Engineered regulatory elements

Abstract

The present disclosure provides, inter alia, methods and compositions comprising engineered nucleic acids, such as engineered regulatory elements, which allow for improved transcriptional activity of operably linked polynucleotides in cells.

Description

Cross-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 incorporated herein by reference in their entirety.

[0002] sequence list This application contains a sequence list, which is incorporated in its entirety by reference. An electronic copy was created on October 9, 2024, named 70012_SeqListing.xml, and is 272,758 bytes in size. Background Technology

[0003] Gene regulatory elements such as promoters and enhancers can possess specific activities and function differently in various environments, such as different cell types. Such regulatory elements are often well-suited for use in therapies, such as cell and gene therapies, to treat diseases that benefit from the expression of a specific gene and / or therapeutic payload. Therefore, regulatory elements used to regulate the expression of therapeutic payloads can ultimately affect the overall functionality of engineered cells or vectors used in gene therapy. However, the issue of transcriptional strength remains with known regulatory elements commonly used to drive payload expression. For example, engineered cell therapy applications may require low integrated transgene copy numbers for regulatory and safety reasons; however, known promoters may not be able to drive sufficient therapeutic payload expression in engineered cell populations at the required copy numbers, particularly in cases of large and / or polycistronic payloads. Therefore, there is a need to identify and develop regulatory elements that can effectively drive payload expression, particularly for therapeutic purposes. Summary of the Invention

[0004] This document provides engineered regulatory elements comprising a nucleotide sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-108. In some aspects, the nucleotide sequence is at least 95% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-108. In some aspects, the nucleotide sequence is 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO: 1-108.

[0005] This document also provides engineered regulatory elements comprising one or more TP63 transcription factor binding sites (TFBS). In some aspects, the one or more TP63 TFBS comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NO 217-220 and its inverse complementary sequence. In some aspects, the one or more TP63 TFBS comprises a sequence that is at least 95% identical to a nucleotide sequence selected from SEQ ID NO 217-220 and its inverse complementary sequence. In some aspects, the one or more TP63 TFBS is selected from SEQ ID NO 217-220 and its inverse complementary sequence. In some aspects, any one of the one or more TP63 TFBS contains a TP63 TFBS half-site motif, wherein the TP63 TFBS half-site motif contains a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NO 221-233 and its reverse complementary sequence. In some aspects, the TP63 TFBS half-site motif contains a sequence that is at least 95% identical to a nucleotide sequence selected from SEQ ID NO 221-233 and its reverse complementary sequence. In some aspects, the TP63 TFBS half-site motif is selected from SEQ ID NO 221-233 and its reverse complementary sequence. In some aspects, the TP63 TFBS contains two TP63 TFBS half-site motifs. In some aspects, the two TP63 TFBS hemisite motifs are operatively linked by a nucleic acid adapter, optionally wherein the adapter is between 1 and 10 base pairs. In some aspects, the two TP63 TFBS hemisite motifs comprise (a) a first hemisite motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO 221-233; and (b) a second hemisite motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an inverse complementary sequence selected from the group consisting of SEQ ID NO 221-233.

[0006] In some aspects, engineered control elements comprise 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 TFBS. In some aspects, engineered control elements comprise at least two TP63 TFBS.

[0007] The engineered control element as described in any one of claims 4 to 13, wherein the engineered control element comprises 1 to 500, 1 to 100, 1 to 50, 2 to 20, or 2 to 10 TP63 TFBSs. In some aspects, the engineered control element further comprises at least one additional non-TP63 TFBS. In some aspects, the at least one additional non-TP63 TFBS is selected from: BARX2 TFBS, NHLH1 TFBS, TP73 TFBS, HOXC10 TFBS, NFE2 TFBS, ATF4 TFBS, HES1 TFBS, FOS TFBS, JUNTFBS, and JUNB TFBS.In some respects, BARX2 TFBS contains 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 sequences to SEQ ID NO: 234, and / or NHLH1 TFBS contains 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 sequences to SEQ ID NO: 235, and / or TP73 TFBS contains 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 sequences to SEQ ID NO: 236, and / or HOXC10 TFBS contains sequences identical to SEQ ID NO: 234. SEQ ID NO: 237 contains 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 sequences to SEQ ID NO: 238, and / or ATF4 TFBS contains 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 sequences to SEQ ID NO: 239, and / or HES1 TFBS contains 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 sequences to SEQ ID NO: 240, and / or FOS TFBS contains 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% of the sequence identical to SEQ ID NO: 241, and / or JUN TFBS contains 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% of the sequence identical to SEQ ID NO: 242, and / or JUNB TFBS contains 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% of the sequence identical to SEQ ID NO: 243.

[0008] In some aspects, the at least one additional non-TP63 TFBS includes BARX2 TFBS, NHLH1 TFBS, or both BARX2 TFBS and NHLH1 TFBS. In some aspects, the at least one additional non-TP63 TFBS includes about 2, 3, 4, 5, or more than 5 BARX2 TFBS. In some aspects, the at least one additional non-TP63 TFBS includes about 2, 3, 4, 5, or more than 5 NHLH1 TFBS.

[0009] In some respects, engineered regulatory elements are operatively linked to the core promoter. In some respects, the core promoter contains sequences selected from the following promoters: minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, SCP3 promoter containing DPR, minP promoter, NFkB responsive element, CREB responsive element, NFAT responsive element, SRF responsive element 1, SRF responsive element 2, API responsive element, TCF-LEF responsive element promoter fusion, hypoxia responsive element, SMAD binding element, STAT3 binding site, YB TATA, minTK, inducer molecule responsive promoter, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaV1, hCAGG, hEFlaV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb, and their tandem repeat sequences. In some respects, the core promoter is selected from the minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, and SCP3 promoter containing DPR.

[0010] This document also provides engineered regulatory elements comprising 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 nucleotide sequences selected from the group consisting of SEQ ID NO: 109-216. In some aspects, the engineered regulatory element comprises at least 95% identical nucleotide sequences selected from the group consisting of SEQ ID NO: 109-216. In some aspects, the engineered regulatory element comprises 100% identical nucleotide sequences selected from the group consisting of SEQ ID NO: 109-216.

[0011] This document also provides a heterogeneous construct comprising: any of the engineered control elements provided herein; and a heterogeneous payload, wherein the engineered control element is operatively connected to the heterogeneous payload.

[0012] In some aspects, the heterologous payload comprises a polynucleotide, optionally wherein the polynucleotide comprises a nucleotide sequence encoding one or more polypeptides. In some aspects, the one or more polypeptides comprises at least one effector molecule. In some aspects, the one or more polypeptides comprises two or more individual polypeptides, the two or more individual polypeptides comprising a first effector molecule, a second effector molecule, optionally a third effector molecule, and 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 E1 is a nucleotide sequence encoding a first effector molecule, L1 is a first adaptor molecule, E2 is a nucleotide sequence encoding a second effector molecule, L2 is a second adaptor molecule, E3 is a nucleotide sequence encoding a third effector molecule, L3 is a third adaptor molecule, and E4 is a nucleotide sequence encoding a fourth effector molecule. In some aspects, L1, L2, L3, and L4 are independently selected from: internal ribosome entry sites (IRES) and one or more nucleotide sequences encoding one or more 2A ribosomal jumping elements. In some aspects, the linker nucleotide sequence encodes one or more 2A ribosomal jumping elements. In some aspects, the one or more 2A ribosomal jumping elements include elements each selected from the following: 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: chimeric receptors, cytokines, chemokines, homing molecules, growth factors, polynucleotide molecules, co-activating molecules, tumor microenvironment modulators, receptors, ligands, transcription factors, antibodies, peptides, and enzymes. In some aspects, the chimeric receptor is a chimeric antigen receptor (CAR).

[0014] In some respects, 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 include a first effector molecule and a 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 a 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), and optionally wherein the first CAR and / or the second CAR is a bivalent CAR.

[0016] In some aspects, the one or more polypeptides include a first effector molecule, a second effector molecule, and a third effector molecule, 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 include a first effector molecule, a second effector molecule, a third effector molecule, and a fourth effector molecule, 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] This paper also provides a vector containing any of the heterogeneous constructs provided herein. This paper also provides a dual-expression vector containing any of the heterogeneous constructs provided herein and a second construct containing an additional payload.

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

[0020] This article also provides immune-response cells comprising any of the heterologous constructs, vectors, or dual-expression vectors provided herein. In some respects, the immune-response cells are selected from: natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, B cells, macrophages, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, monocytes, dendritic cells, erythrocytes, platelets, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some respects, the immune-response cells are NK cells or T cells. In some respects, the immune-response cells express activated immune receptors. In some respects, activated immune receptors include antigen recognition receptors. In some respects, immune response cells are autologous. In other respects, immune response cells are allogeneic.

[0021] This article also provides pharmaceutical compositions comprising any of the vectors, dual-expression vectors or immune-responding cells provided herein, as well as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients or combinations thereof.

[0022] This article also provides methods for increasing the expression of target genes or heterologous payloads, including using any of the engineered regulatory elements, vectors, or dual-expression vectors provided herein to increase the expression of target genes. In some respects, the target genes are immune regulatory genes.

[0023] This article also provides methods for treating subjects in need, including administering a therapeutically effective dose of any of the vectors, dual-expression vectors, immune-response cells, or pharmaceutical compositions provided herein.

[0024] This document also provides kits for the treatment and / or prevention of diseases or conditions, which include any of the immune-response cell or pharmaceutical compositions provided herein. In some aspects, the diseases or conditions include tumors. In some aspects, the kits also include written instructions for using the immune-response cell or pharmaceutical composition to treat and / or prevent a subject's disease or condition. Attached Figure Description

[0025] Figure 1A The diagram illustrates exemplary engineered control element designs and multicistron payload constructs, which include engineered enhancer sequences operatively connected to a core promoter to generate engineered promoter candidates.

[0026] Figure 1B A schematic diagram of a construct for evaluating engineered control element designs, which include engineered enhancer sequences operatively connected to a core promoter, is shown.

[0027] Figure 2 A graph showing the performance metrics of selected first-generation engineered promoter candidates based on transduction efficiency is presented.

[0028] Figure 3 Flow cytometry scatter plots are shown for exemplary selected engineered promoter constructs SB10698 (TP63), 10977 (NHLH1), 10961 (BARX2), 10947 (ERF039), and 10944 (bHLH1).

[0029] Figure 4A A graph showing performance metrics of selected first-generation engineered promoter candidates based on surface CAR protein expression measured on flow cytometry and quantified by fluorescence intensity values ​​is presented.

[0030] Figure 4B A graph showing performance metrics of selected first-generation engineered promoter candidates based on surface CAR protein expression measured on flow cytometry and quantified by fluorescence intensity values ​​is presented.

[0031] Figure 5A graph showing performance metrics of selected first-generation engineered promoter candidates based on surface CAR protein expression measured on flow cytometry and quantified by fluorescence intensity values ​​is presented.

[0032] Figures 6A to 6C A graph shows the percentage by which new promoter constructs that pair enhancers with various core promoters outperform the SV40 promoter as a baseline.

[0033] Figure 7A A graph showing the performance of candidate promoters containing TP63 TFBS is presented.

[0034] Figure 7B The performance of candidate promoters including BARX2, NHLH1, and TP63 TFBS is shown in the figure.

[0035] Figures 8A to 8B A flow cytometry scatter plot showing a performance comparison between the first-generation promoter and the second-generation promoter is presented.

[0036] Figure 8C The graph shows a performance comparison between the first-generation promoter and the second-generation promoter for aCAR (middle) and iCAR (right).

[0037] Figure 9 A schematic diagram of a library / construct for evaluating engineered enhancers and engineered promoters is shown.

[0038] Figure 10 The performance evaluation graphs for MPRA library screening are shown for Library 1 (top) and Library 2 (bottom).

[0039] Figure 11 The diagram shows iCAR MFI (iCAR+ cells) on day 7 and day 14 of NK cells transduced with various constructs.

[0040] Figure 12 An exemplary flow cytometry plot of cells transduced from 25 µl volumes of NV (left), SB12515 (middle), and SB12896 (right) on day 7 is shown, along with the normalized viral copy number (VCN) of the transduced cells.

[0041] Figure 13A The graph shows the transduction efficiency in transduced NK cells (left) and the expression of two payloads, aCAR and iCAR (middle and right).

[0042] Figure 13B The diagram shows the transduction efficiency in transduced NK cells and the expression of two payloads, aCAR (top) and iCAR (bottom).

[0043] Figures 14A to 14BThe surface expression of IL15 in cells transduced by SB12515 and SB12896 is shown. Figure 14A (left and right) and secreted IL15 ( Figure 14B (left) and IL21 ( Figure 14B (Right) of the diagram.

[0044] Figure 14C The diagram shows the copy number of cells with secreted IL15 (top) and IL21 (bottom) in cells transduced by SB12515 and SB12896.

[0045] Figure 15A Exemplary flow cytometry plots of NK cells from different donors, SB12896 and SB12515, are shown.

[0046] Figures 15B to 15D A graph showing promoter strength metrics across NK cells from different donors, SB12896 and SB12515, is presented.

[0047] Figure 16A A schematic diagram of engineered NK cells expressing payload 5 and the DLD-1 target cell line is shown.

[0048] Figures 16B to 16D The results of a comparison of the killing effect of the engineered promoter SB12896 on TA+PA- cells with those of SV40 are shown.

[0049] Figures 16E to 16F The results of a comparison of the killing effect of the engineered promoter SB12896 on TA+PA+ cells with those of SV40 are shown.

[0050] Figure 16G The results of lethality assays comparing the engineered promoter of SB12896 with SV40 are shown in a graph.

[0051] Figure 17A The graph shows the performance of engineered NK promoter candidates (top, middle, and bottom) in terms of payload expression compared to SV40 (SB12515).

[0052] Figure 17B The graph (left and right) shows the performance of the engineered NK promoter candidate compared to SV40 (SB12515) in killing TA+PA- target cells and TA+PA+ “healthy” cells.

[0053] Figure 18A The graph (left and right) shows the performance of the candidate promoter compared to the SV40 promoter (SB12515) in NK cells.

[0054] Figure 18BThe graph (left and right) shows the performance of the candidate promoter compared to the SV40 promoter (SB12515) in T cells.

[0055] Figure 18C The graphs (top and bottom) show the performance correlation between the candidate promoter and the SV40 promoter (SB12515) in NK cells and T cells.

[0056] Figure 18D A single figure depicts the performance of the candidate promoter in NK and T cells.

[0057] Figure 18E The graphs (top and bottom) show the flow cytometry results of SFFV control and SB13498. Detailed Implementation

[0058] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed subject matter pertains. Generally, the nomenclature and techniques described herein in conjunction with immunology, oncology, cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization are those well-known and commonly used in the art. It should be understood that the foregoing general description and the following detailed description are merely exemplary and explanatory and do not limit any subject matter claimed herein or otherwise provided. Section headings used herein are for organizational purposes only and should not be construed as limiting the described subject matter.

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

[0060] As used herein, unless the context clearly indicates otherwise, all numeric values ​​or ranges include integers within or covering such ranges, as well as fractions of values ​​or integers within or covering such ranges. Therefore, for example, references to a range of 90% to 100% include 91%, 92%, 93%, 94%, 95%, 95%, and 97%. wait And 91.1%, 91.2%, 91.3%, 91.4%, 91.5% wait 92.1%, 92.2%, 92.3%, 92.4%, 92.5% wait And so on.

[0061] The use of ordinal terms such as "first," "second," and "third" to modify claim elements does not imply any priority, precedence, or order of one claim element relative to another, or the chronological order of actions of the method. Rather, they serve merely as labels to distinguish one claim element with a specific name from another element with the same name (but using ordinal terms), thereby differentiating the claim elements. Similarly, the use of these terms in the specification does not imply any desired priority, precedence, or order.

[0062] Agent: As used herein, the term "agent" can refer to any chemical class of compound, molecule, or entity, including, for example, polypeptides, nucleic acids (…). For example The agent may contain engineered nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof. In some embodiments, the agent is or comprises a natural product because it is found in and / or obtained from nature. In some embodiments, the agent is or comprises one or more artificial entities because it is designed, engineered, modified, and / or produced through artificial action and / or is not found in nature. In many respects, this disclosure provides for the inclusion of specific engineered transcriptional regulatory elements as described herein (such as engineered nucleic acids, sugars, lipids, small molecules, metals, or combinations thereof). For example The engineered nucleic acids described herein (engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences). In some aspects, the agents can be in isolated or pure form ( For example The agent is used in some embodiments, including isolated polynucleotides; in some embodiments, the agent may be used in crude form. In some aspects, the potential agent is provided as a collection or library, which may be screened to identify or characterize the active agent therein. Some specific embodiments of the agents used according to this disclosure include small molecules, antibodies, antibody fragments, aptamers, nucleic acids (…). For example Agents include siRNA, shRNA, DNA / RNA hybrids, antisense oligonucleotides, ribozymes, peptides, and peptide mimics. In some aspects, agents as described herein encode a coding nucleotide sequence operatively linked to an engineered regulatory element (e.g., an engineered enhancer sequence and / or an engineered promoter containing an engineered enhancer sequence) as provided herein. In some aspects, agents are effector molecules as described herein.

[0063] Approximately: When applied to one or more values ​​of interest, the term "approximately" or "about" refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values ​​falling 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 clearly apparent from the context (unless such figures would exceed 100% of the possible values).

[0064] Bioactivity: The phrase "bioactivity" refers to the activity of a biological system ( For example Isolated cells, cells in specific tissues, cells in cultures, or cells in organisms wait A biologically active agent or substance. For example, an agent or substance that has a biological effect on an organism when applied to that organism is considered biologically active or has biological activity. For example Bioactive agent, bioactive molecule wait Those skilled in the art will understand that typically only a portion or fragment of the bioactive substance is required. For example (This part or fragment is necessary and sufficient) to enable the activity to be present; in this case, the part or fragment is considered a "bioactive" part or fragment.

[0065] Expression cassette: An "expression cassette" is a recombinant or synthetically produced polynucleotide construct that contains regulatory elements ( For example Any engineered regulatory element described herein, such as the engineered enhancer sequence described herein and / or an engineered promoter containing an engineered enhancer sequence, the regulatory element interacts with the selected polynucleotide ( For example The coding sequence (such as a gene) is operatively linked to promote the expression of the selected polynucleotide in a host cell or in a cell-free environment. Heterologous constructs as described herein may contain one or more expression cassettes.

