Synthetic cancer-specific promoters

Abstract

Described herein are synthetic promoters and / or enhancers that are specific for cancer cells, methods of engineering synthetic cancer-specific promoters, and uses thereof.

Description

Attorney Docket No. 53531-724602SYNTHETIC CANCER-SPECIFIC PROMOTERSCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 834,389, filed January 22, 2025, which application is incorporated herein by reference.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 53531-724_601_SL.xml, created January 19, 2026, which is 1,664,478 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.BACKGROUND

[0003] Endogenous cancer-activated promoters are controlled by a wide network of transcription factors (TFs), which can lead to non-ideal basal activity in non-target cells. It is also difficult to reliably predict the activity in a wide variety of cancer models.SUMMARY

[0004] There is a need to develop synthetic cancer-specific promoters with high specificity and sensitivity, for use in delivering polypeptides to cancer cells.

[0005] In an aspect, provided herein is a non-naturally occurring polynucleotide comprising: (a) a core promoter operably linked to an open reading frame (ORF) comprising a gene encoding a peptide therapeutic agent, wherein said core promoter induces expression of said peptide therapeutic agent at a higher level in a cancer cell compared to a non-cancer cell; (b) one or more synthetic response elements comprising one or more enhancers and one or more transcription factor binding sites; and (c) a transcription start site (TSS) upstream of said ORF.

[0006] In some embodiments, said core promoter further comprises one or more promoter elements obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells. In some embodiments, said one or more genes are obtained from a human subject. In some embodiments, said higher levels of expression or activity of said one or more genes in said cancer cell compared to said non-cancer cell is determined by chromatin immunoprecipitation (ChIP). In some embodiments, said core promoter comprises one or more synthetic promoter elements. In some embodiments, said core promoter comprises one or more promoter elements that are endogenous to said subject. In some embodiments, said core promoterAttorney Docket No. 53531-724602comprises one or more promoter elements that are non-endogenous to said subject. In some embodiments, said core promoter comprises two or more promoter elements, wherein at least two promoter elements of said two or more promoter elements are obtained from different genes. In some embodiments, said one or more enhancers comprise a CpG island. In some embodiments, said one or more enhancers does not comprise a CpG island.

[0007] In some embodiments, said peptide therapeutic agent comprises a fusion protein. In some embodiments, said fusion protein comprises a cytokine or a derivative thereof. In some embodiments, said cytokine or derivative thereof comprises a sequence of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, said cytokine or derivative thereof comprises a sequence in Table IL. In some embodiments, said fusion protein comprises: a cytokine or fragment thereof, wherein said fragment of said cytokine comprises a receptor binding domain of said cytokine; or said cytokine and said fragment thereof.

[0008] In some embodiments, said peptide therapeutic agent comprises a toxin, a cytokine, an antibody or antigen binding fragment thereof, or any combination thereof. In some embodiments, said peptide therapeutic agent comprises a cytokine. In some embodiments, said peptide therapeutic agent further comprises an antibody or antigen-binding fragment thereof. In some embodiments, said cytokine is IL-2 or a fragment thereof, IL- 12 or a fragment thereof, IL-7 or a fragment thereof, IL-21 or a fragment thereof, or any combination thereof. In some embodiments, said peptide therapeutic agent comprises a cytokine or fragment thereof, an immune checkpoint inhibitor or fragment thereof, an immune cell activator agonist or fragment thereof, a tumor associated antigen binding moiety, a collagen binding domain or a fragment thereof, or an integrin binding domain or a fragment thereof, or any combination thereof. In some embodiments, (a) said cytokine comprises IL-2, IL-12, IL-7, IL-21, or any combination thereof; (b) said immune checkpoint inhibitor comprises an anti -Programmed Death-Ligand 1 (PD-L1) antibody or an anti- Programmed cell death protein 1 (PD-1) antibody, or any combination thereof; (c) said immune cell activator agonist comprises an anti-CD3 antibody, an anti 4-1BB (CD137) antibody, an anti-OX40 (CD134) antibody, an anti-CD28 antibody, an antiinducible T cell co-stimulator (ICOS) antibody, an anti-Glucocorticoid-Induced TNLR family Related gene (GITR), an anti-CD2 antibody, an anti-NKG2D antibody, an anti-T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) antibody, an anti- Lymphocyte Activation Gene-3 (LAG3) antibody, an anti-CD27 antibody, an anti- Killer-cell Immunoglobulin-like Receptor (KIR) antibody, an anti-V-domain Ig Suppressor of T-cell Activation (VISTA) antibody, an anti-CD7 antibody, an anti-CD44 antibody, or any combination thereof; and (d) said tumor associated antigen binding moiety bind to a tumor associated antigen comprising mesothelin, CD 19, NY-ESO-1, Fibroblast Activation Protein (FAP), Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1), Melanoma Antigen Gene A3 (MAGE-A3), (Disialoganglioside)Cer-Glc-Gal(NeuAc-NeuAc)-GalNAc (GD2), GM2 / GD2 synthase (B4GALNT1), B1-H3 (CD276), CD20, Cancer Antigen 125 (CA125),Attomey Docket No. 53531-724602Carbonic Anhydrase IX (CAIX), Tumor Endothelial Marker 8 (TEM8), Interleukin-3 Receptor Alpha Chain (CD 123), Preferentially Expressed Antigen in Melanoma (PRAME), Epithelial Cell Adhesion Molecule (EPCAM), Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), AXL, Interleukin-13 Receptor Alpha 2 (IL12Ra2), Signaling Lymphocytic Activation Molecule Family 7 (SLAMF7), or any combination thereof.

[0009] In some embodiments, said peptide therapeutic agent comprises a cleavable domain. In some embodiments, said peptide therapeutic agent comprises at least two cytokines. In some embodiments, said at least two cytokines comprise IL-2, IL- 12, IL-7, IL-21, or any combination thereof. In some embodiments, wherein said at least two cytokines comprise IL-2 and IL-12. In some embodiments, said at least two cytokines comprise IL-7 and IL-21.

[0010] In some embodiments, said peptide therapeutic agent comprises at least three cytokines or fragments thereof. In some embodiments, said at least three cytokines comprise IL-2, IL-12, IL-7, IL-21, or any combination thereof. In some embodiments, said at least three cytokines comprises IL-2, IL-21, IL-7, or any combination thereof.

[0011] In some embodiments, said peptide therapeutic agent comprises an amino acid sequence of any one of SEQ ID NOs: 588-621, or a variant thereof. In some embodiments, said peptide therapeutic agent comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, said peptide therapeutic agent comprises an antibody or antigen binding fragment thereof comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 667-686, or a variant thereof.

[0012] In some embodiments, said non-naturally occurring polynucleotide further comprises a spacer element comprising 1-20 nucleotides at a position comprising: (a) between at least two of said one or more synthetic response elements; (b) between at least two of said one or more enhancers; (c) between at least two of said one or more transcription factor binding sites; (d) between said one or more transcription factor binding sites and said one or more enhancers; or (e) any combination thereof.

[0013] In some embodiments, said core promoter comprises two or more promoter elements obtained from two or more of CEACAM5, CEP55, FAM11 IB, CST1, BIRC5, AGR2, UBE2C, KIF20A. In some embodiments, said core promoter comprises two or more promoter elements obtained from FAM11 IB and KIF20A. In some embodiments, said core promoter comprises a region of said non-naturally occurring polynucleotide that is from about -300 bp to about +100 bp relative to said TSS. In some embodiments, a synthetic response element of said one or more synthetic response elements is 5’ to said core promoter in said non-naturally occurring polynucleotide.

[0014] In some embodiments, said cancer cell is a colorectal cancer cell, a hepatocellular carcinoma cell, a lung cancer cell, a liver cancer cell, a breast cancer cell, a prostate cancer cell, a cervix cancerAttorney Docket No. 53531-724602cell, a uterus cancer cell, a pancreas cancer cell, a kidney cancer cell, a stomach cancer cell, a bladder cancer cell, an ovary cancer cell, a brain cancer cell, a head and neck cancer cell, an eye cancer cell, a mouth cancer cell, a throat cancer cell, an esophagus cancer cell, a chest cancer cell, a bone cancer cell, a rectum or other gastrointestinal tract organ cancer cell, a spleen cancer cell, a skeletal muscle cancer cell, a subcutaneous tissue cancer cell, a testicles or other reproductive organ cancer cell, a skin cancer cell, thyroid cancer cell, a blood cancer cell, a lymph nodes cancer cell, or any combination of one or more thereof.

