hour intervals on the Incucyte.Killing of the two engineered DLD- 1 cell lines as were quantified as counts of reporter protein positive cells over time. See FIG. 16A, depicting a schematic of engineered NK cells expressing Payload 5 and the DLD-1 target cell lines.

[0255] Two functional metrics were evaluated: 1) aCAR and cytokine-driven cytotoxicity against TA+PA- target cells, and 2) iCAR-mediated protection of TA+PA+ target cells over TA+PA- cells. These metrics were calculated as below:GFP counts = TA+PA target cell count mCherry counts = TA+PA+target cell countGFP countsTNormalized GFP countsT= -GFP countsTQ100mCherry counts iCAR preference = - — — - — — - - x 100(mCherry counts + GFP counts)ICAR functionT= ICAR preferenceT— ICAR preferenceTQ

[0256] In the formula above, “T” refers to a specific timepoint in the killing assay and “TO” refers to the first timepoint at each round of the killing assay. “Test” refers to CAR-NK cell condition in killing assay from a particular promoter-payload construct, and "No NK” refers to condition with target cells only.

[0257] After the last timepoint in the killing assay, cells in suspension were collected, costained with fluorescent conjugated anti-CD45 antibody to distinguish NK cells from target cells and viability dye to distinguish live from dead cells and ran on flow cytometer to quantify the number of surviving NK cells. Number of surviving NK cells with candidate promotersdescribed in previous Examples were compared to the number of surviving NK cells with SV40 promoter alone (SB 12515).

[0258] The beneficial impact of the engineered promoters on CAR-NK functionality would be indicated by higher aCAR killing function and iCAR protective function, as compared to CAR- NK cells with SV40 promoter-driven payloads at any timepoint in the killing assay, particularly in the last round of killing.

[0259] Results of the killing assay comparing the engineered promoter of SB 12896 to SV40 are shown in FIGS. 16B (depicting killing of TA+PA- cells) and 16C (depicted killing of TA+PA+ cells).

[0260] As shown in FIG. 16C, GFP counts increased for DED-1 TA+PA- cells in the absence of NK cells and also with unengineered NK cells (NV control). Intrinsic (non-aCAR mediated) tumor cell killing was apparent for the NV control in the first round, diminished in the second round, and largely absent from the third round, indicating lack of NK persistence over time. DED-1 TA+PA- cell killing was apparent across the three rounds of killing for the SB 12515- engineered NK cells (expressing the multi-component pay load from the SV40 promoter).SB 12896 engineered NK cells (expressing the same multi-component payload from the engineered promoter) exhibited comparable strong killing in the first round and increased killing in the second and third rounds, as compared to the SB 12515 comparator, demonstrating the engineered promoters improved CAR-NK functionality.

[0261] As shown in FIG. 16F, which shows iCAR function as determined by the formula above, both the No NK and NV controls exhibit no iCAR function. While SB 12515 and SB 12896 exhibited comparable iCAR function over the first two rounds of killing, by the third round, SB 12896 exhibited stronger iCAR function as compared to SB 12515.

[0262] As shown in FIG. 16G, there were greater numbers of surviving SB 12896 engineered cells as compared to SB 12515 engineered cells by the last timepoint of the serial killing assay, indicating improved NK persistence in SB 12896 engineered NK cells as compared to SB 12515 engineered NK cells, demonstrating the engineered promoters improved CAR-NK functionality.Example 7: Validation and functional characterization of 2nd gen promoters

[0263] Engineered promoter candidates described in the previous Examples, and additional engineered promoter candidates including engineered regulatory element variants thereof, were evaluated in another experiment. Briefly, these engineered regulatory element variants were designed by replacing selective TFBS or minimal promoters with ones that were expected to be even stronger based on the learnings from the previous Examples. These engineered promoter candidates were linked upstream of the internal multi-component pay load composed of IL21,crIL-15, aCAR and iCAR linked by 2A peptides (Payload 5). Details of Payload 5 design are described in Example 1.