[0066] Heterogeneous: As used in this article with respect to nucleotide sequences, amino acid sequences, or polypeptides, the term "heterogeneous" refers to something that is foreign to a given host cell. For example Exogenous, such as those not found in nature) or naturally found in a given host cell ( For example (Endogenous) compounds or agents, however, said compounds or agents are in the context of heterologous constructs. For example Heterologous nucleic acids are used, as described in this article. Endogenously discovered heterologous nucleotide sequences can also be used in cells in a non-natural (…) For example (The amount produced is greater than expected or greater than naturally found.) Heteronucleotide sequences, or nucleic acids containing heteronucleotide sequences, may differ in sequence from endogenous nucleotide sequences, but encode the same protein as endogenously found nucleotide sequences. Specifically, heteronucleotide sequences are those nucleotide sequences that have not been found in nature to have the same relationship as the host cell. Any recombinant or artificial nucleotide sequence should be understood as heterologous. Heteropolynucleotides ( For example Non-limiting examples of engineered regulatory elements (such as those described herein) are nucleotide sequences operatively linked to a promoter, such as engineered enhancer sequences that are not naturally associated with the promoter. Heteronucleotides containing such engineered regulatory elements can be further used to replace coding sequences ( For example The expression of the coding sequence is controlled by a natural or wild-type promoter of the gene.

[0067] Identity: The term "identity" refers to the relationship between polymer molecules. For example Nucleic acid molecules ( For example The overall correlation between DNA molecules and / or RNA molecules and / or polypeptide molecules. Therefore, unless the context otherwise indicates, the correlation between specific sequences ( For example nucleotide sequence, amino acid sequence wait In the context of [the above], "identity" or "homology" has the same meaning. For example, the percentage of identity between two nucleic acid sequences can be calculated by aligning two sequences for optimal comparison purposes. For exampleVacancies can be introduced in one or both of the first and second nucleic acid sequences for optimal alignment, and dissimilar sequences can be ignored for comparison purposes. In some respects, the length of the sequences 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. Nucleotides at corresponding nucleotide positions are then compared. The molecules are identical at that position when the position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence. The percentage of identity between two sequences is a function of the number of common positions shared by the sequences, taking into account the number of vacancies introduced for optimal alignment and the length of each vacancy. Sequence comparison and determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. For example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), can be used to determine the percentage of identity between two nucleotide sequences using a PAM120 weighted residue table, a length penalty of 12, and a gap penalty of 4. Alternatively, the GAP program in the GCG package can be used, employing the NWSgapdna.CMP matrix to determine the percentage of identity between two nucleotide sequences. Optimal sequence alignment for comparison can also be performed. For example Local homology algorithms by Smith and Waterman, Adv. Appl. Math. 2:482 (1981), homology comparison algorithms by Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), similarity search methods by Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or visual inspection (see Ausubel for general information). et al. This is done using [the algorithm]. Another example of an algorithm suitable for determining the percentage of sequence identity and the percentage of sequence similarity is the BLAST algorithm, which is described in Altschul. et al.In J. Mol. Biol. 215:403-410 (1990). The software for BLAST analysis is publicly available from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0068] Separated: The term "separated" means a substance and / or entity that is (1) separate from at least some of its components associated with it at the time of its initial production (whether in nature or in an experimental setting), and / or (2) artificially produced, prepared, and / or manufactured. A separated substance and / or entity 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 its originally associated other components. In some embodiments, the separating agent is 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, the calculation of the purity percentage of isolated substances and / or entities should not include excipients ( For example Buffer solution, solvent, water wait ).

[0069] Core promoter: The "core promoter" contains the target coding sequence that sustains the initiation or induces the target ( For example The promoter sequence is the promoter sequence that enables the transcription of genes. Therefore, the 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 incorporated into or potentially incorporated into an oligonucleotide chain. In some embodiments, nucleic acid is a compound and / or substance incorporated into or potentially incorporated into an oligonucleotide chain via a phosphodiester bond. In some respects, "nucleic acid" refers to a single nucleic acid residue ( For example Nucleic acids (nucleotides and / or nucleosides). In some respects, "nucleic acid" refers to an oligonucleotide chain containing a single nucleic acid residue. The terms "oligonucleotide" and "polynucleotide" are used interchangeably. In some respects, "nucleic acid" covers RNA as well as single-stranded and / or double-stranded DNA and / or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and / or similar terms include nucleic acid analogues. Right nowAnalogs having a non-phosphodiester backbone. For example, so-called "peptide nucleic acids" known in the art and having peptide bonds instead of phosphodiester bonds in their backbone are considered within the scope of this disclosure. The terms "nucleotide sequence encoding an amino acid sequence" or "coding sequence" include all nucleotide sequences that are degenerate forms of each other and / or encode the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNA may contain introns. A coding sequence may also refer to a biologically active nucleic acid (…). For example ,mRNA,miRNA,siRNA,shRNA wait The nucleotide sequence of ). Nucleic acids can be isolated or purified from natural sources, produced using recombinant expression systems and optionally isolated or purified, or chemically synthesized. wait Under appropriate circumstances, For example In the case of chemically synthesized molecules, nucleic acids can contain nucleoside analogues, such as chemically modified bases or sugars, and backbone modifications. wait Analogs of [other nucleic acid sequences]. Unless otherwise stated, nucleic acid sequences are presented in a 5' to 3' orientation. The term "nucleic acid segment" is used herein to refer to a nucleic acid sequence that is part of a longer nucleic acid sequence. In many embodiments, the 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, the nucleic acid is or contains a natural nucleoside ( For example Adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine; nucleoside analogues ( For example 2-Aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 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-deazoadenosine, 7-deazoguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine and 2-thiocytidine); chemically modified bases; biologically modified bases ( For example methylated bases); embedded bases; modified sugars ( For example 2'-Fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose; and / or modified phosphate groups ( For example(Thiophosphate and 5'-N-phosphamide bond). In some aspects, this disclosure relates to "unmodified nucleic acids," meaning nucleic acids that have not been chemically modified to facilitate or achieve delivery. For example (Polynucleotides and residues, including nucleotides and / or nucleosides).

[0071] Operable linkage: The term "operable linkage" refers to polynucleotide or amino acid sequences that are functionally related to each other. For example, if a regulatory element ( For example If a regulatory element (as an engineered enhancer sequence and / or an engineered promoter containing an engineered enhancer sequence) regulates or contributes to the regulation of transcription of a coding sequence, then the regulatory element is operatively linked to the coding sequence. The operatively linked DNA sequence encoding the regulatory sequence is typically adjacent to the coding sequence. However, some regulatory elements ( For example (Enhancers) can function even when separated from the promoter by up to several thousand or more bases. Additionally, polycistronic constructs can contain multiple coding sequences, which are achieved by including 2A self-cleaving peptides and IRES elements as described herein. wait However, only one regulatory element is used. Therefore, some polynucleotide elements ( For example The engineered control element provided herein can be operatively connected to one or more coded sequences, but is not adjacent to the one or more coded sequences.

[0072] Protein: The term "protein" refers to polypeptides (…). Right now A protein is a string of at least two amino acids linked together by peptide bonds. Proteins can contain components other than amino acids. For example It can be glycoproteins or proteoglycans. wait And / or may be otherwise processed or modified. Those skilled in the art will understand that a “protein” can be a complete polypeptide chain produced by a cell (with or without a signal sequence), or it can be its biologically active portion. Those skilled in the art will understand that a protein may sometimes contain more than one polypeptide chain, which may be linked by one or more disulfide bonds or otherwise associated. A polypeptide 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. Available modifications include... For example Terminal acetylation, amidation, methylation, etc. In some respects, proteins can contain natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to polypeptides with 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] Essentially: The term “essentially” refers to qualitative conditions that exhibit all or nearly all of the range or degree of the characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if at all) complete and / or proceed to completeness or achieve or avoid absolute results. Therefore, the term “essentially” is used herein to express the inherent potential lack of completeness in many biological and chemical phenomena.

[0074] Subject: The term "subject" refers to a mammal ( For example Humans, in some implementations including prenatal human forms, rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs. In some respects, the subject has a relevant disease, condition, or symptom ( For example (Cancer). In some respects, the subject is susceptible to a disease, condition, or symptom. In some respects, the subject exhibits one or more symptoms or characteristics of a disease, condition, or symptom. In some respects, the subject does not exhibit any symptoms or characteristics of a disease, condition, or symptom. In some respects, the subject is an individual with one or more characteristics that constitute susceptibility or risk to a disease, condition, or symptom. The subject can be a patient, which refers to a person presented to a healthcare provider for the diagnosis or treatment of a disease. In some respects, the subject is an individual to whom a therapy is administered. example like Receptors. In many aspects of this disclosure, the subjects have or are susceptible to diseases or conditions, such as cancer.

[0075] Therapeutic agent: The phrase "therapeutic agent" generally refers to an agent applied to an organism ( For example A therapeutic agent is any agent that induces the desired pharmacological effect when administered to a human subject (or a patient). In some respects, an agent is considered a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some respects, an appropriate population may be a population of model organisms. In some respects, an appropriate population can be defined by various criteria, such as an age group, sex, genetic background, pre-existing clinical symptom, etc. In some respects, a therapeutic agent is a substance that can be used to reduce, improve, alleviate, inhibit, prevent, or delay the onset of one or more symptoms or features of a disease, condition, and / or symptom, reduce its severity, and / or decrease its incidence. In some respects, a "therapeutic agent" is an agent that has been or requires approval by a government agency before it can be marketed for administration to humans. In some respects, a "therapeutic agent" is an agent that requires a medical prescription for administration to humans.

[0076] Therapeutic effective dose: The term "therapeutic effective dose" refers to a therapeutic protein that imparts a therapeutic effect to the treated subject at a reasonable benefit / risk ratio applicable to any medical treatment. For example The therapeutic effect can be objective (the amount of effector molecules). Right now(can be measured through some tests or markers) or subjective ( Right now (The subject gives indications of the effect or feels the effect). Specifically, a "therapeutic effective dose" refers to the amount by which a therapeutic protein or composition effectively treats, improves, or prevents a desired disease or symptom, or exhibits a detectable therapeutic or preventive effect, such as by improving disease-associated symptoms, preventing or delaying the onset of the disease, and / or also reducing the severity or frequency of disease symptoms. Therapeutic effective doses are typically administered in a dosing regimen that may contain multiple unit doses. For any particular therapeutic protein, the therapeutic effective dose (and / or the appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration and combination with other agents. Furthermore, the specific therapeutic effective dose (and / or unit dose) for any particular patient may depend on a variety of factors, including the condition being treated and its severity; the activity of the specific agent used; the specific composition employed; the patient's age, weight, general health condition, sex, and diet; the time of administration, route of administration, and / or the excretion or metabolic rate of the specific fusion protein employed; the duration of treatment; and similar factors well known in the medical field.

[0077] Treatment: The term "treatment" (also known as "treat" or "treating") refers to any application of a substance that partially or completely relieves, improves, reduces, inhibits, delays the onset of, reduces the severity of, and / or reduces the incidence of a particular disease, condition, and / or symptom. Such treatment can be for subjects who do not exhibit signs of the relevant disease, condition, and / or symptom and / or who exhibit only early signs of the disease, condition, and / or symptom. Alternatively or additionally, such treatment can be for subjects who exhibit one or more definitive signs of the relevant disease, condition, and / or symptom. In some aspects, treatment can be for subjects who have been diagnosed with the relevant disease, condition, and / or symptom. In some aspects, treatment can be for subjects known to have one or more susceptibility factors that are statistically associated with an increased risk of developing the relevant disease, condition, and / or symptom.

[0078] Vector: A vector is a nucleic acid molecule capable of transporting another nucleic acid it associates with. The terms "vector" and "plasmid" are used interchangeably. In some respects, a vector can replicate extrachromosomally and / or express the nucleic acid it is associated with in a host cell (such as a eukaryotic and / or prokaryotic cell). It can guide the operatively linked coding sequence (…). For exampleThe vectors used to express genes are referred to as "expression vectors" in this paper. Expression vectors typically contain expression cassettes. Vectors and plasmids include, but are not limited to, replication vectors, probe generation vectors, sequencing vectors, integration vectors, phage particles, prokaryotic plasmids, eukaryotic plasmids, plant synthetic chromosomes, episomes, and viral vectors. For example Animal virus vectors, kinases, and artificial chromosomes.

[0079] II. Engineered Control Components This disclosure provides methods and compositions comprising engineered nucleic acids, the engineered nucleic acids comprising engineered regulatory elements (…). For example Engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences. As used herein, "regulatory element" refers to the regulator ( For example A polynucleotide sequence transcribed into a downstream gene that is operatively linked to a regulatory element (which initiates, induces, activates, increases, or otherwise regulates) for transcription. Such engineered regulatory elements can be used to improve protein expression in cells. As an example, the engineered regulatory elements described herein can be used to drive high expression of multiple proteins encoded in complex polycistronic systems. As other examples, the engineered regulatory elements described herein can allow for the improvement of operatively linked polynucleotide sequences transcribed into cells for transcription (which initiate, induce, activate, increase, or otherwise regulate) for transcription. For example Transcription of genes, such as improving transcription in NK cells and / or T cells. In some cases, it is associated with known regulatory elements (linked to the same polynucleotides) that are operatively linked to the same polynucleotides. For example Compared to SV40 and SFFV, the engineered regulatory elements described in this paper can allow for improvement in cell ( For example The transcription of this polynucleotide is operatively linked in NK cells and / or T cells. In some cases, it is associated with known regulatory elements that drive the expression of the same one or more proteins. For example Compared to vectors containing SV40 and SFFV, vectors containing the engineered regulatory elements described herein can drive higher expression of one or more proteins at the same or lower integration copy number. In some respects, the engineered regulatory elements described herein improve selective transcription in NK cells and / or T cells compared to non-NK cells and / or non-T cells, respectively.

[0080] In many respects, the engineered nucleic acids provided in this disclosure are or contain engineered regulatory elements as described herein. For example (any of SEQ ID NO: 1-216 and / or any of the transcription factor binding sites (TFBS) described herein, such as any of the TP63 TFBS described herein). In some aspects, the engineered regulatory element is provided in an expression cassette, a heterologous construct, a vector, or other polynucleotide sequence.

[0081] In some aspects, the engineered regulatory element comprises an engineered enhancer sequence. The engineered enhancer sequences provided herein comprise 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 with nucleotide sequences selected from the following: SEQ ID NO: 1-108. SEQ ID NO: 1-108 is shown in Table 1. In some aspects, the engineered enhancer sequence comprises a nucleotide sequence that is at least 95% identical with nucleotide sequences selected from the following: SEQ ID NO: 1-108. In some aspects, the engineered enhancer sequence comprises a nucleotide sequence that is 100% identical with nucleotide sequences selected from the following: SEQ ID NO: 1-108.

[0082] The engineered enhancer sequences provided herein include engineered enhancer sequences comprising one or more TP63 transcription factor binding sites (TFBS). In some aspects, the TP63 TFBS comprises a 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 with a nucleotide sequence selected from SEQ ID NO 217-220 and its reverse complementary sequence. SEQ ID NO: 217-220 is shown in Table 3. In some aspects, the TP63 TFBS comprises a sequence that is at least 95% identical with a nucleotide sequence selected from SEQ ID NO 217-220 and its reverse complementary sequence. In some embodiments, the TP63 TFBS is selected from SEQ ID NO 217-220 and its reverse complementary sequence.

[0083] In some aspects, the engineered enhancement subsequence contains 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 TFBS. In some aspects, the engineered enhancement subsequence contains at least two TP63 TFBS. In some aspects, the engineered enhancement subsequence contains at least three TP63 TFBS. In some aspects, the engineered enhancement subsequence contains at least five TP63 TFBS. In some aspects, the engineered enhancement subsequence contains at least ten TP63 TFBS. In some aspects, the engineered enhancement subsequence contains two TP63 TFBS. In some aspects, the engineered enhancement subsequence contains three TP63 TFBS. In some aspects, the engineered enhancement subsequence contains five TP63 TFBS. In some aspects, the engineered enhancement subsequence contains ten TP63 TFBS. In some aspects, the engineered enhancement subsequence contains 1-500, 1-100, 1-50, 2-20, or 2-10 TP63 TFBS.

[0084] In some respects, TP63 TFBS contains a TP63 TFBS half-site motif. In other respects, TP63 TFBS contains two TP63 TFBS half-site motifs. Not wishing to be bound by theory, TP63 generally binds to DNA preferably as a dimer, and its shared binding motif is approximately 20 bp (the “intact” response element), which typically consists of two “half-sites,” each approximately 10 base pairs long. TP63 dimers can bind to half-site pores, and a total of four TP63s (i.e., tetramers) can bind to the intact response element. Two TP63 TFBS half-site motifs are generally operatively linked to each other. Two half-sites can be directly tandemly linked to each other (e.g., without separating the adapter). Two half-sites can be tandemly linked to each other via nucleic acid adapters and still retain TP63 binding. Half-site adapters can have different lengths, such as a single nucleotide or adapters between 2 and 10 nucleotides in length. The two half-sites can also be on the same or different DNA strands (e.g., a half-site in the forward orientation and another half-site in the reverse orientation), so TP63 TFBS can be contained in a first TP63 TFBS half-site motif in one orientation, which is operatively linked (e.g., tandemly) to a second TP63 TFBS half-site motif in the opposite orientation (reverse complementary sequence).

[0085] In some aspects, the TP63 TFBS half-site motif contains a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from SEQ ID NO 221-233 and its reverse complementary sequence. SEQ ID NO: 221-233 is shown in Table 3. In some aspects, the TP63 TFBS half-site motif contains a sequence that is at least 95% identical to a nucleotide sequence selected from SEQ ID NO 221-233 and its reverse complementary sequence. In some aspects, the TP63 TFBS half-site motif is selected from SEQ ID NO 221-233 and its reverse complementary sequence.

[0086] A TP63 TFBS motif with two TP63 TFBS half-site motifs can contain two identical TP63 TFBS half-site motifs. A TP63 TFBS motif with two TP63 TFBS half-site motifs can contain two different TP63 TFBS half-site motifs. A TP63 TFBS motif with two TP63 TFBS half-site motifs can contain a TP63 TFBS half-site motif and an inverse complementary sequence of the same TP63 TFBS half-site motif. In some respects, the two TP63 TFBS half-site motifs include (a) a first half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO 221-233; and (b) a second half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an inverse complementary sequence selected from the group consisting of SEQ ID NO 221-233.

[0087] In some aspects, in addition to the TP63 TFBS, the engineered enhancer sequence also includes at least one additional non-TP63 TFBS. In some aspects, the additional non-TP63 TFBS is selected from: BARX2 TFBS, NHLH1 TFBS, TP73 TFBS, HOXC10 TFBS, NFE2 TFBS, ATF4 TFBS, HES1 TFBS, FOS TFBS, JUN TFBS, and JUNB TFBS. In some aspects, the additional non-TP63 TFBS is selected from: BARX2 TFBS, NHLH1 TFBS, or both BARX2 TFBS and NHLH1 TFBS.

[0088] In some aspects, the additional non-TP63 TFBS is a BARX2 TFBS. In some aspects, the additional non-TP63 TFBS is a BARX2 TFBS containing 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 sequences to SEQ ID NO: 234. In some aspects, the engineered enhancer sequence contains 2, 3, 4, 5, or more than 5 BARX2 TFBSs.

[0089] In some aspects, the additional non-TP63 TFBS is an NHLH1 TFBS. In some aspects, the additional non-TP63 TFBS is an NHLH1 TFBS containing 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 sequence to SEQ ID NO: 235. In some aspects, the engineered enhancer sequence contains 2, 3, 4, 5, or more than 5 NHLH1 TFBS.