[0015] In some embodiments, said one or more synthetic response elements comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 377-397, or 913, or a reverse complement thereof. In some embodiments, said one or more synthetic response elements comprises a sequence having at least 80% sequence identity to at least one of SEQ ID NOs: 386, 388, or 384, or a reverse complement thereof. In some embodiments, said core promoter comprises at least 35 consecutive nucleotides having at least 80% sequence identity to any one of SEQ ID NOs: 558-587, or 1010-1012, or a reverse complement thereof. In some embodiments, said core promoter comprises at least 35 consecutive nucleotides having at least 80% sequence identity to any one of SEQ ID NO: 560 or SEQ ID NO: 570. In some embodiments, said core promoter comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 584-587. In some embodiments, said non-naturally occurring polynucleotide further comprises a nucleic acid sequence comprising any one of SEQ ID NOs: 713, 278-279, 281-285, 289-293, 301-305, 310-312, 315-319, 324-326, 328-335, 342-343, 710-711, 727, 729, 730-731, 733, 736, 739, 742, 745, 748, 751, 757, 761, 764, 767, 770, 773, 776, 779, 782, 785, 788, 791, 794, 797, 800, 803, 806, 809, 812, 815, 818, 825, 828, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 876, 891, 894, 900, 903, 906, 912, 915, 918, 921, 922, 925, 928, 931, 934, 936, 939, 942, 945, 948, 951, 955, 958, 961, 964, 967, 971, 975, 979, 983, 986, 989, 992, 995, 998, 1001-1004, 1006, or 1008.

[0016] In some embodiments, said non-naturally occurring polynucleotide is double-stranded. In some embodiments, said non-naturally occurring polynucleotide is single -stranded. In some embodiments, said non-naturally occurring polynucleotide is a linear polynucleotide. In some embodiments, said non-naturally occurring polynucleotide is a circular polynucleotide. In some embodiments, said non-naturally occurring polynucleotide is synthetic, a recombinant polynucleotide, or a combination thereof. In some embodiments, said non-naturally occurring polynucleotide comprises a ribonucleic acid (RNA) polynucleotide. In some embodiments, said non-naturally occurring polynucleotide comprises a deoxyribonucleic acid (DNA) polynucleotide.

[0017] In some embodiments, said ORF comprises two or more genes encoding two or more peptide therapeutic agents. In some embodiments, said non-naturally occurring polynucleotide further comprises a second ORF comprising a second gene encoding a second peptide therapeutic agent. InAttorney Docket No. 53531-724602some embodiments, said higher level of expression of said peptide therapeutic agent in said cancer cell is at least a 10% increase in expression as compared to said non-cancer cell.

[0018] In another aspect, provided herein is a pharmaceutical formulation, comprising: (a) the non-naturally occurring polynucleotide described herein; and (c) a pharmaceutically acceptable: excipient, carrier, diluent, or any combination thereof.

[0019] In some embodiments, said carrier is a liposome, a micelle, a lipid nanoparticle, a cell, an exosome, or a vesicle. In some embodiments, said cell is an enucleated cell. In some embodiments, said cell is a mammalian cell. In some embodiments, said mammalian cell is a human cell. In some embodiments, said human cell is an immune cell. In some embodiments, said cell is allogenic. In some embodiments, said cell is autologous.

[0020] In another aspect, provided herein is a vector comprising said non-naturally occurring polynucleotide described herein.

[0021] In some embodiments, said vector comprises an adeno-associated viral vector, a lentiviral vector, a retroviral vector, or any combination thereof. In some embodiments, said vector is a non-viral vector. In some embodiments, said vector comprises a bacterial plasmid, a minicircle plasmid, or a nanoplasmid. In some embodiments, said vector is single stranded. In some embodiments, said vector is double stranded. In some embodiments, said vector is linear.

[0022] In another aspect, provided herein is a method comprising: administering said non-naturally occurring polynucleotide described herein, said pharmaceutical formulation described herein, or said vector described herein to a subject with cancer, thereby increasing expression of said peptide therapeutic agent in said cancer cell of said subject compared to a non-cancer cell.

[0023] In some embodiments, the method further comprises administering to said subject an additional therapeutic agent. In some embodiments, said additional therapeutic agent is a nonsteroidal anti-inflammatory drug (NSAID). In some embodiments, said additional therapeutic agent is an anti-histamine drug. In some embodiments, said additional therapeutic agent is a corticosteroid. In some embodiments, said corticosteroid is methyl prednisone. In some embodiments, said additional therapeutic agent is a complement inhibitor. In some embodiments, said additional therapeutic agent is a check-point inhibitor.

[0024] In some embodiments, said administering is performed by intratumoral injection, intravesicular injection, hepatic artery injection, or any combination thereof. In some embodiments, said non-naturally occurring polynucleotide is administered systemically. In some embodiments, said non-naturally occurring polynucleotide is administered in a tissue-specific manner. In some embodiments, said administering is performed locoregionally.

[0025] In some embodiments, said peptide therapeutic agent comprises a toxin, a cytokine, an antibody or antigen binding fragment thereof, or any combination thereof. In some embodiments, saidAttomey Docket No. 53531-724602peptide therapeutic agent comprises a cytokine. In some embodiments, said subject has a tumor comprising said cancer cell.

[0026] In some embodiments, the method further comprises reducing a size of said tumor by at least two-fold as compared to a control subject with a comparable tumor that has not been administered said non -naturally occurring polynucleotide. In some embodiments, the method further comprises reducing a size of said tumor by at least two-fold as compared to said size of said tumor prior to said administering. In some embodiments, said expression of said peptide therapeutic agent is increased in said cancer cell by two-fold to 200-fold. In some embodiments, said expression of said peptide therapeutic agent is increased in said cancer cell by two-fold to 200-fold compared to a reference expression level in said subject prior to said administering. In some embodiments, said expression of said peptide therapeutic agent is increased in said cancer cell by two-fold to 200-fold compared to a reference expression level in a control subject with said cancer that has not been administered said non-naturally occurring polynucleotide.

[0027] In some embodiments, said non-naturally occurring polynucleotide is provided in a liposome. In some embodiments, said liposome comprises one or more of a micelle, a solid lipid nanoparticle, or a combination thereof. In some embodiments, said non-naturally occurring polynucleotide is provided in a vector. In some embodiments, said vector comprises an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a non-viral vector, or any combination thereof. In some embodiments, said non-viral vector comprises a plasmid vector. In some embodiments, said non-naturally occurring polynucleotide is provided by a host cell. In some embodiments, said non-naturally occurring polynucleotide is provided by a host cell, or an exosome or vesicle of said host cell. In some embodiments, said host cell comprises an engineered cell, a natural cell, or a combination thereof. In some embodiments, said host cell comprises a eukaryotic cell, a prokaryotic cell, or a combination thereof. In some embodiments, said eukaryotic cell comprises a yeast cell, a mammalian cell, or a combination thereof. In some embodiments, said mammalian cell is a human cell. In some embodiments, said prokaryotic cell is a bacterial cell. In some embodiments, said host cell is a nucleated host cell, an enucleated host cell, or a combination thereof.

[0028] In another aspect, provided herein is a kit comprising: said non-naturally occurring polynucleotide described herein, said pharmaceutical formulation described herein, or said vector described herein; and instructions for administering said non-naturally occurring polynucleotide to a subject with cancer.

[0029] In some embodiments, said instructions comprise any one of the methods described herein.

[0030] In another aspect, provided herein is a lipid nanoparticle (LNP) comprising said non-naturally occurring polynucleotide described herein.