[0264] These promoter-payload constructs were screened in a similar fashion as Example 6. CAR-NK cells were stained on D = 7, 14 and 21 with fluorescent conjugated antibodies to determine surface expression of aCAR, iCAR and IL 15 by flow cytometry. SV40 promoter was selected as a control promoter for this payload context.

[0265] Similar to Example 6, mixed target serial killing assay was used to evaluate functional implications of high payload expression driven by these engineered promoter candidates. Two rounds of killing at E:T ratio of 1:2 was performed. Images of the assay wells were automatically taken at four-hour intervals on the Incucyte and target cell were quantified as counts of reporter protein positive cells over time. aCAR and iCAR functions were calculated the same way as Example 8 and plotted at the 80-hour timepoint at second round of killing.

[0266] As shown in FIG. 17A (top, middle, and bottom) and 17B (left and right), several engineered NK promoter candidates outperformed SV40 (SB 12515) in payload expression (FIG. 17A) and killing of TA+PA- target cells while offering stronger protection of TA+PA+ “healthy” cells (FIG. 17B). Exemplary engineered promoter candidates outperforming SV40 included: SB 12896, SB 13505, SB 13508, SB 13500, SB 13498, SB 13495, SB 13497, SB 13499, SB 13501, and SB 13491. Enhancer and promoter sequences of these constructs are included in Tables 1 and 2.Example 8: ALH screening of top promoters in NK and T cells

[0267] Engineered T cell-based therapeutics may also benefit from improved promoters for driving expression of multicomponent pay loads. The following experiment was performed to assess engineered promoter candidates that included various engineered regulatory elements from previous Examples in T cells as well as NK cells.

[0268] Top engineered promoter candidates from previous Examples were cloned upstream of the internal multi-component payload composed of IL21, crIL-15, aCAR and iCAR linked by 2A peptides (Payload 5). SV40 promoter was selected as a control promoter for this payload context.

[0269] These promoters were screened clonally in automated fashion on the custom-built ALH, from viral production through NK and T cell transduction and staining for flow cytometry on D = 6. Surface aCAR and iCAR expression were quantified, and promoter strength was presented as iCAR MFI of the %CAR+ cell population. Primary T cells isolated from a donor were thawed and stimulated with Dynabeads for 24 hours prior to transduction. After transduction, the expanded T cells were subsequently cultured in T cell media containing IL-2.

[0270] NK cell results are shown in FIG. 18A (left and right). T cell results are shown in FIG. 18B (left and right). Correlation between NK and T cell results are shown in FIG. 18C (top and bottom). As shown, compared to the SV40 promoter (SB 12515), almost all of the tested promoters were stronger in both NK cells and T cells. Notably, many promoters (e.g., SB #s 13500, 13491, 13213, 13489, 13507, 13488, 13501, 13508, 13498, 13509, 13502, 13495, 13499, 13490, 13497, 13487, 13235, 13233, 13234, 13285) were stronger than SB12896, the 1st generation promoter assessed in Example 6. FIG. 18D depicts NK and T cell results in a single graph. As shown, many of the tested promoters exhibited strong activity in both NK and T cells, and several (e.g., SB #s 13500, 13491, 13489, 13507, 13488, 13501, 13508, 13498, 13509, 13502, 13495, 13499, 13490, 13497, 13487, 13235, 13233, 13234, 13285) exhibited increased activity in T cells as compared to NK cells. FIG. 18E (top and bottom) depicts flow cytometry results for the SFFV control vs. SB 13498. As shown, SB13498-transduced NK cells exhibited much higher aCAR / iCAR double positive population (51.2%) as compared to the SFFV control (16.6%).Other Sequences

[0271] Sequences referred to throughout the disclosure include the following exemplary sequences:Table 4 - Exemplary SequencesINCORPORATION BY REFERENCE

[0272] The entire disclosure of each of the patent and scientific documents referred to herein is incorporated by reference for all purposes.EQUIVALENTS

[0273] The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMSWhat is claimed is:

1. An engineered regulatory element 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 a nucleotide sequence selected from: SEQ ID NOs: 1-108.