[0090] In some respects, the additional non-TP63 TFBS is a TP73 TFBS. In some respects, the additional non-TP63 TFBS is a TP73 TFBS containing 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% of the same sequence as SEQ ID NO: 236.

[0091] In some respects, the additional non-TP63 TFBS is a HOXC10 TFBS. In some respects, the additional non-TP63 TFBS is a HOXC10 TFBS containing 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% of the sequence identical to SEQ ID NO: 237.

[0092] In some respects, the additional non-TP63 TFBS is an NFE2 TFBS. In some respects, the additional non-TP63 TFBS is an NFE2 TFBS containing 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% of the same sequence as SEQ ID NO: 238.

[0093] In some respects, the additional non-TP63 TFBS is an ATF4 TFBS. In some respects, the additional non-TP63 TFBS is an ATF4 TFBS containing 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% of the same sequence as SEQ ID NO: 239.

[0094] In some respects, the additional non-TP63 TFBS is the HES1 TFBS. In some respects, the additional non-TP63 TFBS is the HES1 TFBS, which contains 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% of the sequence identical to SEQ ID NO: 240.

[0095] In some respects, the additional non-TP63 TFBS is a FOS TFBS. In some respects, the additional non-TP63 TFBS is a FOS TFBS containing 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% of the same sequence as SEQ ID NO: 241.

[0096] In some respects, the additional non-TP63 TFBS is a JUN TFBS. In some respects, the additional non-TP63 TFBS is a JUN TFBS containing 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% of the same sequence as SEQ ID NO: 242.

[0097] In some respects, the additional non-TP63 TFBS is a JUNB TFBS. In some respects, the additional non-TP63 TFBS is a JUNB TFBS containing 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% of the same sequence as SEQ ID NO: 243.

[0098] In some respects, TFBSs can be operatively connected. In other respects, TFBSs can be directly cascaded together. For example (Not separated from the adapter). In some respects, TFBS can be tandemly linked to each other via nucleic acid adapters.

[0099] In some respects, engineered regulatory elements are or comprise engineered promoters. The engineered promoters provided herein comprise engineered enhancer sequences (e.g., SEQ ID NO:1-108) operatively linked to a core promoter sequence. The core promoter sequences include, but are not limited to, promoters selected from the following: minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, SCP3 promoter containing DPR, minP promoter, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, API response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, YB TATA, minTK, inducer molecule responsive promoter, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaV1, hCAGG, hEFlaV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb and their tandem repeat sequences. The core promoter sequence includes, but is not limited to, promoters selected from the following: minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, and SCP3 promoter containing DPR.

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

[0101] In some aspects, the engineered promoter comprises 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 with a nucleotide sequence selected from the group consisting of SEQ ID NO: 109-216. SEQ ID NO: 109-216 is shown in Table 1. In some aspects, the engineered promoter comprises a nucleotide sequence that is at least 95% identical with a nucleotide sequence selected from the group consisting of SEQ ID NO: 109-216. In some aspects, the engineered promoter comprises a nucleotide sequence that is 100% identical with a nucleotide sequence selected from the group consisting of SEQ ID NO: 109-216.

[0102] In some aspects, an engineered enhancer sequence is or contains one or more enhancer segments (e.g., a single TP63 TFBS). In some aspects, one or more enhancer segments of an engineered enhancer sequence are continuous. 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 continuous, and one or more engineered enhancer sequences of the same engineered enhancer sequence are non-contiguous. 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 respects, nucleotide sequences 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 are located between the first enhancer segment and the second enhancer segment.

[0103] In some aspects, engineered control elements ( For example The engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences described herein induce operablely linked coding sequences at levels or intensities comparable to alternative regulatory elements. For example Expression of genes For example Such as by using the alternative control element ( For example The percentage of transcriptional activity compared to promoters such as the SFFV promoter, SV40 promoter, or viral LTR is measured. In some respects, it is compared to alternative regulatory elements ( For example Compared to the SFFV promoter, SV40 promoter, or viral LTR, engineered regulatory elements induce the expression of operablely linked coding sequences with intensities of 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or higher.

[0104] In some aspects, engineered control elements ( For exampleThe engineered enhancer sequence (and the engineered promoter and / or enhancer segment containing the engineered enhancer sequence) can contain lengths of approximately 5 base pairs, approximately 10 base pairs, approximately 20 base pairs, approximately 30 base pairs, approximately 40 base pairs, approximately 50 base pairs, approximately 60 base pairs, approximately 70 base pairs, approximately 80 base pairs, approximately 90 base pairs, approximately 100 base pairs, or approximately 200 base pairs. Length, approximately 300 base pairs, approximately 400 base pairs, approximately 500 base pairs, approximately 600 base pairs, approximately 700 base pairs, approximately 800 base pairs, approximately 900 base pairs, approximately 1000 base pairs, approximately 1,100 base pairs, approximately 1,500 base pairs, approximately 2,000 base pairs, approximately 5,000 base pairs, or approximately 10,000 base pairs.

[0105] In some aspects, engineered control elements ( For example The engineered enhancer sequence (and the engineered promoter and / or enhancer segment containing the engineered enhancer sequence) is between 5 and 10,000 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 5,000 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 2,000 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 1,100 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 1,000 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 900 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 800 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 700 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 600 base pairs in length. In some aspects, the engineered regulatory element is between 5 and 500 base pairs in length. In some aspects, the length of engineered regulatory elements is between 5 and 400 base pairs. In some aspects, the length of engineered regulatory elements is between 5 and 300 base pairs. In some aspects, the length of engineered regulatory elements is between 5 and 200 base pairs. In some aspects, the length of engineered regulatory elements is between 5 and 100 base pairs.

[0106] In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 10,000 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 5,000 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 2,000 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 1,100 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 1,000 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 900 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 800 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 700 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 500 and 600 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 600 and 1,100 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 700 and 1,100 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 600 and 1,500 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 700 and 1,200 base pairs. In some aspects, the length of the engineered promoter containing the engineered enhancer sequence is between 700 and 1,500 base pairs.

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

[0108] In some aspects, engineered regulatory elements (e.g., engineered promoters comprising engineered enhancer sequences) include at least one spacer subsequence. In some aspects, engineered regulatory elements comprise an engineered enhancer sequence (e.g., comprising tandem TFBS) and a core promoter ( For example At least one spacer between the minimum promoters described in this article.

[0109] In some aspects, engineered nucleic acids ( For example The heterogeneous constructs described herein are configured to produce multiple agents ( For exampleThese agents (one or more effector molecules) can be encoded in one or more coding sequences that are operatively linked to engineered regulatory elements as described herein (e.g., engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences). For example, engineered nucleic acids can be configured to produce 2-20 different agents.In some respects, 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, 11-1 3. 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 respects, 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. In many cases, the agents provided are therapeutic agents, such as those described herein.

[0110] In some aspects, engineered nucleic acids ( For example (As described in this paper, heterogeneous constructs) can contain polycistronic regions. Right now More than one single polypeptide ( For example Therapeutic agents, effector molecules, etc., can be produced from a single mRNA transcript transcribed from the polycistronic region. For example, a heterologous payload can contain polynucleotides encoding two or more polypeptides, such as a first effector molecule and a second effector molecule, or a first effector molecule, a second effector molecule, and a third effector molecule, or a first effector molecule, a second effector molecule, a third effector molecule, and a fourth effector molecule, wherein each polypeptide and / or effector molecule is a separately expressed peptide. Polycistronic regions can be created using various linkers. For example A linker can be used to connect a first coding sequence to a second coding sequence, for example, to generate a construct from 5' to 3' containing the first coding sequence, a linker, and a second coding sequence. The linker polynucleotide sequence can encode a 2A ribosomal jumping element, such as T2A. Other 2A ribosomal jumping elements include, but are not limited to, E2A, P2A, and F2A. 2A ribosomal jumping elements allow for the generation of separate polypeptides encoded by the first and second genes during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, wherein the cleavable linker polypeptide is cleaved after expression, thereby generating separate polypeptides encoded by the first and second genes. The cleavable linker can include polypeptide sequences, such as flexible linkers that further facilitate cleavage (…). For example , Gly-Ser-Gly sequence). In some respects, the polycistronic region contains up to two, about 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 coding sequence connected by a connector ( For example (Connection of the first connector, second connector, third connector, fourth connector, etc.)

[0111] In some aspects, the heterologous payload comprises polynucleotides of the form E1-L1-E2, E1-L1-E2-L2-E3, and / or E1-L1-E2-L2-E3-L3-E4, wherein E1 is a nucleotide sequence encoding a first effector molecule, L1 is a first adaptor molecule, E2 is a nucleotide sequence encoding a second effector molecule, L2 is a second adaptor molecule, E3 is a nucleotide sequence encoding a third effector molecule, L3 is a third adaptor molecule, and E4 is a nucleotide sequence encoding a fourth effector molecule. In some aspects, L1, L2, L3, and L4 are independently selected from: internal ribosome entry sites (IRES) and one or more nucleotide sequences encoding one or more 2A ribosomal jumping elements.

[0112] In some respects, the engineered nucleic acids disclosed herein ( For example A post-transcriptional regulatory element (PRE), such as the heterologous construct described herein, contains a post-transcriptional regulatory element (PRE). PREs can enhance gene expression by achieving tertiary RNA structural stability and 3' end formation. Non-restrictive examples of PREs include hepatitis B virus PRE (HPRE) and marmot hepatitis virus PRE (WPRE). In some respects, the post-transcriptional regulatory element is the marmot hepatitis virus post-transcriptional regulatory element (WPRE). In some respects, the WPRE contains the α, β, and γ components of a WPRE element. In some respects, the WPRE contains the α grouping of a WPRE element.

[0113] III. Heterogeneous Constructs Certain aspects of this disclosure relate to including one or more engineered control elements ( For example The engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences described herein are used to generate polynucleotides of heterologous constructs. For example (isolated polynucleotides). In some respects, the provided heterologous constructs also contain one or more expression cassettes.

[0114] In some respects, heterologous constructs contain and encode polynucleotide sequences ( For example Engineered regulatory elements (the coding sequences of genes or bioactive molecules) that can be operatively linked For example The engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences described herein. In some aspects, the heterologous construct contains a promoter encoding at least one effector molecule ( For example Engineered regulatory elements are operatively linked from polynucleotide sequences (such as first-effect molecules, second-effect molecules, third-effect molecules, etc.). In some respects, effector molecules comprise biologically active molecules. In some respects, effector molecules comprise polypeptides. In some respects, effector molecules comprise polynucleotides (… For example ,mRNA,miRNA,siRNA,shRNA wait In some respects, the effector molecules are human-derived effector molecules.

[0115] In some respects, the heterogeneous construct includes engineered control elements as disclosed herein. For example The heterologous construct comprises nucleotide sequences encoding two or more effector molecules under the transcriptional control of engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences. In some aspects, the heterologous construct contains nucleotide sequences encoding two or more effector molecules, each encoded under the transcriptional control of a separate engineered regulatory element.

[0116] In some respects, the heterologous construct contains nucleotide sequences encoding two or more effector molecules that are expressed as a single polypeptide chain within the same reading frame. In some respects, the two or more effector molecules expressed as a single polypeptide chain may include one or more peptide cleavage sites. For example A peptide cleavage site (either a self-cleavage site or a cleavage site of an intracellular protease) separates two or more effector molecules upon cleavage. Suitable peptide cleavage sites may include, but are not limited to, T2A, P2A, E2A, and F2A peptide cleavage sites.

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

[0118] In some aspects, the polynucleotide sequence encoding the first effector molecule is linked to the polynucleotide sequence encoding the second effector molecule via a linker polynucleotide sequence. In some aspects, the linker polynucleotide sequence contains a polynucleotide sequence encoding at least one 2A ribosomal jumping element. In some aspects, the 2A ribosomal jumping element is a T2A, P2A, E2A, or F2A element.

[0119] Any suitable effector molecule known in the art can be encoded by or expressed by a polynucleotide in the provided heterologous construct. In some respects, effector molecules ( For example Effector molecules (e.g., first-effect molecules, second-effect molecules, third-effect molecules, etc.) are therapeutic molecules. Appropriate effector molecules can be categorized into therapeutic classes based on structural 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, shRNA, miRNA, and enzymes. Therefore, in some respects, effector molecules (e.g., first-effect molecules, second-effect molecules, third-effect molecules, etc.) belong to a therapeutic class selected from the group consisting of: cytokines, chemokines, homing molecules, growth factors, polynucleotide molecules, co-activating molecules, tumor microenvironment modulators, receptors, chimeric receptors (e.g., chimeric antigen receptors [CAR]), ligands, antibodies, peptides, polynucleotide molecules, co-activating molecules, tumor microenvironment modulators, RNA molecules (e.g., mRNA, miRNA, siRNA, shRNA, etc.), and enzymes.

[0120] In some respects, the effector molecule is a receptor, specifically a chimeric antigen receptor (CAR). In some respects, the CAR is an engineered receptor that grafts or confers specificity of interest onto or confers immune effector cells. In some respects, the CAR can be used to graft the specificity of antibodies onto immune-responding cells, such as T cells or NK cells. In some respects, the CAR of this disclosure comprises an extracellular antigen-binding domain fused to a transmembrane domain and fused to one or more intracellular signaling domains. For example (scFv). In some aspects, the extracellular antigen-binding domain of the CAR disclosed herein binds specifically to one or more antigens expressed on tumor cells. The antigen-binding domain of the CAR disclosed herein may include any domain that binds to an antigen, including but not limited to monoclonal antibodies, polyclonal antibodies, recombinant antibodies, bispecific antibodies, conjugated antibodies, human antibodies, humanized antibodies, and functional fragments thereof, including but not limited to single-domain antibodies (sdAbs), heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH) of camel-derived nanobodies, and alternative scaffolds known in the art for functioning antigen-binding domains, such as recombinant fibronectin domains, T-cell receptors (TCRs), recombinant TCRs with increased affinity or fragments thereof, such as single-chain TCRs, etc. In some cases, it is advantageous for the antigen-binding domain to be derived from the same species from which the CAR will ultimately be used. For example, for use in humans, it may be advantageous for the antigen-binding domain of the CAR to contain human or humanized residues of the antigen-binding domain of an antibody or antibody fragment.

[0121] In some aspects, the transmembrane domain of the CAR disclosed herein comprises a hydrophobic α-helix spanning at least a portion of the cell membrane. Different transmembrane domains have been shown to produce different receptor stabilizations. Following antigen recognition, the receptor aggregates and transmits a signal to the cell. In some aspects, the transmembrane domain of the CAR disclosed herein may include the transmembrane domain of the following peptides: CD8 peptide, CD28 peptide, CD25 peptide, CD7 peptide, CD3-ζ peptide, CD4 peptide, 4-1BB peptide, OX40 peptide, ICOS peptide, CTLA-4 peptide, LAX peptide, LAT peptide, PD-1 peptide, LAG-3 peptide, TIM3 peptide, KIR3DS1 peptide, KIR3DL1 peptide, NKG2D peptide, NKG2A peptide, TIGIT peptide, 2B4 peptide, BTLA peptide, LIR-1 (LILRB1) peptide, SIRPα peptide, or may be a synthetic peptide, or any combination thereof.

[0122] In some aspects, the CAR of this disclosure may also include a spacer region connecting the extracellular antigen-binding domain to the transmembrane domain. The spacer region may be flexible enough to allow the antigen-binding domain to orient itself in different directions, thereby facilitating antigen recognition. In some aspects, the spacer region may be a hinge derived from a human protein. For example, the spacer (also referred to herein as a “hinge”) may be a human Ig (immunoglobulin) hinge, including but not limited to IgG4 hinges, IgG2 hinges, CD8a hinges, or IgD hinges. In some aspects, the spacer region may include IgG4 hinges, IgG2 hinges, IgD hinges, CD28 hinges, KIR2DS2 hinges, LNGFR hinges, or PDGFR-β extracellular adapters. In some aspects, the spacer region is located between the antigen-binding domain and the transmembrane domain.

[0123] In some aspects, the CAR disclosed herein comprises one or more cytoplasmic domains or regions. The cytoplasmic domains or regions of the CAR may include intracellular signal transduction domains. Intracellular signal transduction domains are typically responsible for activating immune cells engineered to express the CAR disclosed herein. For example One or more effector functions of T cells (or NK cells). For example, an effector function of a T cell could be cytolytic activity or helper activity, such as the secretion of cytokines. Therefore, in some aspects, the term "intracellular signal transduction domain" refers to a protein portion that transduces effector function signals and directs the cell to perform specialized functions. Although the entire intracellular signal transduction domain can be used, in many cases it is not necessary to use the entire chain. In aspects where a truncated portion of an intracellular signal transduction domain is used, such a truncated portion can be used instead of the corresponding complete chain, i.e., only the truncated portion transduces effector function signals. Some aspects of this disclosure relate to chimeric inhibitory receptors. Chimeric inhibitory receptors can be used, for example, as NOT logic gates for controlling cellular activities, such as immune cell activities. In some aspects, the chimeric inhibitory receptor of this disclosure binds specifically to one or more antigens 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 this disclosure ( For example (including any suitable transmembrane domains used in conjunction with the chimeric receptors of this disclosure) and intracellular domains. In some respects, chimeric inhibitory receptors can inhibit one or more activities of cells, such as immune response cells.

[0124] In some aspects, the CAR disclosed herein comprises one or more components of natural killer (NK) cells, thereby forming an NKCAR. The NK component may be a transmembrane domain, hinge domain, or cytoplasmic domain derived from any suitable natural killer cell receptor, including but not limited to killer cell immunoglobulin-like receptors (KIRs), such as KIR2DL1, KIR2DL2 / L3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, DIR2DS5, KIR3DL1, KIR3DS1, KIR3DL2, KIR3DL3, KIR2DP1, and KIRS. DPI; natural cytotoxic receptors (NCRs), such as NKp30, NKp44, and NKp46; the signaling lymphocyte-activating molecules (SLAM) family of immune cell receptors, such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10; Fc receptors (FcRs), such as CD16 and CD64; and Ly49 receptors, such as LY49A and LY49C. In some aspects, NK-CARs can interact with adaptor molecules or intracellular signaling domains, such as DAP12. The structural components of CARs described above are also applicable to the structure of NK CARs. Exemplary configurations and sequences of CARs containing NK receptor components are described in International Patent Publication WO2014 / 145252, published on 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 incorporated herein by reference in their entirety.

[0126] In some respects, the effector molecule is a human effector molecule. In some respects, at least one effector molecule is a human effector molecule. In some respects, every effector molecule is a human effector molecule.

[0127] In some aspects, the heterologous payload comprises a first effector molecule and a second effector molecule. In some aspects, the heterologous payload comprises a first effector molecule and a second effector molecule, wherein 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, the heterologous payload comprises a first effector molecule and a second effector molecule, wherein the first effector molecule and the second effector molecule are independently selected from a first CAR and a cytokine. In some aspects, the heterologous payload comprises a first effector molecule and a second effector molecule, wherein the first effector molecule and the second effector molecule are independently selected from a first cytokine and a second cytokine.