[0031] In another aspect, provided herein is an engineered nucleic acid molecule comprising a nucleic acid sequence comprising SEQ ID NOs: 713, 278-279, 281-285, 289-293, 301-305, 310-312,Attorney Docket No. 53531-724602315-319, 324-326, 328-335, 342-343, 710-711, 727, 729, 730-731, 733, 736, 739, 742, 745, 748, 751, 757, 761, 764, 767, 770, 773, 776, 779, 782, 785, 788, 791, 794, 797, 800, 803, 806, 809, 812, 815, 818, 825, 828, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 876, 891, 894, 900, 903, 906, 912, 915, 918, 921, 922, 925, 928, 931, 934, 936, 939, 942, 945, 948, 951, 955, 958, 961, 964, 967, 971, 975, 979, 983, 986, 989, 992, 995, 998, 1001-1004, 1006, or 1008.

[0032] In another aspect, provided herein is a composition comprising an engineered peptide, wherein said engineered peptide comprises: (a) a cytokine or a fragment thereof; (b) an antibody or a fragment thereof; and (c) a collagen binding domain or a fragment thereof, an integrin binding domain or a fragment thereof, a cleavable domain or a fragment thereof, or any combination thereof

[0033] In some embodiments, said engineered peptide is a chimeric peptide or a synthetic peptide. In some embodiments, said engineered peptide is a peptide therapeutic agent. In some embodiments, said engineered peptide is a fusion protein.

[0034] In another aspect, provided herein is a nucleic acid molecule encoding said composition.

[0035] In another aspect, provided herein is a method of detecting an expression product in situ, said method comprising: (a) administering a polynucleotide to a subject, wherein said polynucleotide comprises:: (i) one or more synthetic response elements comprising one or more enhancers and a plurality of transcription factor binding sites; (ii) a core promoter operably linked to an open reading frame (ORF) comprising a gene encoding said expression product; and (iii) a transcription start site (TSS) upstream of said ORF, wherein said one or more synthetic response elements and said core promoter increase expression of said expression product in a target cell of said subject as compared with a non-target cell; and (b) detecting said expression product in situ.

[0036] In some embodiments, expression product is detected in said target cell ex vivo. In some embodiments, said expression product is detected in said subject in vivo. In some embodiments, said expression product is secretable from said target cell. In some embodiments, said detecting comprises using magnetic resonance imaging (MRI) imaging, positron emission tomography (PET) imaging, single-photon emission computed tomography (SPECT) imaging, photoacoustic imaging, fluorescence or luminescence imaging, or any combination thereof. In some embodiments, said detecting comprises said luminescence imaging, and wherein said expression product comprises a luciferase an Enhanced Green Fluorescent Protein (EGFP), a Green Fluorescent Protein (GFP), a Red Fluorescent Protein (RFP), or any combination thereof. In some embodiments, said expression product comprises a polypeptide contrast agent, a lanthanide-binding protein, or an engineered fusion thereof. In some embodiments, said expression product comprises [3-galactosidase, GFP, mCherry or derivatives thereof, aeCP597, cjBlue or derivatives thereof, IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP713 / 256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry, GAF-FP, BphPl-FP / C20S, AphB variants, Dronpa, Dronpa-M159T, BphPl, or variants thereof.Attorney Docket No. 53531-724602

[0037] In some aspects, provided herein is a non-naturally occurring polynucleotide comprising: (a) a core promoter comprising a transcription start site (TSS), wherein the core promoter is obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells and operably linked to an open reading frame (ORF) and (b) a plurality of binding sites for one or more transcription factors (TFs), wherein said one or more TFs are expressed at higher levels or more active in cancer cells compared to non-cancer cells. In some embodiments, the non-naturally occurring polynucleotide further comprises a plurality of enhancers. In some embodiments, said plurality of enhancers are obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells. In some embodiments, said plurality of enhancers are obtained from two or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells, wherein one of said plurality of enhancers comprises: (i) a transcription regulatory element with at least 90% sequence homology to an enhancer consensus sequence of two or more homologous genes, and / or (ii) a sequence capable of binding a transcription associated protein as determined by chromatin immunoprecipitation (ChIP) or an in vitro transfection reporter assay.

[0038] In some embodiments, the non-naturally occurring polynucleotide further comprises a plurality of enhancers. In some embodiments, said plurality of enhancers are obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells. In some embodiments, said plurality of enhancers are obtained from two or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells, wherein one of said plurality of enhancers comprises: (i) a transcription regulatory element with at least 90% sequence homology to an enhancer consensus sequence of two or more homologous genes, and / or (ii) a sequence capable of binding a transcription associated protein as determined by chromatin immunoprecipitation (ChIP) or an in vitro transfection reporter assay. In some aspects, provided herein is a non-naturally occurring polynucleotide comprising: (a) a core promoter comprising a transcription start site (TSS) and two or more promoter elements obtained from two or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells and operably linked to an open reading frame (ORF) and (b) a plurality of binding sites for one or more transcription factors (TFs), wherein said one or more TFs are expressed at higher levels or more active in cancer cells compared to non-cancer cells.

[0039] In some embodiments, the non-naturally occurring polynucleotide further comprises a plurality of enhancers. In some embodiments, said plurality of enhancers are obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells. In some embodiments, said plurality of enhancers are obtained from two or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells, wherein one of said plurality of enhancers comprises: (i) a transcription regulatoryAttorney Docket No. 53531-724602element with at least 90% sequence homology to an enhancer consensus sequence of two or more homologous genes, and / or (ii) a sequence capable of binding a transcription associated protein as determined by chromatin immunoprecipitation (ChIP) or an in vitro transfection reporter assay. In some aspects, provided herein is a non-naturally occurring polynucleotide comprising: (a) a core promoter comprising a transcription start site (TSS), wherein the core promoter is obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells and operably linked to an open reading frame (ORF) and (b) a plurality of enhancers. In some embodiments, said plurality of enhancers are obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells. In some embodiments, said plurality of enhancers are obtained from two or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells, wherein one of said plurality of enhancers comprises: (i) a transcription regulatory element with at least 90% sequence homology to an enhancer consensus sequence of two or more homologous genes, and / or (ii) a sequence capable of binding a transcription associated protein as determined by chromatin immunoprecipitation (ChIP) or an in vitro transfection reporter assay.

[0040] Aspects disclosed herein provide a non-naturally occurring polynucleotide comprising: (a) a core promoter comprising a transcription start site (TSS), wherein the core promoter is obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells and operably linked to an open reading frame (ORF), (b) a plurality of binding sites for one or more transcription factors (TFs), wherein said one or more TFs are expressed at higher levels or more active in cancer cells compared to non-cancer cells, and (c) a plurality of enhancers. In some embodiments, said plurality of enhancers are obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells. In some embodiments, said plurality of enhancers are obtained from two or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells, wherein one of said plurality of enhancers comprises: (i) a transcription regulatory element with at least 90% sequence homology to an enhancer consensus sequence of two or more homologous genes, and / or (ii) a sequence capable of binding a transcription associated protein as determined by chromatin immunoprecipitation (ChIP) or an in vitro transfection reporter assay.

[0041] In some aspects, provided herein is a non-naturally occurring polynucleotide comprising any of the sequences from Table 1A, Table IB, or Table 1C. In some aspects, provided herein is a non-naturally occurring polynucleotide comprising a human alpha-fetoprotein (AFP) promoter sequence comprising a plurality of HNF-1A TF binding sites, wherein each HNF-1A binding site comprises the sequence 5’-GTTAATTATTAAC-3’ (SEQ ID NO: 128).

[0042] In some aspects, provided herein is a vector comprising any of the non-naturally occurring polynucleotide described herein. In some aspects, provided herein is a pharmaceutical compositionAttomey Docket No. 53531-724602comprising any of the non-naturally occurring polynucleotide described herein, or any the vector described herein and a pharmaceutically acceptable excipient, carrier, or diluents, or any combination thereof.