2. An engineered regulatory element comprising one or more TP63 transcription factor binding sites (TFBSs), optionally wherein the one or more TP63 TFBSs comprise a 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: SEQ ID NOs 217-220, and reverse complements thereof.

3. The engineered regulatory element of claim 1 or 2, wherein any of the one or more TP63 TFBSs comprises a TP63 TFBS half-site motif, wherein the TP63 TFBS half-site motif comprises a 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: SEQ ID Nos 221-233, and reverse complements thereof, optionally wherein the TP63 TFBS comprises two TP63 TFBS half-site motifs, optionally, wherein the two TP63 TFBS half- site motifs are operatively linked by a nucleic acid linker, optionally wherein the linker is between 1-10 base pairs, optionally wherein the two TP63 TFBS half-site motifs comprise (a) a first half-site motif 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: SEQ ID Nos 221-233; and (b) a second half-site motif 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 reverse complement of a nucleotide sequence selected from: SEQ ID Nos 221-233.

4. The engineered regulatory element of any one of claims 1-3, comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 TP63 TFBSs, optionally wherein the engineered regulatory element comprises 1-500, 1- 100, 1-50, 2-20, or 2-10 TP63 TFBSs.

5. The engineered regulatory element of any one of claims 1-4, wherein the engineered regulatory element further comprises at least one additional non-TP63 TFBS.

6. An engineered regulatory element comprising one or more TP63 transcription factor binding sites (TFBSs) and comprising at least one additional non-TP63 TFBS.

7. The engineered regulatory element of claim 5 or 6, wherein the at least one additional non-TP63 TFBS is selected from: a BARX2 TFBS, a NHLH1 TFBS, a TP73 TFBS, a HOXCIO TFBS, a NFE2 TFBS, a ATF4 TFBS, a HES1 TFBS, a FOS TFBS, a JUN TFBS, and a JUNB TFBS, optionally wherein:(a) the BARX2 TFBS comprises a 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, and / or(b) the NHLH1 TFBS comprises a 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: 235, and / or(c) the TP73 TFBS comprises a 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: 236, and / or(d) the HOXCIO TFBS comprises a 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: 237, and / or(e) the NFE2 TFBS comprises a 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: 238, and / or(f) the ATF4 TFBS comprises a 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: 239, and / or(g) the HES1 TFBS comprises a 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: 240, and / or(h) the FOS TFBS comprises a 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: 241, and / or(i) the JUN TFBS comprises a 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: 242, and / or(j) the JUNB TFBS comprises a 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: 243;optionally wherein the at least one additional non-TP63 TFBS comprises a BARX2 TFBS, a NHLH1 TFBS, or both a BARX2 TFBS and a NHLH1 TFBS; optionally wherein the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5, or more than 5 BARX2 TFBSs, optionally wherein the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5, or more than 5 NHLH1 TFBSs.

8. The engineered regulatory element of any one of the preceding claims, wherein the engineered regulatory element is operably linked to a core promoter, optionally wherein the core promoter comprises a sequence of a promoter selected from: minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB-SCP3 minimal promoter, a DPR containing SCP3 promoter, a minP promoter, a NFkB response element, a CREB response element, a NFAT response element, a SRF response element 1, a SRF response element 2, an API response element, a TCF-LEF response element promoter fusion, a Hypoxia responsive element, a SMAD binding element, a STAT3 binding site, 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 core promoter is selected from a minCMV minimal promoter, an SV40 promoter, a B2M promoter, an SCP3 minimal promoter, a YB-SCP3 minimal promoter, and a DPR containing SCP3 promoter.