[0129] In some aspects, the heterologous payload comprises a first effector molecule, a second effector molecule, and a third effector molecule, 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. In some aspects, the heterologous payload comprises a first effector molecule, a second effector molecule, and a third effector molecule, wherein 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, the heterologous payload includes a first-effect molecule, a second-effect molecule, a third-effect molecule, and a fourth-effect molecule, wherein the first-effect molecule, the second-effect molecule, the third-effect molecule, and the fourth-effect molecule are independently selected from: a first CAR, a second CAR, a first cytokine, and a second cytokine.

[0131] Typically, heterologous payloads (including the polycistronic payloads described herein) can comprise polynucleotides of any length capable of being expressed using engineered regulatory elements provided herein (e.g., engineered enhancer sequences described herein and / or engineered promoters containing engineered enhancer sequences). In some aspects, the heterologous payload comprises a polynucleotide of at least 1000 nucleotides (nt). In some aspects, the heterologous payload comprises a polynucleotide of at least 1500 nt. In some aspects, the heterologous payload comprises a polynucleotide of at least 2000 nt. In some aspects, the heterologous payload comprises a polynucleotide of at least 2500 nt. In some aspects, the heterologous payload comprises a polynucleotide of at least 3000 nt. In some aspects, the heterologous payload comprises a polynucleotide of at least 3500 nt. In some aspects, the heterologous payload comprises a polynucleotide of at least 4000 nt. In some aspects, the heterologous payload comprises a polynucleotide of at least 4500 nt. In some aspects, the heterologous payload contains polynucleotides of 5000 nt or less in length. In some aspects, the heterologous payload contains polynucleotides of 4800 nt or less in length. In some aspects, the heterologous payload contains polynucleotides of 4700 nt or less in length. In some aspects, the heterologous payload contains polynucleotides of 4500 nt or less in length.

[0132] In some aspects, the heterologous payload contains polynucleotides with a length between 2000 and 4500 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 2000 and 4700 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 2000 and 4800 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 2000 and 5000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 2200 and 4500 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 2200 and 4700 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 2200 and 4800 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 2200 and 5000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 1000 and 4500 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 1000 and 4700 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 1000 and 4800 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 1000 and 5000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 100 and 4500 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 100 and 4700 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 100 and 4800 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 100 and 5000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 100 and 4700 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 4800 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 5000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 6000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 7000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 8000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 9000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 10,000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 25,000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 50,000 nt.In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 100,000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 500,000 nt. In some aspects, the heterologous payload contains polynucleotides with a length between 10 and 1,000,000 nt.

[0133] In some respects, effector molecules are chemokines. Chemokines are small cytokines or signaling proteins secreted by cells that induce directed chemotaxis within the cell. Chemokines can be divided into four main subfamilies: CXC, CC, CX3C, and XC, all of which exert their biological effects by selectively binding to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines encoded by polynucleotides in heterologous constructs of this 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, CCL25, 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 respects, effector molecules are cytokines. Non-limiting examples of cytokines encoded by polynucleotides in the heterologous constructs of this disclosure include: IL-1-α, IL-1-β, 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, IL-17A, IL-17B / C / D, IL-17E (IL-25), IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24 ...A, IL-17B / C / D, IL-17E (IL-25), IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-15, IL-16, IL-17A, IL-17B / C / D, IL-17E (IL-25), IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-15, IL-16, IL-17A IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36-α, IL-36-β, IL-36-γ, IL-37, IL-38, IFN-α, IFN-β, IFN-γ, TGF-β, GM-CSF, CSF-1 / M-CSF, G-CSF, SCF, TNF-α, TNF-β, growth hormone (GH), prolactin (PRL), erythropoietin (EPO), leptin, FLT3 ligand or combinations thereof.

[0135] In some respects, effector molecules are modulators of the tumor microenvironment. Suitable modulators of the tumor microenvironment used as effector molecules include, but are not limited to, adenosine deaminase, TGF-β inhibitors, immune checkpoint inhibitors, and HPGE2, or any combination thereof.

[0136] In some aspects, the heterologous constructs of this disclosure are configured to produce an effector molecule comprising at least one TGF-β inhibitor. Suitable TGF-β inhibitors used as effector molecules include, but are not limited to, anti-TGF-β peptides, anti-TGF-β antibodies, TGF-β-TRAP, or combinations thereof.

[0137] In some aspects, the heterologous constructs of this disclosure are configured to produce an effector molecule comprising at least one immune checkpoint inhibitor. Suitable immune checkpoint inhibitors used as effector molecules include, but are not limited to, anti-PD-1 antibodies, anti-PD-L1 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-α antibodies, anti-TREM1 antibodies, and anti-TREM2 antibodies, or combinations thereof and / or functional fragments thereof.

[0138] Indicative 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-L1; Bavencio®-Pfizer), and durvalumab (anti-PD-L1; MEDI4736 / Imfinzi). Tecentriq®-Medimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq®-Roche / Genentech), BMS-936559 (anti-PD-L1-BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy®-BMS), lirilumab (anti-KIR; BMS), and monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0139] In some aspects, the heterologous constructs of this disclosure are configured to produce effector molecules comprising at least one therapy for treating a disease or condition. In some aspects, the disease or condition includes cancer.

[0140] In some aspects, the heterologous constructs of this disclosure are configured to produce effector molecules comprising at least one gene replacement therapy. Gene therapy is a therapy that replaces a non-functional, partially functional, or missing disease-related gene with an engineered form of the same disease-related gene, thereby providing a therapeutically relevant amount of the disease-related gene product. In some aspects, gene replacement therapy is a therapy for treating diseases or conditions such as cancer.

[0141] In some respects, the effector molecule may contain a secretion signal peptide (also called a signal peptide or signal sequence) at its N-terminus. Without being bound by theory, the secretion signal peptide or signal sequence is understood as directing a newly synthesized protein destined for secretion or membrane insertion to the appropriate protein processing pathway. In the case of two or more effector molecules, each effector molecule may contain a secretion signal.

[0142] In some respects, secretory signal peptides that can be operatively associated with effector molecules can be natural secretory signal peptides. example like (Secretion signal peptides that are naturally associated with a given effector molecule). In some respects, secretion signal peptides that are operatively associated with an effector molecule can be non-natural secretion signal peptides. Non-natural secretion signal peptides can promote improved expression and function, such as maintaining secretion, particularly in contexts of interest, such as those associated with disease or condition (such as cancer).

[0143] IV. Vectors / Plasmids Another aspect of this disclosure relates to a vector comprising a nucleotide sequence encoding an engineered regulatory element (e.g., an engineered enhancer sequence described herein and / or an engineered promoter containing an engineered enhancer sequence) as described herein. For example Heterogeneous constructs. In some respects, the carrier is an expression carrier. Such expression carriers contain encoding any engineered regulatory elements disclosed herein. For example The nucleotide sequence of any NK cell and / or T cell-specific regulatory element in this article, which is operatively linked to a coding nucleotide sequence ( For example For effector molecules, For example (as described herein) to allow the encoding nucleotide sequence to be expressed in cells or cell-free extracts. Various expression vectors can be used to express nucleic acid molecules encoding engineered regulatory elements disclosed herein, including but not limited to viral expression vectors, prokaryotic expression vectors, and eukaryotic expression vectors (as described herein). For example Yeast expression vectors, insect expression vectors, mammalian expression vectors wait ) and cell-free extract expression vectors.

[0144] It should also be understood that the expression vectors used to practice aspects of the methods described herein may contain additional promoters ( For example Expression vectors may contain inducible, constitutive, or cell-specific (i.e., enhancer elements or both). Expression vectors can contain polynucleotides encoding protein tags or epitope tags to aid in the isolation, purification, or selection of (...). For example(Examples include polyhis tags, FLAG tags, hemagglutinin tags, fluorescent protein tags, bioluminescent tags, and nuclear localization tags). As described herein, the coding sequences of such protein tags or epitope tags may be fused with the coding sequence or may be contained in a separate expression cassette. Non-limiting examples of expression vectors, as well as recognized reagents and conditions for the preparation and use of expression constructs from such expression vectors, are readily available from commercial vendors, including but not limited to 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, preparation, and use of suitable expression vectors are entirely within the competence of those skilled in the art and are standard procedures derived from the teachings herein.

[0145] In some aspects, the vector comprises a transposon / transposase system for incorporating the nucleotides of this disclosure into the host cell genome. In some aspects, the transposon system used according to this disclosure is Sleeping Beauty transposon / transposase or piggyBac transposon / transposase.

[0146] In some aspects, the expression vectors of this disclosure can be provided to cells in the form of viral vectors. Suitable viral vector systems are well known in the art. For example, viral vectors can be derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In some aspects, the vectors of this disclosure are lentiviral vectors. Lentiviral vectors are suitable for long-term gene transfer because such vectors allow for the long-term stable integration of transgenes and their dissemination in daughter cells. Lentiviral vectors are also superior to those derived from oncogenic retroviruses (… For example The vector of this disclosure is a murine leukemia virus (MLV), because lentiviral vectors can transduce non-proliferating cells. In some respects, the vector disclosed herein is an adenovirus vector (A5 / 35).

[0147] In some respects, the vectors disclosed herein contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selective biomarkers. For example (WO 01 / 96584; WO01 / 29058; and US 6,326,193). Various virus-based systems have been developed for transferring genes into mammalian cells. Selected genes (…) can be transferred using techniques known in the art. For example(Alone, or in an expression cassette or heterologous construct) inserted into a vector and packaged in retroviral particles. The recombinant virus can then be isolated and... in vivo or In vitro Delivery to mammalian cells. Various retroviral systems are known in the art.

[0148] In some aspects, the vectors disclosed herein contain regulatory elements, including engineered and / or natural regulatory elements, such as engineered enhancer sequences and / or natural enhancers, which regulate the frequency of transcription initiation. Typically, the spacing between regulatory elements can be flexible, such that transcriptional function is preserved when the regulatory elements are inverted or moved relative to each other. Depending on the promoter (e.g., the engineered promoter described herein), the individual elements can act synergistically or independently to activate transcription.

[0149] In some respects, the vectors disclosed herein may also include secretion-promoting signal sequences, polyadenylation signals and transcription terminators, elements that allow appendage replication and / or elements that allow selection.

[0150] In some aspects, the vectors disclosed herein may also contain selective biomarker genes and / or reporter genes to facilitate the identification and selection of certain cells from a cell population that has been transduced with said vector. For example (This includes engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences). In some aspects, the selectivity marker may be encoded by a multinucleotide separate from the vector and used in the co-transfection procedure. The selectivity marker or reporter gene may be side-linked with an appropriate regulatory sequence to allow expression in host cells. In some aspects, selectivity markers include antibiotic resistance genes. Examples of antibiotic resistance genes include, but are not limited to, kanamycin, spectinomycin, streptomycin, ampicillin, carbenicillin, bleomycin, erythromycin, polymyxin B, tetracycline, chloramphenicol, neomycin, and combinations thereof.

[0151] In some respects, reporter genes can be used to identify transduced cells and to assess the function of regulatory sequences. As disclosed herein, a reporter gene is a gene that is not present in or expressed by a recipient organism or tissue and encodes a polypeptide with easily detectable properties, such as enzyme activity or fluorescence. The expression of a reporter gene can be determined at an appropriate time after a polynucleotide containing the reporter gene is introduced into a recipient or host cell capable of expressing the reporter gene. Examples of reporter genes include, but are not limited to, those encoding luciferases (or variants or fragments of luciferases). For example Genes encoding nano-luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, and green fluorescent protein (GFP) (or common variants or fragments of GFP) are included. For exampleRFP, YFP wait The gene is expressed in [the specified gene]. Suitable expression systems are well known in the art and can be prepared or commercially available using known techniques.

[0152] In some aspects, the vector may include a dual-expression vector system, such as a vector encoding multiple boxes, wherein each box encodes a separate heterogeneous construct capable of driving the expression of a heterogeneous payload. For example, a dual-expression vector system may include a vector in which each individual box contains a separate engineered regulatory element (e.g., engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences), each engineered regulatory element driving the expression of a separate heterogeneous payload. The individual engineered regulatory element contains one of the engineered promoters described herein. In some aspects, each engineered regulatory element in a dual-expression vector contains one of the engineered promoters described herein.

[0153] V. Engineered Cells This disclosure also provides methods and compositions for preparing and using engineered cells (e.g., immune-response cells, such as NK cells or T cells) comprising a heterologous construct containing engineered regulatory elements as provided herein (e.g., engineered enhancer sequences described herein and / or engineered promoters containing engineered enhancer sequences).

[0154] In some respects, engineered cells contain engineered regulatory elements (e.g., engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences provided herein).

[0155] This article also provides information for engineering molecules capable of producing one or more effectors. For example Compositions and methods for cellular applications involving first-effect molecules, second-effect molecules, third-effect molecules, etc. The effector molecules disclosed herein may be encoded in one or more heterologous constructs or expression cassettes as described herein or known in the art.

[0156] In some respects, the cells of this disclosure are engineered to introduce ( Right now (delivery) encodes one or more effector molecules ( For example The effector molecules are generated by using one or more polynucleotides (such as those described herein). For example, the polynucleotide heterologous construct or expression cassette encoding one or more effector molecules can be any of the engineered nucleic acids described herein. Delivery methods include, but are not limited to, virus-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. For example (Including all methods described herein). Those skilled in the art will understand that the choice of delivery method can depend on the specific cell type to be engineered.

[0157] Engineered cells may include immune-response cells having any of the heterologous constructs described herein. In some aspects, immune-response cells are selected from: natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, γ-δ T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, B cells, macrophages, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, monocytes, dendritic cells, erythrocytes, platelets, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some aspects, immune-response cells are NK cells. In some aspects, immune-response cells are T cells.

[0158] In some respects, immune-response cells express activated immune receptors, which are antigen recognition receptors, such as CARs.

[0159] In some respects, immune response cells are autologous. In other respects, immune response cells are allogeneic.

[0160] A. Virus-mediated delivery Viral vector-based delivery platforms can be engineered to deliver specific nucleic acids of interest into host cells to generate engineered cells as described herein. Typically, viral vector-based delivery platforms can be used to deliver heterologous nucleic acids (… For example The introduction of transgenic, expression cassette, or heterologous constructs, as described in this article, is ( Right now Viruses are delivered or transduced into specific host cells to generate engineered cells. In many aspects of this disclosure, viral vector-based delivery platforms deliver nucleic acids containing engineered regulatory elements as described herein (e.g., engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences), or heterologous constructs as described herein. In some aspects, the transduced nucleic acid is integrated into the host cell genome. Viruses generated for use in viral vector-based delivery platforms may be referred to as recombinant viruses or engineered viruses. It should be understood that, in addition to the nucleic acid to be delivered to the host cell, recombinant viruses may also encode one or more viral genes required for viral infectivity and / or viral production. For example Capsid proteins, envelope proteins, viral polymerase, viral transcriptase wait In some cases, these are called cis-acting elements or cis-acting genes.

[0161] In some respects, recombinant viruses can be used to deliver genes encoding one or more genes. For example The delivered nucleic acid, expression cassette, or heterologous construct (transgenic). In many respects, the delivered nucleic acid comprises one or more nucleotide sequences that contain engineered regulatory elements as described herein (e.g., engineered enhancer sequences and / or engineered promoters containing engineered enhancer sequences). In some respects, the delivered nucleic acid (transgenic) For example (Heterogeneous constructs) are configured to express one or more effector molecules.

[0162] Viral vector-based delivery platforms may include more than one viral vector, such as genes encoding the trans-acting element or trans-acting gene described herein. For example Separate viral vectors encoding transgenic expression cassettes or heterologous constructs. For example, in addition to vectors encoding transgenic expression cassettes or heterologous constructs, helper-dependent viral vector-based delivery platforms can deliver additional genes required for viral infectivity and / or viral production on one or more additional separate vectors. The number of viral vectors used can depend on the packaging capacity of the aforementioned viral vector-based platforms, and those skilled in the art can select an appropriate number of viral vectors.

[0163] Typically, any viral vector-based system can be used for engineered nucleic acids ( For example (heterogeneous constructs) in vitro Express, For example Used to generate molecules, such as effector molecules, or for in vivo and In vitro Gene therapy procedures, For example , used for body Inside Delivery of engineered nucleic acids containing nucleotide sequences encoding one or more effector molecules under transcriptional control of engineered regulatory elements (e.g., engineered enhancer sequences described herein and / or engineered promoters containing engineered enhancer sequences). The selection of a system based on an appropriate viral vector will depend on a variety of factors, such as cargo / payload size, immunogenicity of the viral system, target cells of interest, gene expression intensity and timing, and other factors understood by those skilled in the art.

[0164] Virus vector-based delivery platforms can utilize RNA-based or DNA-based viruses. Exemplary virus vector-based delivery platforms include, but are not limited to, herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana vesicular virus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, Malaba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, measles virus, lentivirus, replicating retrovirus, rhabdovirus, Seneca Valley virus, Sindbis virus, and any variants or derivatives thereof. Other exemplary virus vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, Malaba virus, adenovirus (see [link to documentation]). ， For example Tatsis et al. Adenoviruses, Molecular Therapy (2004) 10, 616–629), or lentiviruses, including but not limited to second-generation lentiviruses, third-generation lentiviruses, or hybrid second-generation / third-generation lentiviruses, and any first-generation recombinant lentivirus designed to target a specific cell type or receptor (see, ... For example Hu et al. , ImmunizationDelivered by Lentiviral Vectors for Cancer and Infectious Diseases, ImmunolRev. (2011) 239(1): 45-61, Sakuma et al. , Lentiviral vectors: basic totranslational, Biochem J. (2012) 443(3):603-18, Cooper et al. , Rescue ofsplicing-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] The provided engineered nucleic acid sequence may be preceded by one or more nucleic acid sequences, which, either individually or via their encoded polypeptide sequences, target subcellular compartments. The engineered nucleic acid is then introduced into (… Right now After being delivered to the host cell, the infected cells ( Right now Engineered cells can express polypeptides encoded by the engineered nucleic acids. For example (one or more effector molecules), and in some cases secrete the polypeptide. Vacciniocytosis vectors and methods for use in immunization programs are described in [the relevant section]. For example In U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guérin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). According to the description herein, it can be used to introduce ( Right now (delivery) various other vectors of engineered nucleic acids ( For example (Salmonella typhi vectors, etc.) will be obvious to those skilled in the art.

[0166] The viral vector-based delivery platform described herein can utilize viruses that target tumor cells, referred to herein as oncolytic viruses. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indiana vesicular virus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic Malaba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic measles virus, oncolytic lentivirus, oncolytic replicating retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variants or derivatives thereof. Any oncolytic virus described herein may contain one or more engineered nucleic acids (e.g., genes, expression cassettes, heterologous constructs). wait Recombinant oncolytic virus.

[0167] In some respects, recombinant viruses are produced by viruses selected from the following: lentiviruses, retroviruses, oncolytic viruses, adenoviruses, adeno-associated viruses (AAVs), and virus-like particles (VLPs).