[0043] Also provided, in some aspects, are lipid nanoparticles (LNP) comprising any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, or any of the pharmaceutical composition described herein. In some aspects, provided herein is a cell comprising any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, any of the pharmaceutical composition described herein, or any of the LNP described herein. In some aspects, provided herein is a method of selectively expressing a reporter protein in a cancer or tumor cell, comprising contacting said tumor cell with any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, any of the pharmaceutical composition described herein, or any of the LNP described herein, wherein the non-naturally occurring polynucleotide further comprises an open reading frame (ORF) encoding said reporter protein, wherein said ORF is operatively linked to said synthetic promoter.

[0044] In some aspects, provided herein is a method comprising: (a) administering to a subject any of the pharmaceutical composition described herein; or a composition any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, or any of the LNP described herein; wherein the non-naturally occurring polynucleotide further comprises an open reading frame (ORF) encoding a reporter protein, wherein said ORF is operatively linked to a synthetic promoter in said non-naturally occurring polynucleotide, and (b) detecting said reporter protein, wherein said pharmaceutical composition or said composition induces expression of said reporter protein preferentially in diseased cells in said subject compared to in non-disease cells, and wherein a relative ratio of said reporter protein expressed in said diseased cells over said non-diseased cells is greater than 1.0.

[0045] In some aspects, provided herein is a method for treating a subject having or suspected of having a disease, comprising administering to said subject any of the pharmaceutical composition described herein; or a composition any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, or any of the LNP described herein; wherein the non-naturally occurring polynucleotide further comprises an open reading frame (ORF) encoding a therapeutic protein, wherein said ORF is operatively linked to a synthetic promoter in said non-naturally occurring polynucleotide, wherein said pharmaceutical composition or said composition induces expression of said therapeutic protein preferentially in diseased cells in said subject compared to in non-disease cells, and wherein a relative ratio of said therapeutic protein expressed in said diseased cells over said non-diseased cells is greater than 1.0.

[0046] In some aspects, provided herein is a method comprising: (a) administering to a subject any of the pharmaceutical composition described herein; or a composition any of the non-naturally occurringAttorney Docket No. 53531-724602polynucleotide described herein, any of the vector described herein, or any of the LNP described herein; wherein the non-naturally occurring polynucleotide further comprises an open reading frame (ORF) encoding a reporter protein, wherein said ORF is operatively linked to a synthetic promoter in said non-naturally occurring polynucleotide, and (b) localizing a tumor or an absence thereof in a body of said subject via expression of said reporter protein using an imaging technique performed on said body of said subject.

[0047] In some aspects, provided herein is a method comprising: (a) introducing to a subject suspected of having a cancer via intravenous administration any of the pharmaceutical composition described herein; or a composition any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, or any of the LNP described herein; wherein said non-naturally occurring polynucleotide further comprises an open reading frame (ORF) encoding a reporter protein, wherein said ORF is operatively linked to a synthetic promoter in said non-naturally occurring polynucleotide, and (b) detecting said reporter protein from said subject.

[0048] In some aspects, provided herein is a method comprising: (a) introducing to a subject suspected of having a cancer via intravenous administration a plurality non-naturally occurring polynucleotides, wherein: said plurality of non-naturally occurring polynucleotides comprises a plurality of different promoters of genes overexpressed in a tumor cell versus a normal tissue or functional fragments thereof operably linked to genes encoding reporter proteins, wherein said plurality of different promoters of genes overexpressed in said tumor cell versus said normal tissue drive expression of said corresponding reporter proteins in a cell affected by said cancer, wherein said DNA molecules are selected from the group consisting of nanoplasmids and linear double-stranded DNA molecules; and (b) detecting said reporter proteins from said subject.INCORPORATION BY REFERENCE

[0049] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.Attomey Docket No. 53531-724602

[0051] The novel features of the inventive concepts are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present inventive concepts will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the inventive concepts are utilized, and the accompanying drawings of which:

[0052] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0053] FIG. 1 shows a schematic of synthetic promoter architecture and design including, for example, a fragment of SEQ ID NO: 378.

[0054] FIG. 2 describes, in some embodiments, the coreCEACAM5 design, including, for example, a fragment of SEQ ID NO: 121. FIG. 2 discloses SEQ ID NO: 1021.

[0055] FIG. 3 describes, in some embodiments, the coreCEP55 design.

[0056] FIG. 4 describes, in some embodiments, the coreFAMl 1 IB design.

[0057] FIG. 5 describes, in some embodiments, the coreAGR2 design.

[0058] FIG. 6 shows the comparison of the reporter gene expression by endogenous promoter and synthetic promoter in Hl 299 cells.

[0059] FIG. 7 shows the reporter gene expression performance by synthetic promoters in human PDX models. Bar graphs from left to right: BIRC5, FOSLl-coreBIRC5, FOSL1-CEACAM5, FOSL1-FAM11 IB, FOSL1-KIF20A, FOSL1-AGR2, and FOSL1-TATA, respectively.

[0060] FIG. 8 shows signal-to-noise profdes of the reporter gene expression by synthetic promoters. Bar graphs from left to right: BIRC5, FOSLl-coreBIRC5, FOSL1-FAM11 IB, FOSL1-KIF20A, FOSL1-AGR2, FOSL1-CST1, and FOSL1-TATA, respectively.

[0061] FIG. 9 shows the reporter gene expression by synthetic promoters in H1299 cells.

[0062] FIG. 10 describes the workflow of synthetic promoter design and construction according to some embodiments.

[0063] FIG. 11 describes the workflow of synthetic promoter design and construction with coreAGR2 according to some embodiments.

[0064] FIG. 12 describes the synthetic promoter architecture, design, discovery, and validation pipeline according to some embodiments.

[0065] FIG. 13 describes Transcription Factor Tile Design (top) and how to measure synthetic element expression (bottom). Each synthetic DNA sequence was designed as a series of repeated transcription factor (TF) binding sites obtained from the consensus binding motif for the TF of interest (blue). To test the impact of the different relative positioning of these sites around the helical nature ofAttorney Docket No. 53531-724602the double stranded DNA (one helical turn is equivalent to -10.5 base pairs), the repeated binding sites are separated by a variable length of nucleic acid spacer sequences (yellow). Lastly, the synthetic DNA sequence contains a short fdler sequence (grey) to maintain consistent total length of the candidate enhancer sequence block.

[0066] FIG. 14 shows Expression Score Distribution Across Lung Cancer Models. The expression score distribution varies across different lung cancer models. The PDX cell line LXFL430 had the widest distribution and outliers with the highest expression scores.

[0067] FIG. 15 shows the reporter gene expression by HOXC10 tiles. Using a luciferase reporter assay lead candidates representing the MNX1, HOXC10 and CREB3L1 transcription factors were tested across seven lung cancer cell line models (H1299, PDX430, PDX1121, PDX629, PDX529, PDX586, and PDX2184) and one lung normal cell line (IMR90). Higher expression compared to FOSL-coreBIRC5 lead synthetic promoter with up to 50-80-fold improvement was observed.

[0068] FIG. 16 shows the reporter gene expression by TCF7L1 TF tiles in PDX430 cell line.

[0069] FIG. 17 shows Wnt-driven cell lines identified by PCA (LK2 and NCI-H520) driving the expression by TCF7 and TCF7L1 promoters. In a transient transfection of two TCF7 variant promoters across five cell lines, H520 and LK-2 show the same high levels of activation as PDX430, which was predicted by the PCA analysis. As expected, H1299 and A549 cell lines do not show substantial expression by the TCF7 promoters, and are much better represented by the FOS-coreBIRC5 promoter.

[0070] FIG. 18 shows the expression of the reporter gene by TP53 elements. Addition of TP53 elements to TATA-TSS core results in significantly increased expression of the reporter gene in PDX586 as predicted by HTS-002.

[0071] FIG. 19 shows the expression of the reporter gene by TP53 variants in A549 cells.

[0072] FIG. 20 shows PCA analysis in Hl 944 and H2023 cells.

[0073] FIGs. 21A-C show tables comparing mutation statuses of select genes. FIG. 21A shows a table comparing mutation status of P53, key gene set expression, and TP63 expression in different cancer cell lines. FIGs. 21B and 21C show mutation profile in Clinical Proteomic Tumor Analysis Consortium (CPTAC) Lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), respectively.