9. An engineered regulatory element 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 a nucleotide sequence selected from: SEQ ID NOs: 109-216.

10. A heterologous construct comprising:(a) the engineered regulatory element of any one of claims 1-9; and(b) a heterologous payload, wherein the engineered regulatory element is operably linked to the heterologous payload.

11. The heterologous construct of claim 10, wherein the heterologous payload comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding one or more polypeptides, optionally wherein the one or more polypeptides comprise a) at least one effector molecule or b) two or more separate polypeptides comprising a first effector molecule, a second effector molecule, optionally a third effector molecule, optionally afourth effector molecule, optionally wherein the at least one effector molecule or each effector molecule is selected from one or more therapeutic classes, wherein the one or more therapeutic classes are selected from: a chimeric receptor, 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, optionally wherein the chimeric receptor is a chimeric antigen receptor (CAR).

12. The heterologous construct of claim 11, wherein the polynucleotide comprises El-Ll- E2, optionally wherein the polynucleotide comprises E1-L1-E2-L2-E3-L3-E4, wherein El is a nucleotide sequence encoding the first effector molecule, LI is a first linker molecule, E2 is a nucleotide sequence encoding the second effector molecule, L2 is a second linker molecule, E3 is a nucleotide sequence encoding the third effector molecule, L3 is a third linker molecule, and E4 is a nucleotide sequence encoding the fourth effector molecule, optionally wherein LI, L2, L3, and L4 are independently selected from: an internal ribosome entry site (IRES) and one or more nucleotide sequences encoding one or more 2A ribosome skipping elements, 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: P2A, T2A, E2A, and F2A.

13. The heterologous construct of claim 10 or 11, wherein the one or more polypeptides comprise: a) the first effector molecule and the second effector molecule, and wherein: i) the first effector molecule and the second effector molecule are independently selected from a first CAR and a second CAR, or ii) the first effector molecule and the second effector molecule are independently selected from a first CAR and a cytokine, or iii) the first effector molecule and the second effector molecule are independently selected from the first cytokine and a second cytokine, and optionally wherein the first CAR is an activating CAR (aCAR) and the second CAR is an inhibitory CAR (iCAR), optionally wherein the first CAR and / or the second CAR is a bivalent CAR; orb) the first effector molecule, the second effector molecule, and the third effector molecule, and wherein the first effector molecule, the second effector molecule, and the third effector molecule are independently selected from: i) a first CAR, a second CAR, and a cytokine, or ii) a first CAR, a first cytokine, and a second cytokine; or c) the first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule, and wherein the first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule are independently selected from: a first CAR, a second CAR, a first cytokine, and a second cytokine.

14. A vector comprising the heterologous construct of any one of claims 10-13, optionally wherein the vector is a viral vector, optionally wherein the viral vector is a retroviral vector.

15. A dual expression vector comprising the heterologous construct of any one of claims 10- 13 and a second construct comprising an additional pay load, optionally wherein the dual expression vector is a retroviral vector.

16. An immunoresponsive cell comprising the heterologous construct of any one of claims 10-13, the vector of claim 14, or the dual expression vector of claim 15, optionally wherein the immunoresponsive cell is selected from: a Natural Killer (NK) cell, 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 B cell, a macrophage, 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 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.

17. A pharmaceutical composition comprising the vector of claim 14, the dual expression vector of claim 15, or the immunoresponsive cell of claim 16, and a pharmaceutically acceptable carrier, pharmaceutically acceptable excipient, or a combination thereof.

18. A method of treating a subject in need thereof, the method comprising administering a therapeutically effective dose of the vector of claim 14, the dual expression vector of claim 15, the immunoresponsive cell of claim 16, or the pharmaceutical composition of claim 17.

19. A kit for treating and / or preventing a disease or disorder, comprising the immunoresponsive cell of claim 16 or the pharmaceutical composition of claim 17, optionally wherein the disease or disorder comprises a tumor, optionally 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.

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