[0168] The viral vector-based delivery platform used according to this disclosure can be retroviral. Generally, retroviral vectors consist of cis-acting long terminal repeat sequences, wherein the encapsulation capacity is a maximum of 6-10 kb of foreign sequence. A minimal cis-acting LTR is sufficient for replicating and packaging the vector, which is then used to deliver one or more engineered nucleic acids (e.g., genes, expression cassettes, heterologous constructs). wait The engineered nucleic acid is integrated into target cells to provide permanent integration and / or expression. Retrovirus-based delivery systems include, but are not limited to, those based on murine leukemia virus (MuLV), gibberish leukemia virus (GaLV), simultaneous immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see [link to relevant documentation]). For example 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 wait people , J. Virol. 63:2374-2378 (1989); Miller et al. (J, Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retroviral system.

[0169] The viral vector-based delivery platform used according to this disclosure can be lentivirus-based. Generally, lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing high viral titers. Lentiviral-based delivery platforms can be HIV-based, such as the ViraPower system (Thermo Fisher Scientific) or the pLenti system (Cell Biolabs). Lentiviral-based delivery platforms can also be SIV or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Patents 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, and 6,955,919, each of which is incorporated herein by reference for all purposes.

[0170] The viral vector-based delivery platform used in this disclosure can be adenovirus-based. Generally, adenovirus-based vectors can achieve very high transduction efficiency in many cell types, without requiring cell division, achieving high titers and expression levels, and can be mass-produced in relatively simple systems. Generally, adenoviruses can be used for transient expression of transgenes in infected cells because adenoviruses typically do not integrate into the host 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 92:7700 7704, 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 incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Patent Nos. 5,585,362; 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 incorporated herein by reference for all purposes.

[0171] The viral vector-based delivery platform used according to this disclosure can be based on adeno-associated virus (AAV). An AAV vector can be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). The AAV system can be used for... in vitro To produce effector molecules, or to be used for in vivo and In vitro Gene therapy procedures, For example For in vivo Delivery of engineered nucleic acids, For example Engineered nucleic acids that encode one or more effector molecules (see, For example West et al. Virology 160:38-47 (1987); U.S. Patent Nos. 4,797,368, 5,436,146, 6,632,670, 6,642,051, 7,078,387, 7,314,912, 6,498,244, and 7,906,111; U.S. 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, HumanGene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each incorporated herein by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Patent No. 5,173,414; Tratschin et al. , Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin And others. , Mol. Cell, Biol. 4:2072-2081 (1984); Muzyczka, PNAS 81:64666470 (1984); and Samuiski et al.In J. Virol. 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes. Generally, AAV-based vectors contain a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, and their variants.

[0172] The viral vector-based delivery platform used in this disclosure can be a virus-like particle (VLP) platform. Generally, a VLP is constructed by generating viral structural proteins and purifying the resulting viral particles. Then, after purification, the cargo / payload ( For example (Any engineered nucleic acid described in this article) In vitro The virus is encapsulated within purified particles. Therefore, VLP production maintains the separation of nucleic acids encoding viral structural proteins from those encoding the cargo / payload. Viral structural proteins used for VLP production can be generated in a variety of expression systems, including mammals, yeast, insects, bacteria, or... in vivo Translation expression system. Purified viral particles can be denatured and reformed in the presence of the desired cargo using methods known to those skilled in the art to generate VLPs. The generation of VLPs is described in more detail in Seow. et al. (Mol Ther. 2009 May; 17(5):767–777), which is incorporated herein by reference for all purposes.

[0173] The viral vector-based delivery platform used in this disclosure can be engineered to target ( Right now Viral vector-based delivery platforms can infect or transduce a range of cells, target a narrow subset of cells, or target specific cells. Typically, the choice of envelope protein for a viral vector-based delivery platform determines viral tropism. Viruses used in viral vector-based delivery platforms can be pseudotyped to target specific cells of interest. Viral vector-based delivery platforms can be pantropy and infect a range of cells. For example, a pantropy-based viral vector delivery platform may include a VSV-G envelope. Viral vector-based delivery platforms can be amphiphilic and infect mammalian cells. Therefore, those skilled in the art can select appropriate tropism, pseudotype, and / or envelope proteins to target the desired cell type.

[0174] B. Lipid Structure Delivery System The engineered nucleic acids of this disclosure can be delivered using a lipid-mediated delivery system. For exampleLipid-mediated delivery systems typically utilize structures consisting of an outer lipid membrane encapsulating the internal compartments. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. Lipid-based delivery systems can... in vitro , in vivo or In vitro Delivery of goods / payload ( For example (any engineered nucleic acid described in this article).

[0175] Lipid-based nanoparticles can include, but are not limited to, monolayer liposomes, multilayer liposomes, and lipid formulations. As used herein, "liposome" is a generic term that encompasses the process of transporting desired goods ( For example Lipid mediators formed by encapsulating engineered nucleic acids (such as any engineered nucleic acids as described herein) in a lipid shell or lipid aggregate. in vitro Formulations. Liposomes are characterized by a vesicle structure with a bilayer membrane, typically containing phospholipids, and an internal medium that typically contains an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes can be monolayer liposomes. Liposomes can be multilayer liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or uncharged. In some respects, liposomes are charge-neutral. Liposomes can be formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The selection of lipids is usually guided by consideration of the desired purpose. For example, in the body Delivery criteria, such as liposome size, acid instability, and stability of liposomes in the bloodstream, are important considerations. Several methods can be used to prepare liposomes, described in [the document / section / etc.]. For example Szoka et al. , Ann. Rev. Biophys. Bioeng. 9; 467 (1980); U.S. Patent Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028 and 5,019,369, each incorporated herein by reference for all purposes. When phospholipid-containing lipids are suspended in an excess of aqueous solution, allowing multiple lipid layers to be separated by the aqueous medium, multilayered liposomes spontaneously form. Following self-rearrangement of the lipid components, water and dissolved solutes are trapped within the closed structure between the lipid bilayers. (Required goods) For example Polypeptides, nucleic acids, small molecule drugs, engineered nucleic acids such as any engineered nucleic acids described herein, viral vectors, and virus-based delivery systems. waitThese molecules can be encapsulated within the aqueous interior of liposomes, attached to liposomes via linkers that associate with both the liposome and the peptide / nucleic acid, dispersed within the lipid bilayer of the liposome, embedded in the liposome, complexed with the liposome, or otherwise associated with the liposome, allowing them to be delivered to the target. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0176] As is known to those skilled in the art, liposomes used according to this disclosure can be prepared by various methods. The preparation of liposomes is further described in detail in WO 2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706, each of which is incorporated herein by reference for all purposes.

[0177] Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent Nos. 5,962,016, 5,030,453, and 6,680,068, U.S. Application 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO 04029213A2, and WO 02 / 100435A1, each of which is incorporated herein by reference. Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Patent No. 5,279,833; Rose U.S. Patent No. 5,279,833; WO91 / 06309; and Felgner et al. , Proc. Natl. Acad. Sci. USA84: 7413-7414 (1987), each incorporated herein by reference for all purposes.

[0178] As used herein, the term "exosome" refers to a small, cell-derived vesicle (with a diameter between 20 and 300 nm, more preferably between 40 and 200 nm) containing a membrane enclosing its internal space, and generated from the cell via direct plasma membrane budding or fusion of late endosomes with the plasma membrane. Exosomes contain lipids or fatty acids and peptides, and optionally include a payload ( For example Therapeutic agents), recipients ( For example Targeted portion), polynucleotides ( For exampleNucleic acids, RNA, or DNA, such as any engineered nucleic acids as described herein, and sugars ( For example Exosomes (monosaccharides, polysaccharides, or glycans) or other molecules. Exosomes can be derived from production cells and isolated from production cells based on their size, density, biochemical parameters, or combinations thereof. An exosome is an extracellular vesicle. Typically, the production / biogenesis of exosomes does not lead to the destruction of production cells. Exosomes and their preparation are described in further detail in WO 2016 / 201323, which is incorporated herein by reference in its entirety. Exosomes that can be used to deliver nucleic acids are known to those skilled in the art. For example Exosomes are described in more detail in U.S. Patent No. 9,889,210, which is incorporated herein by reference for all purposes.

[0179] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle containing a membrane enclosing its internal space. Generally, extracellular vesicles encompass all membrane-bound vesicles, which are smaller in diameter than the cells from which they originate. Typically, extracellular vesicles range in diameter from 20 nm to 1000 nm and can contain various macromolecular cargoes residing within their internal space, displayed on their outer surface, and / or spanning the membrane. The cargo may include nucleic acids (…). For example Extracellular vesicles include, but are not limited to, any engineered nucleic acids as described herein, proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. As examples, and not limitations, extracellular vesicles include apoptotic bodies, cell debris, and those manipulated directly or indirectly (…). For example Vesicles, vesicular organelles, and other structures derived from cells (through continuous extrusion or treatment with an alkaline solution) are formed from living cells. For example Extracellular vesicles are produced through direct budding of the cytoplasmic membrane or fusion of late endosomes with the cytoplasmic membrane. Extracellular vesicles can originate from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0180] As used herein, the term "nanovesicle" (also known as "microvesicles") refers to small, cell-derived vesicles (with a diameter between 20 and 250 nm, more preferably between 30 and 150 nm) containing a membrane enclosing their internal space, and which are generated from cells by direct or indirect manipulation such that the producing cells do not generate the nanovesicles without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Appropriate manipulation of the producing cells includes, but is not limited to, continuous squeezing, treatment with an alkaline solution, sonication, or combinations thereof. In some cases, the generation of nanovesicles can lead to the destruction of the producing cells. Preferably, the nanovesicle population is substantially free of vesicles obtained from the producing cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. Nanovesicles contain lipids or fatty acids and peptides, and optionally contain a payload ( For exampleTherapeutic agents), recipients ( For example Targeted portion), polynucleotides ( For example Nucleic acids, RNA, or DNA, such as any engineered nucleic acids as described herein, and sugars ( For example (monosaccharides, polysaccharides, or glycans) or other molecules. Once derived from the production cell according to the aforementioned manipulation, nanovesicles can be isolated from the production cell based on their size, density, biochemical parameters, or combinations thereof.

[0181] Generally, lipid nanoparticles (LNPs) are engineered lipid structures that rely on the amphiphilic nature of lipids to form membrane and vesicle-like structures (Riley 2017). Typically, these vesicles deliver cargo / payloads, such as any engineered nucleic acid or viral systems described herein, by taking up into the membrane of a target cell and releasing cargo into the cytosol. Lipids used for LNP formation can be cationic, anionic, or neutral. Lipids can be engineered or naturally derived and, in some cases, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates, including but not limited to polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins. Lipid compositions typically comprise a defined mixture of materials, such as cationic lipids, neutral lipids, anionic lipids, and amphiphilic lipids. In some cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional chemical groups that promote attachment of additional portions. Lipid compositions can influence the overall size and stability of the LNP. In one example, the lipid composition contains dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids. Furthermore, 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 understood by those skilled in the art.

[0182] Micelles are typically engineered, spherical lipid structures formed using single-chain lipids, where the hydrophilic heads of the single-chain lipids form the outer layer or membrane, and the hydrophobic tails form the micelle centers. Micelles generally refer to lipid structures containing only a lipid monolayer. Micelles are described in more detail in Quader... et al. (Mol Ther. 2017 Jul 5; 25(7): 1501–1513), which is incorporated herein by reference for all purposes.

[0183] Nucleic acid vectors (such as expression vectors) directly exposed to serum can have several undesirable consequences, including degradation of nucleic acids by serum nucleases or off-target stimulation of the immune system by free nucleic acids. Similarly, viral delivery systems directly exposed to serum may trigger undesirable immune responses and / or neutralization. Therefore, encapsulation of engineered nucleic acid and / or viral delivery systems can be used to avoid degradation, as well as potential off-target effects. In some instances, engineered nucleic acid and / or viral delivery systems are completely encapsulated within a delivery medium, such as within an aqueous LNP or other vesicle or lipid system as described herein. Encapsulation of engineered nucleic acid and / or viral delivery systems within an LNP or other lipid system can be performed using techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation on a microfluidic droplet generation device. Such devices include, but are not limited to, standard T-connector devices or flow focusing devices. In one example, the desired lipid formulation (such as a composition containing MC3 or MC3-like substances) is supplied in parallel to a droplet generating device along with an engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery carrier and desired agents are completely encapsulated within the MC3-based or MC3-like LNP. In one example, the droplet generating device can control the size range and size distribution of the generated LNPs. For example, the size of the LNPs can range from 1 to 1000 nanometers in diameter. For example 1, 10, 50, 100, 500, or 1000 nanometers. After droplet formation, the cargo / payload can be further processed or engineered for encapsulation. For example Delivery media (engineered nucleic acid and / or virus delivery systems) for their preparation for application.

[0184] C. Nanoparticle delivery Nanomaterials can be used to deliver engineered nucleic acids ( For example, (Any engineered nucleic acid described herein). Importantly, the nanomaterial mediators can be made of non-immunogenic materials and generally avoid inducing immunity against the delivery carrier 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 on Riley. et al. (Recent Advances in Nanomaterials for GeneDelivery—A Review. Nanomaterials 2017, 7(5), 94), each incorporated herein by reference for all purposes.

[0185] D. Genome editing system Genome editing systems can be used to engineer host genomes to encode engineered nucleic acids, such as any engineered nucleic acids described herein. Generally, "genome editing system" refers to any system used to integrate exogenous genes into the host cell genome. Genome editing systems include, but are not limited to, transposon systems, nuclease genome editing systems, and viral vector-based delivery platforms. For example (Those mentioned in this article).

[0186] The transposon subsystem can be used to transfer engineered nucleic acids ( For example Transposons integrate cargo / payload (e.g., engineered nucleic acids, as described herein) into the host genome. Transposons typically consist of terminal inverted repeats (TIRs) flanking the cargo / payload nucleic acid and the transposase. Transposon systems can provide transposons in either cis or trans positions with the cargo flanked by the TIRs. Transposon systems can be retrotransposon systems or DNA transposon systems. Generally, transposon systems randomly integrate cargo / payload (e.g., engineered nucleic acids) into the host genome. Examples of transposon systems include systems using transposons from the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, which is 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 incorporated herein by reference for all purposes. Another example of a transposon subsystem includes the PiggyBac transposon subsystem, which is described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each incorporated herein by reference for all purposes.

[0187] Nuclease-mediated genome editing systems can be used to engineer host genomes to encode engineered nucleic acids, such as those disclosed herein. Not wishing to be bound by theory, nuclease-mediated gene editing systems for introducing exogenous genes or nucleic acids typically utilize the cell's natural DNA repair mechanisms, particularly the homologous recombination (HR) repair pathway. In short, after genomic DNA is damaged (usually a double-strand break), the cell can resolve the damage by using another DNA source with the same or substantially identical sequences at its 5' and 3' ends as a template during DNA synthesis. In nature, HR can use other chromosomes present in the cell as templates. In gene editing systems, exogenous polynucleotides are introduced into the cell to serve as homologous recombination templates (HRTs or HR templates). Generally, any additional exogenous sequence contained between the 5' and 3' complementary ends within the HRT that was not initially found in the chromosome with the damage ( For example A gene or a portion thereof, or an engineered nucleic acid as described herein, can be incorporated during templated HDR. Right now "Integration" into a given genomic locus. Therefore, a typical HR template for a given genomic locus has the same nucleotide sequence as the first region of the endogenous genomic target locus, the same nucleotide sequence as the second region of the endogenous genomic target locus, and the nucleic acid encoding the cargo / payload. For example As described in this article, any engineered nucleic acid For example Engineered nucleic acids that encode one or more effector molecules.

[0188] In some instances, the HR template can be linear. Examples of linear HR templates include, but are not limited to, linearized plasmid vectors, ssDNA, synthetic DNA, and PCR-amplified DNA. In certain instances, the HR template can be circular, such as plasmids. Circular templates can include supercoiled templates.

[0189] The identical or substantially identical sequences found at the 5' and 3' ends of the HR template relative to the foreign sequence to be introduced are usually called arms (HR arms). HR arms can be identical to regions of target loci in the endogenous genome. Right now (100% identical). In some instances, the HR arm can be substantially identical to the region of the endogenous genome target locus. While substantially identical HR arms can be used, HR arm identity may be advantageous because the efficiency of the HDR pathway can be affected by HR arms with less than 100% identity.

[0190] Each HR arm, Right now The 5' and 3' HR arms can be the same or different sizes. The length of each HR arm can be greater than or equal to 50, 100, 200, 300, 400, or 500 bases. While HR arms can generally be of arbitrary length, practical factors such as the impact of HR arm length and overall template size on overall editing efficiency can be considered. HR arms can be identical or substantially identical to the region of the endogenous genome target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genome target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genome target locus within a certain distance from the cleavage site, such as a distance of 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.

[0191] Nuclease genome editing systems can use a variety of nucleases to cut target genomic loci, including but not limited to CRISPR family nucleases or their derivatives, transcription activator-like effector nucleases (TALENs) or their derivatives, zinc finger nucleases (ZFNs) or their derivatives, and homing endonucleases (HEs) or their derivatives.

[0192] CRISPR-mediated gene editing systems can be used to engineer host genomes to encode engineered nucleic acids as described in this article. For example Engineered nucleic acids that encode one or more effector molecules described herein. The CRISPR system is described in more detail in M. Adli (“The CRISPR toolkit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), which is incorporated herein by reference for all purposes. Generally, a CRISPR-mediated gene editing system comprises a CRISPR-associated (Cas) nuclease and RNA that guides cleavage to a specific target sequence. An exemplary CRISPR-mediated gene editing system is a CRISPR / Cas9 system consisting of a Cas9 nuclease and RNA having a CRISPR RNA (crRNA) domain and a trans-activation CRISPR (tracrRNA) domain. crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that guides specificity to the target sequence (“defined nucleotide sequence”) via base pair hybridization. For example The genome sequence; and the RNA domain, which hybridizes to tracrRNA. tracrRNA can bind to nucleases (…). For example The crRNA and tracrRNA polynucleotides interact with Cas9, thereby facilitating the recruitment of nucleases to genomic loci. The crRNA and tracrRNA polynucleotides can be individual polynucleotides, also known as single guide RNAs (sgRNAs). While the Cas9 system is described here, other CRISPR systems, such as the Cpf1 system, can also be used. Nucleases can include their derivatives, such as Cas9 functional mutants. For example The Cas9 “nicking enzyme” mutant typically mediates only single-strand cleavage of a defined nucleotide sequence, rather than the complete double-strand breaks that are usually produced by the Cas9 enzyme.