[0074] FIG. 22 shows the reporter gene expression by p53 in A549, H1944, and H358 cell lines.

[0075] FIG. 23 shows a table comparing TP53 status and reporter gene expression in different cell lines.

[0076] FIG. 24 shows the reporter gene expression by TP53 and TCF7. Pathway specific TP53 and TCF7 response elements pair well and get higher signal using new non-coreBIRC5 cores. As observed with the FOS response element, TP53 and TCF7 response elements combined withAttorney Docket No. 53531-724602coreCSTl, coreAGR2, and coreFAMl 1 IB show up to a 10-fold signal increase compared to the same promoters constructed with coreBIRC5.

[0077] FIG. 25 shows the reporter gene expression by coreBIRC5 and coreAGR2 combined with different response elements in H1299, PDX430, and PDX586 cell lines.

[0078] FIG. 26 shows the reporter gene expression by coreBIRC5, coreAGR2, coreFAMl 1 IB combined with different response elements in different cell lines.

[0079] FIG. 27 shows fold change in expression of reporter genes from constructs comprising combination of FOSL and CREB3L1.

[0080] FIG. 28 shows fold change in expression of reporter genes from constructs comprising combination of TCF7 and TP53.

[0081] FIG. 29 shows validation of top ranked TF tiles with the coreBIRC5 promoter. Using a luciferase reporter assay various TF tiles that were highly ranked in the MPRA screens for Hl 299 and LXFL430 were tested. Many of the TF tiles showed stronger expression than the base expression of the coreBIRC5 and the FOSL-coreBIRC5. The TCF7L1 TF tiles showed specific expression in the LXFL430 cell line.

[0082] FIGs. 30A and 30B show expression of synthetic promoter FOS-coreBIRC5 in PDX cell lines and normal lung cell lines. Compared to endogenous promoters, including the Survivin (BIRC5) promoter and other first-generation endogenous promoters used in multiplexes, the synthetic promoter FOS-coreBIRC5 outperformed in terms of strength and sensitivity in 8 PDX cell lines that represent different patients’ genomic profiles (FIG. 30A). FIG. 30B shows that the synthetic promoter also demonstrates lack of expression in normal human fibroblast cell line (IMR-90), small airway epithelial cells (SAEC) and normal human bronchial epithelial cells (NHBE).

[0083] FIG. 31 shows the top 30 contributing features that make up a factor of MOFA analysis.

[0084] FIG. 32 shows comparison of reporter gene expression by FOSL2 in Normal Adjacent Tissues (NAT) and tumor.

[0085] FIG. 33 shows the binding of FOSL2 and C-Jun TFs to the FOS element in the FOS-coreBIRC5 promoter. Chromatin immunoprecipitation (ChIP) was performed on two different cell lines transfected with the FOS-coreBIRC5 promoter construct (e.g., SEQ ID NO: 169). Pulldowns for FOSL2 and c-Jun showed significant enrichment of the coreBIRC5 element compared to nonspecific pulldown, by 14X for FOSL2 in H1299 and 5x for FOSL2 in A549. With the comparison to the control construct of solely coreBIRC5, this makes it clear that the FOS response element is responsible for the association of FOSL2 and C-Jun with the synthetic promoter.

[0086] FIG. 34 shows demonstration of high sensitivity and specificity in primary-derived and commercial cell lines by chimeric promoters using core-BIRC5. Response elements for different TFs (FOSL2, TWIST1, ETV4) in combination with the coreBIRC5 promoter showed variable sensitivityAttorney Docket No. 53531-724602across different PDX cell lines, H1299 NSCLC cell line, and a lack of expression in IMR-90 (normal human fibroblast) cell line.

[0087] FIG. 35 shows the activity of TCF7 & TCFL1 variants in different cell lines. TCF7 & TCFL1 variants were only active in PDX LXFL430 among cell lines tested. Two variants of the TCF7-response element promoter, as compared to the minimal coreBIRC5 and positive control FOS-coreBIRC5 promoter, demonstrated extremely high levels of expression in the large cell lung cancer PDX430.

[0088] FIG. 36 shows that alternative core promoters to coreBIRC5 demonstrate high utility in synthetic promoter constructs. The full-length endogenous promoters, core promoters, and FOS-core promoters using BIRC5, FAM11 IB, AGR2 and CST1 were tested in two lung cancer cell lines -H1299 and PDX629. The use of the new cores with FOS demonstrated up to 20-fold improvement in signal compared to the original FOS-coreBIRC5 promoter described previously. On the bottom, experiments using three primary normal lung cell lines (small airway epithelial cells from two donors and normal human lung fibroblasts) demonstrated the FOS-coreAGR2 and FOS-coreCSTl constructs still maintain high specificity for cancer, while FOS-coreFAMl 1 IB appears to have significant noise in lung fibroblasts.

[0089] FIG. 37 shows reporter gene expression derived by different synthetic promoters in cancer epithelial cells, cancer associated fibroblast cells, and normal adjacent tissue (NAT) cells from patient derived cell lines (LU057: 63 / F / White, Stage IIIB Adeno-squamous pT4, N2). *: not tested, dotted line: CAG, constitutive promoter.

[0090] FIGs. 38A and 38B show AFP-3, an engineered variant of the human alpha-fetoprotein (AFP) promoter that can drive strong and highly specific expression in HCC. In FIG. 38A, the primary changes to the AFP promoter sequence are shown, changing the HNF-1A sites to the consensus sequence for the transcription factor binding site. FIG. 38A discloses SEQ ID NOs: 553-554, and 128, respectively, in order of appearance. FIG. 38B shows that engineered AFP-3 (SEQ ID NO: 554) drives up to 200-fold higher expression in liver cancer cell lines than the wildtype AFP promoter (SEQ ID NOs: 553), while still maintaining high specificity against lung normal (IMR-90, MRC-9), lung cancer (Hl 299) and melanoma (MeWo) cell lines, as compared to the Survivin (BIRC5) promoter which shows some cancer-activated activity in both liver and non-liver cancer cell lines.

[0091] FIG. 39 shows signal-to-noise ratio of SEAP in Hep3B orthotopic tumor model. Secreted alkaline phosphatase (SEAP) was measured from the serum of tumor-bearing and normal animals dosed with the BIRC5-SEAP construct versus the AFP-3-SEAP construct. At the day 0 bleed (predosing), background levels of SEAP in all mice were below the lower limit of quantification (LLOQ) of the assay (0.4pg / 12.5uL), as expected. At 3 days post-dose, the BIRC5-SEAP construct dosed animals showed a 7-fold increase of SEAP reporter in the serum over the LLOQ, with no backgroundAttorney Docket No. 53531-724602expression at all in non-tumored animals. The AFP-3 construct promoted expression in tumored animals approximately 97-fold higher than non-tumored animals.

[0092] FIGs. 40A, 40B, and 40C show immunohistochemistry (IHC) results for AFP-3-sr39tk, using HA epitope. FIGs. 40A and 40B show representative serial sections from the tumor-bearing left lobe of a mouse in Group 6 (AFP-3 -sr39tk) dosed at 2.8mpk of EM-40 stained by H&E and by HA antibody for the reporter expression. The tumor boundary has been outlined in the H&E slide. Reporter expression is confined to the tumor cells only. In FIG. 40C, the same mouse’s right liver lobe, devoid of tumor is shown to have no positive cells.

[0093] FIGs. 41A, 41B, 41C, 41D, 41E, and 41F show IHC results for positive control CAG-sr39tk. Serial sections of the tumor-containing left lobe from a mouse in Group 10 show positive staining in the tumor (FIGs. 41A and 41B; stained dark purple by H&E). Left and right lobe sections from the same mouse show occasional disperse signal from individual cells (FIGs. 41C and 41D). Serial sections stained by H&E and by IHC for the -HA tag for a second mouse’s tumor also show many positive-stained cells throughout the tumor tissue, as outlined in the H&E figure (FIGs. 41E and 41F).

[0094] FIG. 42 shows images of animal bioluminescence.

[0095] FIGs. 43A, 43B, 43C, and 43D show muti-omics data on benign cell lines.