[0193] Generally, 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 generated independently and used to form the RNP complex. In other CRISPR systems, the individual components can... in vitro Produced separately, and in vitro Contact with each other ( Right now "Complexing" to form an RNP complex. Subsequently, it can be... in vitro The resulting RNP import ( Right now "Delivered" to the cytoplasm and / or nucleus of the cell. For example In the cytoplasm and / or nucleus of T cells. in vitro The resulting RNP complex can be delivered to cells via various means, including but not limited to electroporation, lipid-mediated transfection, cell membrane deformation via physical means, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In one specific example, in vitro The generated RNP complex can be delivered to cells using the Nuclefactor / Nucleofection® electroporation delivery system (Lonza®). Other electroporation systems include, but are not limited to, the MaxCyte electroporation system, the Miltenyi CliniMACS electroporation system, the Neon electroporation system, and the BTX electroporation system. A variety of protein production techniques known to those skilled in the art can be used to... in vitro produce( Right now (Synthesis and purification) CRISPR nuclease, For example Cas9. A variety of RNA production techniques known to those skilled in the art (e.g., in vitro transcription or chemical synthesis) can be used to produce (i.e., synthesize and purify) CRISPR system RNAs, such as sgRNA, in vitro.

[0194] in vitro The resulting RNP complex can be complexed with gRNA at different ratios of nucleases. in vitro The resulting RNP complexes can also be used in different amounts in CRISPR-mediated editing systems. For example, the total amount of added RNPs can be adjusted depending on the number of cells to be edited, such as reducing the amount of added RNP complexes when editing a large number of cells in a reaction.

[0195] In some CRISPR systems, each component ( For example Cas9 and sgRNA can be encoded by individual polynucleotides, with each polynucleotide introduced into the cell either together or individually. In some CRISPR systems, individual components can be encoded by a single polynucleotide (CRISPR, Cas9, and sgRNA). Right now(Multi-promoter or polycistronic vectors, see the description of exemplary polycistronic systems below) encode and are introduced into cells. Each polynucleotide-encoded CRISPR component is expressed in the cell ( For example After the translation of nucleases and the transcription of CRISPRRNA, the RNP complex can be formed in the cell and then guide site-specific cleavage.

[0196] Some RNPs can be engineered to have portions that facilitate RNP delivery to the cell nucleus. For example, the Cas9 nuclease can have a nuclear localization signal (NLS) domain, which allows the NLS to facilitate further transport of the Cas9 RNP to the cell nucleus if the Cas9 RNP complex is delivered into the cytoplasm of the cell or after Cas9 translation and subsequent RNP formation.

[0197] The engineered cells described in this article can be engineered using non-viral methods. For example Non-viral methods can be used to deliver the nucleases and / or CRISPR-mediated gene editing systems described herein into cells. Viral methods can be used to engineer the engineered cells described herein. For example The nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using viral methods (such as adenovirus, retrovirus, lentivirus, or any other virus-based delivery method described herein).

[0198] In some CRISPR systems, more than one CRISPR composition can be provided, each targeting the same gene or general genomic locus at more than one target nucleotide sequence. For example, two separate CRISPR compositions can be provided to guide cleavage at two different target nucleotide sequences at a certain distance from each other. In other CRISPR systems, more than one CRISPR composition can be provided, each targeting the opposite strand of the same gene or general genomic locus individually. For example, two separate CRISPR "nicking enzyme" compositions can be provided to guide cleavage at the opposite strand of the same gene or general genomic locus.

[0199] Generally, the characteristics of the CRISPR-mediated editing system described herein can be applied to other nuclease-based genome editing systems. TALENs are engineered site-specific nucleases consisting of a DNA-binding domain of TALE (transcription activator-like effector) and a catalytic domain of the restriction endonuclease Fokl. Different artificial TALENs can be generated to target a variety of nucleotide sequences by altering amino acids in highly variable residue regions present in the DNA-binding domain monomer. The DNA-binding domain then directs the nuclease to the target sequence and generates a double-strand break. TALEN-based systems are described in more detail in U.S. Serial No. 12 / 965,590; U.S. Patent No. 8,450,471; U.S. Patent No. 8,440,431; U.S. Patent No. 8,440,432; U.S. Patent No. 10,172,880; and U.S. Serial No. 13 / 738,381, all of which are incorporated herein by reference in their entirety. The ZFN-based editing system is 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, and 7,253,273; and U.S. Patent Publications Nos. 2005 / 0064474, 2007 / 0218528, and 2005 / 0267061, all of which are incorporated herein by reference in their entirety for all purposes.

[0200] E. Other engineered delivery systems engineered nucleic acids ( For example Various other means of introducing (such as any engineered nucleic acid described herein) into cells or other target receptor entities (such as any lipid structure described herein).

[0201] Electroporation can be used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing cargo / payload into the internal compartments of a target cell or entity by applying an electric field to temporarily permeate the outer membrane or shell of the target cell or entity. Typically, this method involves placing the cell or target entity containing the cargo of interest (…). For example Between two electrodes in a solution of any engineered nucleic acid (as described herein). Then, a temporarily set voltage is applied to disrupt ( Right now (Permeable) to the lipid membrane of a cell, which allows cargo to enter the interior of the entity, such as the cytoplasm of a cell. In cellular instances, at least some (if not most) cells remain alive. Cells and other entities can... in vitro , in vivo or In vitroPerform electroporation. Electroporation conditions ( example like Cell count, cargo concentration, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cup length, and electroporation solution composition. wait The specific requirements vary depending on several factors, including, but not limited to, the type of cell or other receptor entity, the cargo to be delivered, the required internalization efficiency, and the required viability. Optimization of such criteria is within the capabilities of those skilled in the art. A variety of devices and protocols are available for electroporation. Examples include, but are not limited to, the Neon® transfection system, the MaxCyte® FlowElectroporation™, the Lonza® Nucleofector™ system, and the Bio-Rad® electroporation system.

[0202] Other means of introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into cells or other target receptor entities include, but are not limited to, sonication, gene gun, hydrodynamic injection, and cell membrane deformation by physical means.

[0203] Compositions and methods for in vivo delivery of engineered mRNA (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. (GenomeMed. 2017; 9: 60.), each of which is incorporated herein by reference for all purposes.

[0204] VI. How to Use This disclosure covers methods and compositions for treating a subject suffering from a disease or condition. The provided methods include administering to a subject suffering from a disease or condition a therapeutically effective amount of any engineered nucleic acid described herein that contains an engineered regulatory element (e.g., an engineered enhancer sequence described herein and / or an engineered promoter containing an engineered enhancer sequence). For example Heterologous constructs, vectors, dual expression vectors), engineered cells ( For example (isolated engineered cells) and / or pharmaceutical compositions.

[0205] In some respects, the methods and compositions provided herein can be used to treat diseases or conditions. In some respects, diseases or conditions include cancer.

[0206] This article also describes methods for increasing the expression of target genes or heterologous payloads, methods including the use of engineered regulatory elements described herein (e.g., engineered enhancer sequences described herein and / or engineered promoters containing engineered enhancer sequences), such as any vector or dual-expression vector containing engineered regulatory elements described herein. In some respects, the target gene is an immune regulatory gene.

[0207] VII. Pharmaceutical Composition The provided engineered nucleic acids or engineered cells can be formulated into pharmaceutical compositions. In addition to one or more engineered nucleic acids or engineered cells, these compositions may also contain pharmaceutically acceptable excipients, carriers, buffers, stabilizers, 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 exact properties of the carrier or other materials may depend on the route of administration. For example It can be administered orally, intravenously, through the skin or subcutaneously, via the nose, intramuscularly, or intraperitoneally.

[0208] Pharmaceutical compositions intended for oral administration may be in tablet, capsule, powder, or liquid form. Tablets may include a solid carrier, such as gelatin or an adjuvant. Liquid pharmaceutical compositions typically include a liquid carrier, such as water, petroleum, animal or vegetable oil, mineral oil, or engineered oil. They may include physiological saline, dextran or other sugar solutions, or glycols, such as ethylene glycol, propylene glycol, or polyethylene glycol.

[0209] For intravenous, skin, or subcutaneous injection, or injection at the site of pain, the active ingredient will be in the form of a parenteral-acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art can prepare suitable solutions using, for example, isotonic mediators (such as sodium chloride injection, Ringer's solution, lactated Ringer's solution). Preservatives, stabilizers, buffers, antioxidants, and / or other additives may be included, if necessary.

[0210] The compositions provided herein can be applied alone or in combination with other treatments, simultaneously or sequentially, depending on the condition to be treated.

[0211] VIII. Reagent Kit Some aspects of this disclosure relate to reagent kits for treating and / or preventing diseases or conditions. In some aspects, the disease or condition is cancer (…). For example (Tumors, such as solid tumors). In some respects, the kit includes a therapeutic or prophylactic composition comprising an effective amount of one or more engineered nucleic acids disclosed herein. For exampleThe kit includes isolated engineered nucleic acids, vectors, and / or engineered cells of this disclosure, wherein the engineered nucleic acids, vectors, and / or engineered cells contain the engineered regulatory elements described herein (e.g., engineered enhancer sequences described herein and / or engineered promoters containing engineered enhancer sequences). In some aspects, the kit includes a sterile container. In some aspects, such a container may be a box, ampoule, bottle, vial, tube, bag, pouch, blister pack, or other suitable container forms known in the art. The container may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing pharmaceuticals.

[0212] In some respects, therapeutic or preventative compositions (e.g., engineered cells, such as engineered immune response cells described herein) are associated with the application of said therapeutic or preventative compositions to individuals who have or are at risk of developing a particular disease or condition (e.g., cancer The instructions for use are provided together with the subject's information. In some aspects, the instructions for use may include information about the use of the composition to treat and / or prevent a disease or condition. In some aspects, the instructions for use include, but are not limited to, a description of the therapeutic or preventive composition, dosage regimen, administration regimen for the treatment or prevention of a disease or condition or its symptoms, precautions, warnings, indications, contraindications, overdose information, adverse reactions, animal pharmacology, clinical studies, and / or references. In some aspects, the instructions for use may be printed directly on the container (where present), applied as a label to the container, or provided as a separate sheet of paper, brochure, card, or folder in or with the container.

[0213] Throughout the instruction manual, the terms "agent," "compound," and "entity" are used. For example When the engineered nucleic acid provided is described as having, including, or containing a specific component, or when the process and method are described as having, including, or containing a specific step, it is anticipated that there will be additional agents, compounds, entities, etc. of the invention that are substantially composed of or composed of the said component, and that there will be processes and methods of the invention that are substantially composed of or composed of the said processing steps.

[0214] Alternative solutions ( For example The use of “or” should be understood as meaning one, both, or any combination of alternatives.

[0215] The practice of the invention will be more fully understood from the foregoing embodiments, which are presented herein for illustrative purposes only and should not be construed as limiting the invention in any way.

[0216] List of implementation plans: Implementation Scheme 1 An engineered regulatory element comprising 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 nucleotide sequences selected from the following: SEQ ID NO: 1-108.

[0217] Implementation Scheme 2: The engineered regulatory element as described in Implementation Scheme 1, wherein the nucleotide sequence is at least 95% identical to a nucleotide sequence selected from the following: SEQ ID NO: 1-108.

[0218] Implementation Scheme 3: The engineered regulatory element as described in Implementation Scheme 2, wherein the nucleotide sequence is 100% identical to a nucleotide sequence selected from the following: SEQ ID NO: 1-108.

[0219] Implementation Scheme 4: An engineered regulatory element comprising one or more TP63 transcription factor binding sites (TFBS).

[0220] Implementation Scheme 5: The engineered regulatory element as described in Implementation Scheme 4, wherein one or more TP63 TFBSs contain a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from the following: SEQ ID NO 217-220 and its reverse complementary sequence.

[0221] Implementation Scheme 6: The engineered regulatory element as described in Implementation Scheme 5, wherein the one or more TP63 TFBSs contain at least 95% identical sequences to nucleotide sequences selected from: SEQ ID NO 217-220 and their reverse complementary sequences.

[0222] Implementation Scheme 7: The engineered control element as described in Implementation Scheme 6, wherein one or more TP63 TFBS are selected from SEQ ID NO 217-220 and their inverse complementary sequences.

[0223] Implementation Scheme 8: The engineered regulatory element as described in Implementation Scheme 4, wherein any one of the one or more TP63 TFBS contains a TP63 TFBS half-site motif, wherein the TP63 TFBS half-site motif contains a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the nucleotide sequences selected from: SEQ ID NO 221-233 and their reverse complementary sequences.

[0224] Implementation Scheme 9: The engineered regulatory element as described in Implementation Scheme 8, wherein the TP63 TFBS half-site motif comprises a sequence that is at least 95% identical to a nucleotide sequence selected from: SEQ ID NO 221-233 and its reverse complementary sequence.

[0225] Implementation Scheme 10: The engineered control element as described in Implementation Scheme 9, wherein the TP63 TFBS half-site motif is selected from SEQ ID NO 221-233 and its reverse complementary sequence.

[0226] Implementation Scheme 11: The engineered control element as described in any one of Implementation Schemes 8 to 10, wherein the TP63TFBS comprises two TP63 TFBS half-site motifs.

[0227] Implementation Scheme 12: The engineered regulatory element as described in Implementation Scheme 11, wherein the two TP63 TFBS half-site motifs are operatively linked by a nucleic acid adapter, optionally wherein the adapter is between 1 and 10 base pairs.

[0228] Implementation Scheme 13 The engineered regulatory element as described in Implementation Scheme 11 or 12, wherein the two TP63 TFBS half-site motifs comprise (a) a first half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from the group consisting of SEQ ID NO 221-233; and (b) a second half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an inverse complementary sequence selected from the group consisting of SEQ ID NO 221-233.

[0229] Implementation Scheme 14: The engineered control element as described in any one of Implementation Schemes 4 to 13, wherein the engineered control 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 ten TP63 TFBS.

[0230] Implementation Scheme 15: An engineered control element as described in any one of Implementation Schemes 4 to 13, wherein the engineered control element comprises at least two TP63 TFBSs.

[0231] Implementation Scheme 16: The engineered control element according to any one of claims 4 to 13, wherein the engineered control element comprises 1 to 500, 1 to 100, 1 to 50, 2 to 20, or 2 to 10 TP63 TFBS.

[0232] Implementation Scheme 17 The engineered control element as described in any one of claims 4 to 16, wherein the engineered control element further comprises at least one additional non-TP63 TFBS.

[0233] Implementation Scheme 18: The engineered control element of claim 17, wherein the at least one additional non-TP63 TFBS is selected from: BARX2 TFBS, NHLH1 TFBS, TP73 TFBS, HOXC10 TFBS, NFE2 TFBS, ATF4 TFBS, HES1 TFBS, FOS TFBS, JUN TFBS, and JUNB TFBS.

[0234] Implementation Scheme 19: The engineered control element as described in Implementation Scheme 18, wherein: The BARX2 TFBS contains 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 sequences to SEQ ID NO: 234, and / or The NHLH1 TFBS contains 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 sequence to SEQ ID NO: 235, and / or The TP73 TFBS contains 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 sequences to SEQ ID NO: 236, and / or The HOXC10 TFBS contains 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 sequence to SEQ ID NO: 237, and / or The NFE2 TFBS contains 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 sequences to SEQ ID NO: 238, and / or The ATF4 TFBS contains 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 sequences to SEQ ID NO: 239, and / or The HES1 TFBS contains 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 sequences to SEQ ID NO: 240, and / or The FOS TFBS contains 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 sequences to SEQ ID NO: 241, and / or The JUN TFBS contains 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 sequences to SEQ ID NO: 242, and / or The JUNB TFBS contains 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 sequences to SEQ ID NO: 243.

[0235] Implementation Scheme 20: The engineered control element as described in any one of Implementation Schemes 18 to 19, wherein the at least one additional non-TP63 TFBS includes BARX2 TFBS, NHLH1 TFBS, or both BARX2 TFBS and NHLH1 TFBS.

[0236] Implementation Scheme 21: The engineered control element as described in Implementation Scheme 20, wherein the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5 or more BARX2 TFBS.

[0237] Implementation Scheme 22: The engineered control element as described in Implementation Scheme 20, wherein the at least one additional non-TP63 TFBS comprises about 2, 3, 4, 5 or more NHLH1 TFBS.

[0238] Implementation Scheme 23: An engineered control element as described in any of the preceding implementation schemes, wherein the engineered control element is operatively connected to the core promoter.

[0239] Implementation Scheme 24: The engineered regulatory element as described in Implementation Scheme 23, wherein the core promoter comprises a sequence of promoters selected from the following: minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, SCP3 promoter containing DPR, minP promoter, NFkB responsive element, CREB responsive element, NFAT responsive element, SRF responsive element 1, SRF responsive element 2, API responsive element, TCF-LEF responsive element promoter fusion, hypoxia responsive element, SMAD binding element, STAT3 binding site, YB TATA, minTK, inducer molecule responsive promoter, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaV1, hCAGG, hEFlaV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb, and their tandem repeat sequences.

[0240] Implementation Scheme 25: An engineered control element as described in Implementation Scheme 23 or 24, wherein the core promoter is selected from the minCMV minimum promoter, SV40 promoter, B2M promoter, SCP3 minimum promoter, YB-SCP3 minimum promoter, and SCP3 promoter containing DPR.

[0241] Implementation Scheme 26 An engineered regulatory element comprising 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 nucleotide sequences selected from the group consisting of: SEQ ID NO: 109-216.

[0242] Implementation Scheme 27: The engineered regulatory element as described in Implementation Scheme 26, wherein the engineered regulatory element comprises at least 95% identical nucleotide sequences to nucleotide sequences selected from the group consisting of SEQ ID NO: 109-216.

[0243] Implementation Scheme 28: The engineered regulatory element as described in Implementation Scheme 27, wherein the engineered regulatory element comprises a nucleotide sequence that is 100% identical to the nucleotide sequence selected from the following: SEQ ID NO: 109-216.

[0244] Implementation Scheme 29 A heterogeneous construct, the heterogeneous construct comprising: a. The engineered control element as described in any one of embodiments 1 to 28; and b. Heterogeneous payload, Implementation Scheme 30: A heterogeneous construct as described in Implementation Scheme 29, wherein the engineered control element is operatively connected to the heterogeneous payload.

[0245] Implementation Scheme 31: A heterologous construct as described in Implementation Scheme 29, wherein the heterologous payload comprises a polynucleotide, and optionally the polynucleotide comprises a nucleotide sequence encoding one or more polypeptides.

[0246] Implementation Scheme 32: A heterologous construct as described in Implementation Scheme 30, wherein the one or more polypeptides comprise at least one effector molecule.

[0247] Implementation Scheme 33: A heterologous construct as described in any one of Implementation Schemes 30 to 31, wherein the one or more polypeptides comprise two or more separate polypeptides, the two or more separate polypeptides comprising a first effector molecule, a second effector molecule, optionally a third effector molecule, and optionally a fourth effector molecule.

[0248] Implementation Scheme 34: The heterologous construct as described in Implementation Scheme 32, wherein the polynucleotide comprises E1-L1-E2, optionally wherein the polynucleotide comprises E1-L1-E2-L2-E3-L3-E4, wherein E1 is a nucleotide sequence encoding the first effector molecule, L1 is a first adapter molecule, E2 is a nucleotide sequence encoding the second effector molecule, L2 is a second adapter molecule, E3 is a nucleotide sequence encoding the third effector molecule, L3 is a third adapter molecule, and E4 is a nucleotide sequence encoding the fourth effector molecule.