[0096] FIG. 44 shows that there is no reporter expression by synthetic promoter constructs in granulomatous lesions caused by Mycobacterium tuberculosis (M. tb) infection in CBA / J mice despite high disease burden.

[0097] FIG. 45 shows the reporter gene expression performance by different synthetic promoters in various cancer and non-cancer cell lines. Combining the FOS element with new core promoters resulted in significant increases in expression across NSCLC cell lines & PDX CL models. Bar graphs from left to right: HIGH-coreBIRC5, FOS-coreBIRC5, FOS-CEACAM5, FOS-FAM111B, FOS-KIF20A, FOS-AGR2, FOS-CST, and FOS-TATA, respectively.

[0098] FIG. 46 shows the reporter gene expression performance by different synthetic promoters in various cancer and non-cancer cell lines. Some FOS-newCores combinations had elevated noise in Normal Lung Fibroblasts. Bar graphs from left to right: FOS-BIRC5, FOS-CEACAM5, FOS-FAM11 IB, FOS-KIF20A, FOS-AGR2, FOS-CST1, and FOS-TATA, respectively.

[0099] FIG. 47 shows an exemplary workflow of diagnostic medical sonography (DMS) study.

[0100] FIG. 48 shows a schematic of adding activating elements to the core promoters.

[0101] FIG. 49 shows the reporter gene expression performance by different synthetic promoters in H1299 and PDX430 cell lines. HIGH element was observed to be functional in vitro when combined with alternate core promoters. Bar graphs from left to right: BIRC5, CEACAM5, FAM11 IB, KIF20A, AGR2, and FOS-TATA, respectively.Attorney Docket No. 53531-724602

[0102] FIG. 50 shows the reporter gene expression performance by different synthetic promoters in normal small airway epithelial cells and normal lung fibroblasts. In vitro specificity models were predictive of lung noise with HIGH-CEA CAM5, HIGH-FAM11 IB and HIGH-KIF20A. Bar graphs from left to right: HIGH-BIRC5, HIGH-CEACAM5, HIGH-FAM11 IB, HIGH-KIF20A, HIGH-AGR2, FOS-AGR2, and FOS-TATA, respectively.

[0103] FIG. 51 shows the reporter gene expression performance by different synthetic promoters in various PDX cell lines. Synthetic promoters described herein outperform endogenous promoter in PDX cell lines. Bar graphs from left to right: Survivin (endogenous BIRC5 promoter), FOS-coreBIRC5, HIGH-coreBIRC5, FOS-coreAGR2, FOS-coreCSTl, HIGH-FAM111B, FOS-TATA-TSS, and EFl A (positive control), respectively.

[0104] FIG. 52 shows the reporter gene expression performance by different synthetic promoters in various primary cell lines derived from PDX or primary tissue. Bar graphs from left to right: Survivin (endogenous BIRC5 promoter), FOS-coreBIRC5, HIGH-coreBIRC5, FOS-coreAGR2, FOS-coreCSTl, HIGH-FAM111B, FOS-TATA-TSS, and CAG (positive control), respectively.

[0105] FIG. 53 shows the reporter gene expression performance by different synthetic promoters in primary lung normal cells (Lonza). Bar graphs from left to right: Survivin (endogenous BIRC5 promoter), FOS-coreBIRC5, HIGH-coreBIRC5, FOS-coreAGR2, FOS-coreCSTl, HIGH-FAM111B, FOS-TATA-TSS, and EFl A (positive control), respectively.

[0106] FIG. 54 shows the reporter gene expression performance by different synthetic promoters in different primary lung normal cells derived from the same patient.

[0107] FIG. 55 shows the comparison of the reporter gene expression performance by synthetic promoters in EMT state cells and wild type A549 cells.

[0108] FIG. 56 shows a table of top 10 enhancer candidates.

[0109] FIG. 57 shows the reporter gene expression performance by synthetic promoters comprising enhancer elements in various cancer and non-cancer cells. Constructs were tested in vitro across panel of 5 LUAD cell lines, 3 HCC cell lines, and IMR90 lung normal cells for expression profiles of enhancer elements paired with each core promoter (including 7x CRL PDX cell lines and 2x Lonza normal cells).

[0110] FIG. 58 shows comparison of the reporter gene expression performance by different synthetic promoters comprising enhancer elements in various cancer cell lines.

[0111] FIG. 59 shows the reporter gene expression performance by different synthetic promoters in various cell lines. Bar graphs from left to right: BIRC5, Canscript, FOSL1, GATA1, MYC MAX, SOX9, AFP, AFP3, Enhancer+AFP3, and NT EFla, respectively.

[0112] FIG. 60 shows a two-step promoter amplification utilizing the yeast GAL4-VP system.

[0113] FIG. 61 shows comparison of the reporter gene expression performance by different synthetic promoters and the yeast GAL4-VP system in H1299, LXFA 629, and LXFA 737 cell lines. TSTA:Attorney Docket No. 53531-724602two-step transcriptional activation. Bar graphs from left to right: EF1A, CMV, BIRC5, FOSL1, AFP3, TSTA PR-GAL4 only, BIRC5, FOSL1, AFP3, respectively.

[0114] FIG. 62 shows comparison of the reporter gene expression performance by different synthetic promoters and the yeast GAL4-VP system in SNU-475, PLC / PRF / 5, and C3A cell lines. TSTA: two-step transcriptional activation. Bar graphs from left to right: EF1A, CMV, BIRC5, FOSL1, AFP3, TSTA PR-GAL4 only, BIRC5, FOSL1, AFP3, respectively.

[0115] FIG. 63 shows exemplary core promoters with annotations. FIG. 63 discloses SEQ ID NO: 555.

[0116] FIGs. 64A-B show elements of coreFAMl 1 IB. FIG. 64 A shows a diagram of an annotated coreFAMl 1 IB promoter with predicted TF binding sites. FIG. 64B shows activating and repressing elements within coreFAMl 1 IB identified from core promoter element deletion studies.

[0117] FIG. 65 shows top 10 ranked response elements from H1299 (Large Cell Carcinoma), LXFA586 (Adenocarcinoma), and LXFL430 (Large Cell Carcinoma). Control response elements containing FOS / CREB (H1299), TP53 / TP73 (LXFA586), or TCF (LXFL430) drive strong expression of reporter gene in H1299, LXFA586, and LXFL430 cell lines respectively, and there are several additional hits.

[0118] FIGs. 66A, 66B, 66C, and 66D show in vitro low throughput validation of response elements from FIG. 112 using Firefly luciferase (FLuc) assay.

[0119] FIGs. 67-68 show a DNA binding consensus sequence of Forkhead Box Protein 01 (FOXO1;FIG. 67, left, e.g., a fragment of SEQ ID NO: 202), ELK3 (FIG. 67, middle, e.g., a fragment of SEQ ID NO: 150), FOXO::ELK (FIG. 67, right, e.g, a fragment of SEQ ID NO: 150), XBP1 (FIG. 68, top left, e.g., a fragment of SEQ ID NO: 155), NFE2L2 (FIG. 68, top right, e.g., a fragment of SEQ ID NO: 152), and MTF1 (FIG. 68, bottom, e.g., a fragment of SEQ ID NO: 151).

[0120] FIG. 69 shows validation of response elements with FOS and CREB using Firefly luciferase (FLuc) assay.

[0121] FIG. 70 shows Firefly luciferase (FLuc) assay results of combination of TCF and FOS elements.

[0122] FIG. 71 shows Firefly luciferase (FLuc) assay results of different elements in patient-derived cancer cells (cancer epithelia and cancer fibroblasts) and normal adjacent tissues. Bar graphs from left to right: Cancer Epithelia, Cancer Fibroblasts, and Normal Adjacent Tissues, respectively.

[0123] FIG. 72 shows Synthetic Response Sensors (SRS) that drive cancer specific expression where the SRS comprises a series of Synthetic Response Elements (SREs), or enhancers, and a cancer activated core promoter. TF: Transcription Factor.