[0249] Implementation Scheme 35: A heterologous construct as described in Implementation Scheme 33, wherein L1, 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 jumping elements.

[0250] Implementation Scheme 36: A heterologous construct as described in Implementation Scheme 34, wherein the linker nucleotide sequence encodes one or more 2A ribosomal jumping elements.

[0251] Implementation Scheme 37. A heterogeneous construct as described in Implementation Scheme 34 or 35, wherein the one or more 2A ribosome hopping elements comprise elements selected from the group consisting of P2A, T2A, E2A and F2A.

[0252] Implementation Scheme 38. A heterologous construct as described in any one of Implementation Schemes 31 to 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: chimeric receptors, cytokines, chemokines, homing molecules, growth factors, polynucleotide molecules, coactivators, tumor microenvironment modulators, receptors, ligands, transcription factors, antibodies, peptides, and enzymes.

[0253] Implementation Scheme 39: Heterogeneous construct as described in Implementation Scheme 37, wherein the chimeric receptor is a chimeric antigen receptor (CAR).

[0254] Implementation Scheme 40: A heterologous construct as described in any one of Implementation Schemes 31 to 38, wherein the at least one effector molecule or each effector molecule is a human effector molecule.

[0255] Implementation Scheme 41: A heterologous construct as described in any one of Implementation Schemes 31 to 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 the first CAR and the second CAR, or ii. The first effector molecule and the second effector molecule are independently selected from the first CAR and cytokines, or iii. The first effector molecule and the second effector molecule are independently selected from the first cytokine and the second cytokine, and Optionally, the first CAR is an activating CAR (aCAR) and the second CAR is an inhibitory CAR (iCAR), and optionally, the first CAR and / or the second CAR is a bivalent CAR.

[0256] Implementation Scheme 42: A heterologous construct as described in any one of Implementation Schemes 31 to 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. First CAR, second CAR and cytokines, or ii. First CAR, first cytokine and second cytokine.

[0257] Implementation Scheme 43: A heterologous construct as described in any one of Implementation Schemes 31 to 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 the fourth effector molecule are independently selected from: a first CAR, a second CAR, a first cytokine, and a second cytokine.

[0258] Implementation Scheme 44 A carrier comprising a heterogeneous construct as described in any one of Implementation Schemes 29 to 42.

[0259] Implementation Scheme 45 A dual expression vector comprising a heterogeneous construct as described in any one of Implementation Schemes 29 to 42 and a second construct comprising an additional payload.

[0260] Implementation Scheme 46 The vector or dual expression vector as described in Implementation Scheme 43 or 44, wherein the vector or dual expression vector is a viral vector, and optionally wherein the viral vector is a retroviral vector.

[0261] Implementation Scheme 47 An immune response cell comprising a heterologous construct as described in any one of Implementation Schemes 29 to 42, a vector as described in Implementation Scheme 43 or 45, or a dual expression vector as described in Implementation Scheme 44 or 45.

[0262] Implementation Scheme 48. Immune response cells as described in Implementation Scheme 46, wherein the immune response cells are selected from: natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, γ-δ T cells, cytotoxic T lymphocytes (CTL), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, B cells, macrophages, tumor-infiltrating lymphocytes (TIL), congenital lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESC), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSC), induced pluripotent stem cells (iPSC), and iPSC-derived cells.

[0263] Implementation Scheme 49: Immune response cells as described in Implementation Scheme 47, wherein the immune response cells are NK cells or T cells.

[0264] Implementation Scheme 50: An immune response cell as described in any one of Implementation Schemes 46 to 48, wherein the immune response cell expresses an activated immune receptor.

[0265] Implementation Scheme 51: Immune response cells as described in Implementation Scheme 49, wherein the activated immune receptor includes an antigen recognition receptor.

[0266] Implementation Scheme 52: Immune response cells as described in any one of Implementation Schemes 46 to 50, wherein the immune response cells are autologous.

[0267] Implementation Scheme 53: Immune response cells as described in any one of Implementation Schemes 46 to 51, wherein the immune response cells are allogeneic.

[0268] Implementation Scheme 54 A pharmaceutical composition comprising a carrier as described in Implementation Scheme 43 or 45, a dual expression carrier as described in Implementation Scheme 44 or 45, or an immune response cell as described in any one of Implementation Schemes 46 to 52, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

[0269] Implementation Scheme 55 A method for increasing the expression of a target gene or a heterologous payload, the method comprising using an engineered regulatory element as described in any one of Implementation Schemes 1 to 28, a vector as described in Implementation Scheme 43 or 45, or a dual expression vector as described in Implementation Scheme 44 or 45 to increase the expression of the target gene.

[0270] Implementation Scheme 56: The method described in Implementation Scheme 54, wherein the target gene is an immune regulatory gene.

[0271] Implementation Scheme 57 A method of treating a subject in need, the method comprising administering a therapeutically effective dose of a carrier as described in Implementation Scheme 43 or 45, a dual-expression carrier as described in Implementation Scheme 44 or 45, an immune-response cell as described in any one of Implementation Schemes 46 to 52, or a pharmaceutical composition as described in Implementation Scheme 53.

[0272] Implementation Scheme 58 A kit for treating and / or preventing diseases or conditions, said kit comprising immune response cells as described in any one of Implementation Schemes 46 to 52 or a pharmaceutical composition as described in Implementation Scheme 53.

[0273] Implementation Scheme 59: The kit described in Implementation Scheme 57, wherein the disease or condition includes tumors.

[0274] Implementation Scheme 60: A kit as described in Implementation Scheme 57 or 58, wherein the kit further includes written instructions for using the immune response cells or the pharmaceutical composition to treat and / or prevent the disease or condition of the subject.

[0275] Example This disclosure will be further illustrated in the following embodiments, which are given for illustrative purposes only and are not intended to limit this disclosure in any way.

[0276] Example 1: Construction of polycistronic engineered nucleic acids for expressing multiple payloads The following multicistronic payload constructs are designed to express multiple payload components from a single open reading frame. These payload constructs are used to test the performance of various engineered control element designs that include engineered enhancer sequences operatively connected to a core promoter to generate engineered promoter candidates, as described in the following embodiments. For a schematic diagram of exemplary engineered promoter candidate designs and multicistronic payload constructs, see [link to schematic diagram]. Figure 1A .

[0277] The payload number, design, and total length (nt and aa) are shown in Table A below.

[0278] Table A - Characteristics of Multicistronic Payload Constructions

[0279] Example 2: Nomination and screening of first-generation strong enhancers in NK cells Candidate enhancer sequences were nominated using bioinformatics analysis of internal pooling screening data from NK cells from multiple donors using the MPRA library. Candidate transcription factors upregulated in NK cells were bioinformatically identified, and their corresponding transcription factor binding sites (TFBSs) were arranged in various combinations to generate candidate enhancer sequences. These candidate enhancer sequences were then operatively ligated to the minCMV core promoter sequence to generate candidate engineered promoters. See also Figure 1B .

[0280] Two methods were used to interpret promoter strength and nominate the best engineered promoter candidates: 1) RNA to DNA ratio, where the best candidate was selected based on a fixed RNA / DNA ratio cutoff value, and 2) RNA-only method, where the best candidate was selected based on the highest strength measured by the average normalized RNA count.

[0281] Promoter-payload constructs were designed by linking candidate engineered promoters to payload 1. The aCAR and iCAR payloads contain peptide tags for downstream detection purposes. See also... Figures 1A to 1B .

[0282] Promoter activity of candidate engineered promoters was evaluated clonally. Briefly, promoter-payload constructs were packaged into self-inactivating (SIN) γ-retroviruses (sinvec viruses) and transduced into NK cells (D=0). In short, sinvec viruses were generated by co-transfecting the test construct and envelope plasmid into GP2-293 cells, followed by collection and concentration. After transduction, NK cells were allowed to stand in culture, and at one or more later time points, typically D=3 to 7, surface expression of aCAR and iCAR proteins was detected by flow cytometry using fluorescently conjugated antibodies recognizing specific peptide tags. Where applicable, the integrated viral copy number (VCN), representative of the integrated transgene copies, was also assessed from these transduced NK cells. Promoter strength was assessed by measuring payload expression relative to the SFFV promoter, which served as a control promoter.

[0283] Surface expression of aCAR and iCAR was used as an indicator of promoter strength. These expression data were extracted from raw flow cytometry data gated on live single NK cells. Measures included 1) total %aCAR+ of the live NK cell population, 2) total %iCAR+ of the live NK cell population, 3) %aCAR+iCAR (%++) of the live NK cell population, 4) mean or median fluorescence intensity (MFI) of aCAR from the total %aCAR+ and %++ populations, and 5) MFI of iCAR from the total %iCAR+ and %++ populations. Promoter strength was primarily quantified by aCAR and iCAR MFI (with or without VCN normalization), and secondarily by the degree of %transduction (i.e., %CAR+) population. VCN-normalized MFI values ​​were calculated as follows: Figure 2 Performance metrics for selected first-generation engineered promoter candidates based on transduction efficiency are shown. Figure 3 Flow cytometry scatter plots of exemplary selected engineered promoter constructs SB10698, 10977, 10961, 109457 and 10944 are shown. Figures 4A to 4BA graph shows performance metrics of selected first-generation engineered promoter candidates based on surface CAR protein expression measured by flow cytometry and quantified by fluorescence intensity values. Engineered promoter constructs selected based on, for example, higher performance compared to SFFV in at least one of the above metrics include: SB10968, SB10977, SB10961, SB10941, SB10945, SB10974, SB10978, SB10980, SB10981, SB10966, SB10963, SB10956, SB10953, SB10944, and SB10947. In particular, SB10968, SB10977, SB10961, SB10947, and SB10944 exhibited a higher %CAR+ population than the SFFV control construct (SB10984). The enhancer sequences and complete promoter sequences from these engineered promoter constructs are shown in Tables 1 and 2, respectively. For design details of the evaluated first-generation promoters, see Table B below. The sequences of exemplary TFBSs are shown in Table 3.

[0284] Table 1 – Enhancer Subsequences

[0285] Table 2 – Complete Starter Sequence

[0286] Table B – First-generation promoter configurations

[0287] Example 3: Design and screening of cell promoters using combined TFBS arrays To further engineer more robust promoters, novel promoters were designed based on combinations of selective TFBS from Example 1 linked to different core promoter sequences, such as minCMV, SV40, B2M, and NKG7. These novel engineered promoters were then linked to a multi-component payload (payload 2) consisting of crIL-15, aCAR, and iCAR linked via 2A peptides. The aCAR and iCAR payloads contained peptide tags for downstream detection purposes.

[0288] Engineered promoters were screened using a clonal approach as described in Example 2 by transducing NK cells with packaged sinvec virus and flow cytometry (as downstream readout), but the entire process was performed on a custom-designed, internally customized automated liquid processor (ALH) for high-throughput screening. Flow cytometry evaluation of surface aCAR and iCAR expression was performed at D=6, and the SV40 promoter was selected as a control promoter for this payload background.

[0289] Surface expression of aCAR and iCAR was quantified using %CAR+ population and CAR MFI with and without VCN normalization, serving as a representative of promoter strength. For each expression metric, the test promoter was directly compared to the SV40 control promoter (details below), and engineered promoter candidates were nominated as strong engineered promoter candidates if they showed an SV40 fold greater than 1 for both aCAR and iCAR MFI metrics and an SV40 fold of at least 0.8 for the %CAR+ population metric.

[0290] Given that the expression of the multi-component payload (payload 2) is driven by a single promoter, and we experimentally observed strong correlations between various MFI expression metrics, we selected iCARMFI of the iCAR+ and aCAR+iCAR+(++) NK cell populations as the primary metric to rank promoters by intensity for the purpose of nominating the best promoter candidates.

[0291] Many second-generation promoters outperform SV40 on at least one expression metric (e.g., %CAR+ population, MFI without VCN normalization), such as Figure 5 and Figures 6A to 6C As shown, these figures depict the percentage by which new promoter constructs that pair enhancers with various core promoters outperform the SV40 promoter as a baseline.

[0292] Several engineered promoters were selected as optimal engineered promoter candidates and are shown in Table C. All of the optimal engineered promoter candidates contain TP63 TFBS (2, 3, or 5 TP63 TFBS). Further analysis of the ALH screening results showed that, compared to the SV40 benchmark, most promoter designs with >5 TP63 TFBS or 2 TP63 TFBS performed well, such as... Figure 7A As shown. For example, compared to SV40, five promoter candidates with ten TP63 TFBSs connected to various core promoters (e.g., SB12295, SB12324, SB12353, SB12382, SB12440) improved payload expression (data not shown).

[0293] In addition, many optimal engineered promoter candidates were found to contain BARX2, NHLH1, and / or TP63 TFBS, such as SB12305, SB12310-SB12315, SB12319, SB12372, SB12343, SB12342, SB12344, SB12370, SB12371, SB12300, SB12373, SB12304, SB12309, SB12496, SB12377, SB12368, SB12339, and SB12348. Figure 7B As shown, BARX2, NHLH1, and TP63 TFBSs containing enhancers connected to minCMV are the strongest selected engineered promoter candidates. Further details regarding the design of the optimal promoter from this embodiment are shown in Table C below. The relevant TFBS sequences are shown in Table 3.

[0294] Table C – Optimal Combination of TFBS Array Promoters

[0295] Table 3 – TP63 and other non-TP63 sequences

[0296] A performance comparison between the first-generation promoter described in Example 2 and the second-generation promoter selected in this example is shown in the figure. Figures 8A to 8C (Left, middle, and right) Middle. Compared with the SV40 promoter control (SB10985), the best second-generation promoter showed 2-4 times the effective payload expression.

[0297] Example 4: Design of cell promoters and pooling MPRA screening using combined TFBS arrays Based on a combined array of TFBS paired with the core promoter (minCMV), a separate set of 16,000 synthetic engineered promoters was designed and calculated. TFBS were selected based on strong promoters from Example 1 and considerations from the literature. These promoters were ligated upstream of a DNA barcode at 12 base pairs (bp), followed by a multi-component payload as a readout. In summary, two libraries with different payloads were generated. SFFV, SV40, and LTR were included in this library as viral control promoters. The control viral promoters were approximately 300-400 bp in length, while the MPRA library promoters were approximately 200 bp in length.

[0298] Document 1 (SB11858): aCAR targeting CEA, iCAR targeting VSIG2 (payload 3) Library 2 (SB11859): aCAR and crIL-15 targeting CEA (payload 4) For each library, each component of the multi-component payload is separated by a 2A peptide. See Example 1 for details on payload design. Primer binding sites are included for downstream next-generation sequencing (NGS) applications.

[0299] The engineered promoters were synthesized into pooled oligonucleotides and cloned into plasmids with corresponding payloads to generate libraries 1 and 2. These libraries were then screened using a massively parallel reporter assay (MPRA) method. Figure 9 The diagram is shown below. In short, pooled plasmids were packaged into sinvec viruses and transduced into NK cells with two biological repeats (D=0). At D=3, NK cells were harvested, and RNA and DNA samples were extracted, analyzed by NGS, and promoter strength was assessed.

[0300] Based on the average promoter strength and the variation among biological replicates, the promoter score of each MPRA library member was calculated.

[0301] The best engineered promoter candidates for downstream cloning validation were selected based on high promoter scores from two MPRA libraries. Other untested engineered promoter candidates with strong predictive scores from proprietary analytical procedures were also selected for evaluation.

[0302] The results are shown in Figure 10 (Top and bottom) In the middle. The larger solid circles correspond to the viral promoter control. As shown in the figure, although significantly smaller, several candidates have promoter strengths comparable to the viral control promoter. Statistical analysis nominated strong TFBSs, which can be used to guide subsequent rationally designed engineered regulatory elements derived from machine learning.

[0303] Example 5: Cloning and Verification of the Second-Generation Promoter Derived from MPRA The engineered promoter candidate from Example 4 was linked to an internal multi-component payload (payload 5) consisting of IL21, crIL-15, aCAR, and iCAR linked via 2A peptides. The aCAR and iCAR payloads contained peptide tags for downstream detection purposes. Expression of these payloads was used to evaluate promoter strength.

[0304] Similar to Example 3, these promoter-payload constructs were packaged into sinvec virus and transduced into NK cells using ALH, with flow cytometry performed at D=6 as a downstream readout of promoter strength. The SV40 promoter was selected as a control promoter for this payload background. Promoter strength was measured by the surface expression of aCAR and iCAR, which was quantified as %CAR+ population and CAR MFI without VCN normalization.

[0305] The best engineered promoter candidates were selected based on the higher aCAR and iCAR MFI of the CAR+ population compared to the SV40 promoter (i.e., a fold increase of more than 1 over SV40). Approximately half of the MRPA engineered promoter candidates and most of the well-designed engineered promoter candidates derived from machine learning outperformed the SV40 promoter (data not shown). Exemplary engineered regulatory element sequences from the best engineered promoter candidates screened from the MPRA library, including engineered enhancer sequences and engineered promoter sequences, are shown in Tables 1 and 2.

[0306] Example 6: Validation and functional characterization of engineered promoter candidates for expressing multiple CARs and armored cytokines from tetracistronic constructs The promoter strength of the best engineered promoter candidates from previous embodiments was re-evaluated in a larger payload system, and their associated CAR-NK cells were functionally characterized. Specifically, they were ligated upstream of a tetracistronic construct for expressing IL21, crIL-15, a CEA-targeting aCAR, and a VSIG2-targeting iCAR (payload 5) linked via a 2A peptide. Details of the payload 5 design are provided in Example 1.

[0307] These promoter-payload constructs were screened using a cloning method similar to that of Example 1. Briefly, the constructs were packaged into sinvec virus and used to transduce NK cells at three different viral doses (in µl volumes). After transduction, CAR-NK cells were cultured and stained with fluorescently conjugated antibodies at D=7, 14, and 21 to determine the surface expression of aCAR, iCAR, and IL15 by flow cytometry. The supernatant from CAR-NK cells was collected for Luminex assays to determine the levels of secreted IL-15 and IL-21 payloads. The VCN value of transduced NK cells was quantified by qPCR. The SV40 promoter was selected as a control promoter for this payload background.

[0308] Overall promoter strength was determined by the surface expression of aCAR, iCAR, and crIL-15, as well as the secretion levels of IL-15 and IL-21, quantified by MFI values ​​in live NK cells. Secreted IL-15 and IL-21 were quantified in pg / mL, normalized by cell density, and calculated back from Luminex standards.

[0309] Figure 11 The graphs depict iCAR MFI (iCAR+ cells) on days 7 and 14 for unengineered NK cells (NV control), NK cells transduced with SB12515 (SV40), NK cells transduced with SB12894 (B2M promoter), NK cells transduced with SB12895 (NKG7 promoter), and cells transduced with SB12896 (4xTP63 TFBS-minCMV). As shown, at all time points and at all viral doses, SB12896 drove higher iCAR expression compared to the SV40, B2M, and NKG7 promoters.