[0124] FIG. 73 shows a graph of gene expression activated by SRS-G comprising the core promoter specific for lung cancer and a single SRE. A luciferase reporter expression system was used to evaluate the strength of activation in cell lines that represent the three main Non-Small Cell LungAttorney Docket No. 53531-724602Cancer (NSCLC) subtypes. The expression values are shown as the fold change over a strong constitutive promoter. SRS-G was able to achieve expression that is 10-20% on the expression of the constitutive promoter.

[0125] FIGs. 74A, 74C, 74E, 74G, 741, and 74K show graphs of gene expression activated by different SRSs (SRS-A, SRS-B, SRS-C, SRS-D, SRS-E, and SRS-F) designed to drive gene expression in lung cancers. A luciferase reporter expression system was used to evaluate the strength of activation in cell lines that represent the three main NSCLC subtypes. The expression values are shown as the fold change over a strong constitutive promoter. SRS-A was able to achieve expression that is 5-50% on the expression of the constitutive promoter (FIG. 74A). SRS-B was able to achieve expression that is 20-50% on the expression of the constitutive promoter (FIG. 74C). SRS-C was able to achieve expression similar to or 3-fold above the constitutive promoter (FIG. 74E). SRS-D was able to achieve expression similar to or 2-10-fold above the constitutive promoter (FIG. 74G). SRS-E was able to achieve expression similar to or 2-8-fold above the constitutive promoter (FIG. 741). SRS-F was able to achieve expression similar to or 3-5-fold above the constitutive promoter. (FIG.74K).

[0126] FIGs. 74B, 74D, 74F, 74H, 74 J, and 74L show graphs of gene expression activated by an SRS designed to drive gene expression in lung cancers (SRS-A, SRS-B, SRS-C, SRS-D, SRS-E, and SRS-F). A luciferase reporter expression system was used to evaluate the strength of activation in cell lines that represent the NSCLC subtypes as well as normal primary lung cells. Expression values are shown as the fold change over a strong constitutive promoter on the left. Same data plotted as an ROC curve is presented on the right.

[0127] FIG. 75 shows graphs of expression pattern of a reporter gene activated by a constitutive or non-cancer specific promoter, Cytomegalovirus (CMV). A luciferase reporter expression system was used to evaluate the strength of activation in cell lines that represent the NSCLC subtypes as well as normal primary lung cells. Expression values are shown as the fold change over a strong constitutive promoter on the left. Same data plotted as an ROC curve is presented on the right.

[0128] FIG. 76 shows graphs of gene expression activated by SRSs, demonstrating that SRSs can be active in both lung and liver cancer models, or selectively active in a target model. H358 lung cancer cells, HepG2 liver cancer cells, and Hep3B liver cancer cells were seeded in 96-well plates at a density of 10,000 cells per well, with each plasmid containing luciferase reporter expression system tested in triplicate. Transfection was performed using Lipofectamine™ 3000, a transfection agent comprising DOSPA (2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propaniminium trifluoroacetate) and DOPE (dioleoyl phosphatidylethanolamine), following the manufacturer's protocol. After 24 hours of incubation, expression levels were measured using the Promega Luciferase Assay System (E1501). The expression values are shown as the fold change over a strong constitutive promoter, where greater than 10% expression is set as a threshold for positiveAttorney Docket No. 53531-724602signal. The results demonstrate that SRS-G and SRS-B are active in both lung and liver cancer cell lines, whereas SRS-H, a liver-specific promoter, is active only in liver cancer cell lines.

[0129] FIG. 77 shows a graph of gene expression activated by SRSs in different tissues, illustrating the in vivo performance of several SRSs when administered via intravenous (i.v.) bolus to tumorbearing mice. Quantification of firefly bioluminescence of tissues ex vivo was taken 24 hours after compound dosing normalized to the average bioluminescence imaging (BLI) of PBS dosed animals (n = 3, dotted line set at 1). Plotted by dosing group with each tissue in column. Each point represents a tissue from a unique animal. Circles: CAG constitutive promoter; squares: SRS-F; triangles: SRS-I; diamonds: SRS-E; stars: SRS-J. Error bars represent standard error of the mean (SEM). Tables on the bottom show calculated signal to noise ratios (SNR) for a given promoter over potential background noise tissues (liver, spleen) demonstrating improved SNR and selectivity for synthetic promoters relative to constitutively active CAG promoter.

[0130] FIG. 78 shows a graph of reporter gene expression under different SRSs compared to a constitutive promoter. A FLUC reporter readout was used to assess specificity of SRSs comprising combinations of different promoters and SREs in lung cancer (H1299) and two different normal lung cell lines (Lung Normal 1 and Lung Normal 2). Reporter expression under SRS-K (using the nonspecific promoter TATA-TSS) was high in both lung cancer and normal cell lines. Reporter expression under SRS-L and SRS-M was lower in all cell lines compared to that under SRS-K, especially in normal cell lines. Specifically, reporter gene expression under SRS-L was reduced 2X in cancer cell line and 10-2 OX in normal cell lines compared to reporter gene expression under SRS-K, which comprises non-specific promoter TATA-TSS, indicating that core promoters provide selectivity and specificity for cancer cells compared to normal cells.

[0131] FIGs. 79A-79B show expression levels after treatment in B 16.F10-tumor bearing mice. 79A shows firefly luciferase mRNA expression after SEQ ID NO: 704 treatment, and IL-2 mRNA expression after SEQ ID NO: 703 treatment, compared to phosphate buffered saline (PBS) treatment, across treatment groups at days 2, 6, and 11, in tumor tissue of B16.F10-tumor bearing mice. mRNA expression in tumor tissues was measured as mRNA copies per 20 ng of total mRNA. FIG. 79B shows IL-2 mRNA expression after SEQ ID NO: 703 treatment compared to phosphate buffered saline (PBS) treatment, at days 2, 6, and 11 of the dosage schedule, as disclosed herein, in tumor tissue of B16.F10-tumor bearing mice.

[0132] FIGs. 80A-80B show certain IL-2 concentrations (pg / mL) in serum samples from B16.F10-tumor bearing mice. FIG. 80A shows a quantification of IL-2 concentration (pg / mL) in serum samples from B16.F10-tumor bearing mice treated with PBS, SEQ ID NO: 703, or SEQ ID NO: 704, across treatment groups, as described herein, at days 1, 2, 3, 5, 7, 9, and 11. FIG. 80B shows IL-2 concentration (pg / mL) across time post first dose in serum samples from B16.F10-tumor bearing mice treated with a single dose of SEQ ID NO: 703 or SEQ ID NO: 704.Attomey Docket No. 53531-724602

[0133] FIGs. 81A-81P depict various FACS analysis. FIG. 81A shows FACS analysis of CD45-, CD45+CD3-, and CD45+CD3+ cells as a percentage of total live cells from spleens of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703, across dosing groups, as described herein. FIG. 81B shows FACS analysis of CD45-, CD45+CD3-, and CD45+CD3+ cells as a percentage of total live cells from tumors of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81C shows FACS analysis of T cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and non-NK cells as a percentage of total leukocytes from the spleens of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81D shows FACS analysis of T cells, CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and non-NK cells as a percentage of total leukocytes from the tumors of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81E shows FACS analysis of CD4+ T Cells, CD8+ T Cells, and Tregs as a percentage of total T cells from the spleens of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81F shows FACS analysis of CD4+ T Cells, CD8+ T Cells, and Tregs as a percentage of total T cells from the tumors of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 low, across dosing groups, as described herein. FIG. 81G shows FACS analysis of CD44-CD62L+ cells, CD44+CD62L+ cells, CD44+CD62L- cells, and CD44-CD62L- cells as a percentage of total CD8+ T cells from the spleens of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81H shows FACS analysis of CD44-CD62L+ cells, CD44+CD62L+ cells, CD44+CD62L- cells, and CD44-CD62L- cells as a percentage of total CD8+ T cells from the tumors of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 811 shows FACS analysis of CD4+ T cells, CD8+ T cells, and CD4-CD8- T cells as a percentage of total CD25+ cells from the spleens of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein.FIG. 81 J shows FACS analysis of CD4+ T cells, CD8+ T cells, and CD4-CD8- T cells as a percentage of total CD25+ cells from the spleens of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81K shows FACS analysis of CD45- cells, CD4+ T cells, CD8+ T cells, NK1.1+ cells, and NK1.1- cells as a percentage of total Ki67+ cells from the spleens of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81L shows FACS analysis of CD45- cells, CD4+ T cells, CD8+ T cells, NK1.1+ cells, and NK1.1- cells as a percentage of total Ki67+ cells from the tumors of MB49 tumor bearing mice treated with PBS, SEQ ID NO: 704, and SEQ ID NO: 703 across dosing groups, as described herein. FIG. 81M shows the ratio of CD8+ cells to Tregs from the spleens of MB49 tumor bearing mice, by FACS analysis, across treatment groupsAttorney Docket No. 53531-724602(Groups 1-9), as disclosed herein. FIG. 81N shows the ratio of CD8+ T cells to Tregs from the tumors of MB49 tumor bearing mice, by FACS analysis, across treatment groups (Groups 1-9), as disclosed herein. FIG. 810 shows a P value matrix from unpaired T tests comparing the ratio of CD8+ T cells to Tregs across treatment groups. FIG. 81P shows the ratio of CD8+ T cells (effector T cells) to Tregs for the PBS-dosed group (Group 1), the SEQ ID NO: 704-dosed group (Group 2), and the SEQ ID NO: 703 mid-dosed group (Group 4).