[0310] Figure 12 Exemplary flow cytometry plots of cells transduced from 25 µl volumes of NV (left), SB12515 (middle), and SB12896 (right) on day 7, along with the normalized VCN of the transduced cells, are depicted. As shown, cells transduced from SB12515 (right) exhibited a higher population of iCAR+, aCAR+, and aCAR iCAR++% at a lower VCN compared to cells transduced from SB12515. Figure 13A (Center and right) and Figure 13B As shown (top and bottom), compared with cells transduced by SB12515, transduction efficiency and expression of both aCAR and iCAR payloads were enhanced in cells transduced by SB12896 at all viral doses and across all NK cell donors.

[0311] Figure 14A (left and right), Figure 14B (left and right) and Figure 14C (Top and bottom) (Depicts surface expression of IL15 and secreted IL15 and IL21 in cells transduced with SB12515 and SB12896. As shown, SB12896 drove higher cytokine expression levels compared to SB12515 at all viral doses and normalized VCN.)

[0312] Figures 15A to 15D Promoter strength metrics for SB12896 and SB12515 across NK cells from different donors are shown. As illustrated, SB12896 drives higher transduction efficiency across all NK donors compared to SB12515. Figure 15B ) and aCAR and iCAR payload expression ( Figures 15C to 15D ).

[0313] Furthermore, promoters incorporating 4X TP63 and 5X TP63 outperformed SV40 across multiple experiments and in various multicistron payload constructs. See Table D below. Details of the designs for payloads 2 and 5 are provided in Example 1.

[0314] Table D – TP63-based promoter evaluation

[0315] Further analysis was performed on the identified TP63 TFBS sites. TP63 typically binds to DNA preferentially as a dimer, and without being bound by theory, its shared binding motif is approximately 20 bp (“intact” response element), which typically consists of two 10 bp “half-sites.” TP63 dimers can bind to half-site pores, and a total of four TP63s (i.e., tetramers) can bind to the intact response element. The two half-sites may not be separated by adapters, or may be separated by adapters of different lengths (such as 2 and 10 bp adapters), and still retain TP63 binding. The two half-sites may also be on the same or different DNA strands (e.g., one half-site in the forward orientation and the other half-site in the reverse orientation). Therefore, the TP63 “half-site” sequences and motifs of the identified “intact” TP63 TFBS sites were evaluated. The results of the analysis identifying the half-sites and half-site motifs are shown in Table 3.

[0316] Furthermore, the cytotoxic function of NK cells engineered with these promoter-payload constructs was evaluated in a mixed-target sequential kill assay. For each round of killing, two engineered DLD-1 (colorectal adenocarcinoma) cell lines were mixed at a 1:1 ratio and pre-coated in flat-bottomed tissue culture plates. The TA+PA-DLD-1 target cell line was engineered to express: GFP reporter protein, the CEA target antigen recognized by aCAR (TA+), and the off-target protective antigen Her2 (PA-) not recognized by iCAR. The TA+PA+DLD-1 target cell line was engineered to express: mCherry reporter protein, the same CEA target antigen recognized by aCAR (TA+), and the on-target protective antigen VSIG2 (PA+) recognized by iCAR.

[0317] Effector cells (unengineered NK cells (NV), SB12515 engineered NK cells (expressing payload 5 from the SV40 promoter), or promoter candidate-payload 5 engineered NK cells (e.g., SB12896 engineered NK cells) were added to pre-coated DLD-1 target cells at an E:T ratio of 1:4. (For clarity, the E:T ratio refers to the ratio of effector cells (NK cells) to pre-coated DLD-1 target cells.) After approximately 45 hours of co-culture, the NK cells in suspension were transferred to newly coated target cells for the next round of killing, for a total of three rounds of killing. Images of the assay wells were automatically captured on Incucyte at four-hour intervals. Killing of the two engineered DLD-1 cell lines was quantified by the count of reporter protein-positive cells over time. See also Figure 16A It depicts a schematic diagram of engineered NK cells expressing payload 5 and the DLD-1 target cell line.

[0318] Two functional measures were evaluated: 1) aCAR and cytokine-driven cytotoxicity against TA+PA- target cells, and 2) iCAR-mediated protection against TA+PA+ target cells relative to TA+PA- cells. These measures were calculated as follows: In the above formula, "T" refers to a specific time point in the kill assay, and "T0" refers to the first time point of each round of kill assay. "Test" refers to the CAR-NK cell conditions from a specific promoter-payload construct in the kill assay, and "no NK" refers to conditions with only target cells.

[0319] After the final time point in the kill assay, cells in the suspension were collected, co-stained with a fluorescently conjugated anti-CD45 antibody to distinguish NK cells from target cells, and co-stained with a viability dye to distinguish live cells from dead cells. Flow cytometry was then used to quantify the number of surviving NK cells. The number of surviving NK cells having the candidate promoter described in the previous embodiments was compared with the number of surviving NK cells having the SV40 promoter (SB12515) alone.

[0320] Compared to CAR-NK cells with payloads driven by the SV40 promoter, the beneficial effects of engineered promoters on CAR-NK function at any time point in the killing assay, particularly in the final round of killing, will be indicated by higher aCAR killing function and iCAR protective function.

[0321] The results of the comparison between the engineered promoter of SB12896 and the lethality assay of SV40 are shown in the figure. Figure 16B (Describes the killing effect on TA+PA- cells) and Figure 16C (Describing the killing effect on TA+PA+ cells)

[0322] like Figure 16C As shown, DLD-1 TA+PA- cells exhibited increased GFP counts in the absence of NK cells and in the absence of engineered NK cells (NV control). Endogenous (non-aCAR-mediated) tumor cell killing was significant in the first round for the NV control, decreased in the second round, and essentially absent in the third round, indicating a lack of NK persistence over time. DLD-1 TA+PA- cells showed significant killing activity across three rounds of SB12515-engineered NK cells (expressing a multi-component payload from the SV40 promoter). Compared to the SB12515 comparator, SB12896-engineered NK cells (expressing the same multi-component payload from the engineered promoter) demonstrated considerably stronger killing activity in the first round and increased killing activity in the second and third rounds, demonstrating that the engineered promoter improves CAR-NK function.

[0323] like Figure 16F As shown, it illustrates the iCAR function determined by the above formula. Neither SB12515 nor SB12896 exhibited iCAR function in the NK and NV controls. Although SB12515 and SB12896 showed comparable iCAR function in the first two rounds of lethality, SB12896 showed stronger iCAR function compared to SB12515 in the third round.

[0324] like Figure 16GAs shown, at the final time point of the continuous kill assay, there were a greater number of surviving SB12896 engineered cells compared to SB12515 engineered cells, indicating that NK persistence in SB12896 engineered NK cells was improved compared to SB12515 engineered NK cells, demonstrating that the engineered promoter improved CAR-NK function.

[0325] Example 7: Verification and Functional Characterization of the Second-Generation Promoter In another experiment, the engineered promoter candidates described in the previous embodiments, as well as additional engineered promoter candidates, including their engineered regulatory element variants, were evaluated. In short, these engineered regulatory element variants were designed by replacing the selective TFBS or minimal promoter with a TFBS or minimal promoter anticipated based on knowledge from the previous embodiments, or even stronger. These engineered promoter candidates are linked upstream of an internal multi-component payload (payload 5) consisting of IL21, crIL-15, aCAR, and iCAR linked via 2A peptides. Details of the payload 5 design are described in Example 1.

[0326] These promoter-payload constructs were screened in a manner similar to that in Example 6. CAR-NK cells were stained with fluorescently conjugated antibodies at D=7, 14, and 21 to determine the surface expression of aCAR, iCAR, and IL15 by flow cytometry. The SV40 promoter was selected as a control promoter for this payload background.

[0327] Similar to Example 6, a hybrid target sequential kill assay was used to evaluate the functional impact of high payload expression driven by these engineered promoter candidates. Two rounds of killing were performed at an E:T ratio of 1:2. Images of the assay wells were automatically captured on Incucyte at four-hour intervals, and target cells were quantified by the count of reporter protein-positive cells over time. aCAR and iCAR functions were calculated in the same manner as in Example 8, and plotted at the 80-hour time point after the second round of killing.

[0328] like Figure 17A (Top, Middle, and Bottom) and Figure 17B As shown (left and right), several engineered NK promoter candidates express (the payload) Figure 17A While killing TA+PA- target cells, it also provides stronger protection for TA+PA+ "healthy" cells. Figure 17BIn terms of performance, it outperforms SV40 (SB12515). Exemplary engineered promoter candidates that outperform SV40 include: SB12896, SB13505, SB13508, SB13500, SB13498, SB13495, SB13497, SB13499, SB13501, and SB13491. The enhancer and promoter sequences of these constructs are listed in Tables 1 and 2.

[0329] Example 8: ALH screening of optimal promoters in NK and T cells Therapeutic agents based on engineered T cells can also benefit from promoters that drive improved expression of multi-component payloads. The following experiments were performed to evaluate engineered promoter candidates, including various engineered regulatory elements from previous embodiments, in T cells and NK cells.

[0330] The best engineered promoter candidate from the previous embodiments was cloned upstream of an internal multi-component payload (payload 5) consisting of IL21, crIL-15, aCAR, and iCAR linked by a 2A peptide. The SV40 promoter was selected as a control promoter for this payload background.

[0331] From virus generation to NK and T cell transduction and staining at D=6 for flow cytometry, promoters were automatically cloned and screened on a custom-made ALH. Surface aCAR and iCAR expression was quantified, and promoter strength was indicated by the iCAR MFI of the %CAR+ cell population. Primary T cells isolated from the 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 culture medium containing IL-2.

[0332] NK cell results are shown in Figure 18A (Left and right) Center. T cell results are shown in... Figure 18B (Left and right) Center. The correlation between NK and T cell results is shown in... Figure 18C (Top and bottom) In the figure, almost all tested promoters were stronger in NK and T cells compared to the SV40 promoter (SB12515). Notably, many promoters (e.g., SB numbers 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 first-generation promoter evaluated in Example 6). Figure 18DThe results for NK and T cells are depicted in a single figure. As shown, many of the tested promoters exhibited strong activity in both NK and T cells, and several (e.g., SB numbers 13500, 13491, 13489, 13507, 13488, 13501, 13508, 13498, 13509, 13502, 13495, 13499, 13490, 13497, 13487, 13235, 13233, 13234, 13285) showed increased activity in T cells compared to NK cells. Figure 18E (Top and bottom) depict the flow cytometry results of SFFV control and SB13498. As shown in the figure, SB13498-transduced NK cells showed a much higher aCAR / iCAR double-positive population (51.2%) compared to the SFFV control (16.6%).

[0333] Other sequences The sequences mentioned throughout the disclosure include the following exemplary sequences: Table 4 – Exemplary Sequences

[0334] By incorporating references The full disclosure of each patent and scientific document mentioned in this article is incorporated herein by reference for all purposes.

[0335] equivalent This disclosure may be implemented in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the foregoing embodiments are to be considered illustrative in all respects and not limiting of the invention described herein. Accordingly, the scope of this disclosure is indicated by the appended claims rather than the foregoing description, and all changes falling within the equivalent meaning and scope of the claims are intended to be included therein.

Claims

1. An engineered regulatory element comprising 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 nucleotide sequences selected from the group consisting of SEQ ID NO: 1-108.

2. An engineered regulatory element comprising one or more TP63 transcription factor binding sites (TFBS), optionally wherein the one or more TP63 TFBS comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from: SEQ ID NO 217-220 and its inverse complementary sequence.

3. The engineered regulatory element of claim 1 or 2, wherein any one of the one or more TP63 TFBSs comprises a TP63 TFBS hemisite motif, wherein the TP63 TFBS hemisite motif comprises a sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from: SEQ ID NO 221-233 and its reverse complementary sequence, optionally wherein the TP63 TFBS comprises two TP63 TFBS hemisite motifs, optionally wherein the two TP63 TFBS hemisite motifs are operatively linked by a nucleic acid adapter, optionally wherein the adapter is between 1 and 10 base pairs, optionally wherein the two TP63 The TFBS half-site motif comprises (a) a first half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a nucleotide sequence selected from the following: SEQ ID No 221-233; and (b) a second half-site motif that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an inverse complementary sequence selected from the following: SEQ ID No 221-233.

4. The engineered control element as described in any one of claims 1 to 3, wherein the engineered control 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 ten TP63 TFBS, optionally wherein the engineered control element comprises 1-500, 1-100, 1-50, 2-20, or 2-10 TP63 TFBS.

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

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

7. The engineered control element as described in claim 5 or 6, wherein the at least one additional non-TP63 TFBS is selected from: BARX2 TFBS, NHLH1 TFBS, TP73 TFBS, HOXC10 TFBS, NFE2 TFBS, ATF4 TFBS, HES1 TFBS, FOS TFBS, JUN TFBS, and JUNB TFBS, optionally wherein: (a) The BARX2 TFBS contains 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 sequences to SEQ ID NO: 234, and / or (b) The NHLH1 TFBS contains 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 sequence to SEQ ID NO: 235, and / or (c) The TP73 TFBS contains 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 sequences to SEQ ID NO: 236, and / or (d) The HOXC10 TFBS contains 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 sequences to SEQ ID NO: 237, and / or (e) The NFE2 TFBS contains 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 sequences to SEQ ID NO: 238, and / or (f) The ATF4 TFBS contains 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 sequences to SEQ ID NO: 239, and / or (g) The HES1 TFBS contains 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 sequences to SEQ ID NO: 240, and / or (h) The FOS TFBS contains 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 sequences to SEQ ID NO: 241, and / or (i) The JUN TFBS contains 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 sequences to SEQ ID NO: 242, and / or (j) The JUNB TFBS contains 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 sequences to SEQ ID NO: 243; Optionally, the at least one additional non-TP63 TFBS may include BARX2 TFBS, NHLH1 TFBS, or both BARX2 TFBS and NHLH1 TFBS; optionally, the at least one additional non-TP63 TFBS may include about 2, 3, 4, 5 or more 5 BARX2 TFBS; optionally, the at least one additional non-TP63 TFBS may include about 2, 3, 4, 5 or more 5 NHLH1 TFBS.

8. The engineered control element as claimed in any of the preceding claims, wherein the engineered control element is operatively connected to a core promoter, optionally wherein the core promoter comprises a sequence of promoters selected from: minCMV minimal promoter, SV40 promoter, B2M promoter, SCP3 minimal promoter, YB-SCP3 minimal promoter, SCP3 promoter containing DPR, minP promoter, NFkB responsive element, CREB responsive element, NFAT responsive element, SRF responsive element 1, SRF responsive element 2, API responsive element, TCF-LEF responsive element promoter fusion, hypoxia responsive element, SMAD binding element, STAT3 binding site, YB TATA, minTK, inducer molecule responsive promoters, CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaV1, hCAGG, hEFlaV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, hUBIb and their tandem repeat sequences, Optionally, the core promoter is selected from the minCMV minimum promoter, SV40 promoter, B2M promoter, SCP3 minimum promoter, YB-SCP3 minimum promoter, and SCP3 promoter containing DPR.

9. An engineered regulatory element comprising 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 nucleotide sequences selected from the group consisting of SEQ ID NO: 109-216.

10. A heterogeneous construct, the heterogeneous construct comprising: (a) the engineered control element as described in any one of claims 1 to 9; and (b) Heterogeneous payload, The engineered control element is operatively connected to the heterogeneous 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 comprises a) at least one effector molecule or b) two or more separate polypeptides, wherein the two or more separate polypeptides comprise a first effector molecule, a second effector molecule, optionally a third effector molecule, optionally a fourth 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: chimeric receptors, cytokines, chemokines, homing molecules, growth factors, polynucleotide molecules, coactivators, tumor microenvironment modulators, receptors, ligands, transcription factors, antibodies, peptides, and enzymes, optionally wherein the chimeric receptor is a chimeric antigen receptor (CAR).

12. The heterologous construct of claim 11, wherein the polynucleotide comprises E1-L1-E2, optionally wherein the polynucleotide comprises E1-L1-E2-L2-E3-L3-E4, wherein E1 is a nucleotide sequence encoding the first effector molecule, L1 is a first adapter molecule, E2 is a nucleotide sequence encoding the second effector molecule, L2 is a second adapter molecule, E3 is a nucleotide sequence encoding the third effector molecule, L3 is a third adapter molecule, and E4 is a nucleotide sequence encoding the fourth effector molecule, optionally wherein L1, L2, L3, and L4 are independently selected from: internal ribosome entry sites (IRES) and one or more nucleotide sequences encoding one or more 2A ribosome jumping elements, optionally wherein the adapter nucleotide sequence encodes one or more 2A ribosome jumping elements, optionally wherein the one or more 2A ribosome jumping elements comprise elements 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, wherein: i) The first effector molecule and the second effector molecule are independently selected from the first CAR and the second CAR, or ii) The first effector molecule and the second effector molecule are independently selected from the first CAR and cytokines, or iii) The first effector molecule and the second effector molecule are independently selected from the first cytokine and the second cytokine, and Optionally, the first CAR is an activating CAR (aCAR) and the second CAR is an inhibitory CAR (iCAR); alternatively, the first CAR and / or the second CAR is a bivalent CAR; or b) The first effector molecule, the second effector molecule, and the third effector molecule, wherein the first effector molecule, the second effector molecule, and the third effector molecule are independently selected from: i) First CAR, second CAR and cytokines, or ii) First CAR, first cytokine, and second cytokine; or c) The first effector molecule, the second effector molecule, the third effector molecule, and the fourth effector molecule, 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 a heterologous construct as described in any one of claims 10 to 13, optionally wherein the vector is a viral vector, and optionally wherein the viral vector is a retroviral vector.

15. A dual expression vector comprising a heterologous construct as described in any one of claims 10 to 13 and a second construct comprising an additional payload, optionally wherein the dual expression vector is a retroviral vector.

16. An immune-response cell, said immune-response cell comprising a heterologous construct as described in any one of claims 10 to 13, a vector as described in claim 14, or a dual-expression vector as described in claim 15, optionally wherein said immune-response cell is selected from: natural killer (NK) cells, T cells, CD8+ T cells, CD4+ T cells, γ-δ cells, etc. T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, B cells, macrophages, tumor-infiltrating lymphocytes (TILs), congenital lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells, optionally wherein the immune response cells express activated immune receptors, optionally wherein the activated immune receptors include antigen recognition receptors, and optionally wherein the immune response cells are autologous or allogeneic.

17. A pharmaceutical composition comprising the carrier as claimed in claim 14, the dual expression carrier as claimed in claim 15, or the immune-responding cell as claimed in claim 16, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

18. A method of treating a subject in need, the method comprising administering a therapeutically effective dose of the carrier as claimed in claim 14, the dual-expression carrier as claimed in claim 15, the immune-response cells as claimed in claim 16, or the pharmaceutical composition as claimed in claim 17.

19. A kit for treating and / or preventing a disease or condition, said kit comprising immune-response cells as claimed in claim 16 or a pharmaceutical composition as claimed in claim 17, optionally wherein said disease or condition includes a tumor, optionally wherein said kit further comprises written instructions for using said immune-response cells or said pharmaceutical composition to treat and / or prevent said disease or condition in a subject.

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