[0134] FIGs. 82A-82B show a schematic illustration of IL- 12 DNA copies per cell (FIG. 82A) and RNA copies (FIG. 82B) across tissues in MB49 tumor-bearing mice administered the DNA nanoplasmid comprising a TCF7-Split-coreKIF20A promoter expressing IL- 12, corresponding to the data provided in Table 10.

[0135] FIGs. 83A-83C show total tumor volumes over time in MB49 tumor-bearing mice treated with PBS, LNPs comprising SEQ ID NO: 712, or LNPs comprising SEQ ID NO: 713 at a dosing concentration of 0.175 mg / kg (FIG. 83A), PBS, LNPs comprising SEQ ID NO: 712, or LNPs comprising SEQ ID NO: 713 at a dosing concentration of 0.35 mg / kg (FIG. 83B), and PBS, LNPs comprising SEQ ID NO: 712, or LNPs comprising SEQ ID NO: 713 at a dosing concentration of 0.70 mg / kg (FIG. 83C), corresponding to the data provided for individual mice in Table 11.

[0136] FIGs. 84A-84B show IL-12 protein concentration in tumor tissue (FIG. 84A) and in serum (FIG. 84B) across treatment conditions.

[0137] FIGs. 85A-85C provide the percentage of CD8 T cells expressing Granzyme B in the spleen or tumors of B16.F10 tumor bearing mice (FIG. 85A), percentage of proliferating Ki-67 expressing CD8 T cells in the spleen or tumors of B16.F10 tumor bearing mice (FIG. 85B), percentage of proliferating Ki-67 expressing CD8 T cells in the spleen or tumors of B16.F10 tumor bearing mice (FIG. 85C), expressed as a percentage of total CD8 or CD4 T cells in those tissues from B16.F10 tumor-bearing mice intravenously administered PBS, LNPs carrying CAP-STOP-mIL-12 (control DNA construct), or LNPs carrying CAP-mIL-12 (test article).

[0138] FIGs. 86A-86F provide MHC-I or MHC-II expression intensity (mean fluorescent intensity; MFI) in splenocytes of B16.F10-tumor bearing mice at Day 6 or Day 13 post-intravenous administration of PBS, LNPs carrying CAP-STOP-mIL-12 (control DNA construct), or LNPs carrying CAP-mIL-12 (test article) for Ly6C++ inflammatory macrophages (FIGs. 86A-86B), Ly6C++ monocytes (FIG. 86C), Ly6C+ tissue resident macrophages (FIGs. 86D-86E), and dendritic cells (FIG. 86F).

[0139] FIGs. 87A-87B show tumor volume (mm3) in wild-type (WT) and STING7MB49-tumor bearing mice across days post-dosing with PBS or a control construct (cancer-activated-promoter (CAP)-STOP-mIL-12).Attomey Docket No. 53531-724602

[0140] FIG. 87C shows the percentage of CD8+ splenocytes expressing Ki-67 out of the total CD8+ splenocytes at Day 5, Day 7, and Day 9 post-dosing with PBS, a control DNA construct (CAP-STOP-mIL-12), or a test article (CAP -IL-12), as described herein.

[0141] FIGs. 88A-88B show tumor volume (mm3) across days post first dose in the injected tumor (FIG. 88A) and in the non-injected tumor (FIG. 88B), following dosage with PBS, or LNPs carrying a control construct or a test article.

[0142] FIG. 89 shows total tumor volume over time in MB49 tumor-bearing mice treated with PBS, an anti-PDl antibody (aPDl), or with a combination treatment with LNP carrying SEQ ID NO: 712 and aPDl or LNP carrying SEQ ID NO: 713 and aPDl, as described herein.

[0143] FIG. 90 shows total tumor volume (mm3) over time in Bl 6F 10 tumor-bearing mice after treatment with PBS, or with LNPs carrying SEQ ID NO: 712, SEQ ID NO: 713, or SEQ ID NO: 727, as described herein.

[0144] FIG. 91 shows lung weight of lungs harvested from B16F10 tumor bearing mice seventeen days post first dose with PBS or with LNPs carrying SEQ ID NO: 712 or SEQ ID NO: 727.

[0145] FIG. 92 shows total tumor volume (mm3) over time across for MB49-tumor bearing mice treated with PBS, a combination of LNPs carrying SEQ ID NO: 728 and LNPs carrying SEQ ID NO: 729, a combination of LNPs carrying SEQ ID NO: 728 and SEQ ID NO: 713, or a combination of LNPs carrying SEQ ID NO: 729 and LNPs carrying SEQ ID NO: 713, as described herein.

[0146] FIGs. 93A-93C show cytokine serum levels of MB49-tumor bearing mice nine days post IV administration of PBS, LNPs carrying SEQ ID NO: 728, or LNPs carrying SEQ ID NO: 713, measuring interferon gamma (IFNg) (FIG. 93A), IL-6 (FIG. 93B), and tumor necrosis factor alpha (TNFa) (FIG. 93C).

[0147] FIGs. 94A-94B show that treatment of MB49-tumor bearing mice with an IV LNP carrying a DNA nanoplasmid encoding a cancer-activated promoter controlling expression of IL- 12 (SEQ ID NO: 713) increases level of tumor CD8+ proliferating cells in tumors (FIG. 94A) and increases the percentage of tumor Granzyme B+ CD8 T cells (FIG. 94B) as compared to mice dosed with PBS or a control nanoplasmid (SEQ ID NO: 728).

[0148] FIG. 95 shows levels of inflammatory Ly6C+ macrophages in the tumors of mice with MB49 subcutaneous tumors nice days post IV administration with LNPs carrying DNA encoding a cancer-activated promoter to express IL-12 (SEQ ID NO: 713) as compared to IV administration of LNPs carrying DNA encoding SEQ ID NO: 728, or PBS.

[0149] FIGs. 96A-96D show concentrations of IL-2 or IL-12 from supernatants of cancer cells including non-small cell lung cancer NCI-H1299 cells (FIGs. 96A-96B), non-small cell lung cancer NCI-H358 cells, hepatocellular carcinoma Huh7 cells (FIG. 96C), or mouse melanoma B16F10 cells and mouse MB49 bladder cancer cells (FIG. 96D), transfected with CAP nanoplasmids encoding IL-12 or IL-2 in multiple configurations and fusion orientations including SEQ ID NO: 733 (encodingAttorney Docket No. 53531-724602LAIR1 collagen binding domain fused by a GS linker to mouse IL-2), SEQ ID NO: 736 (encoding WT mIL2), SEQ ID NO: 739 (encoding WT mIL12), SEQ ID NO: 742 (encoding mouse IL-12 fused by a GS linker to a LAIR1 collagen binding domain to enhance tumor retention and target IL- 12 to the collagen rich tumor microenvironment), SEQ ID NO: 745 (encoding a fusion protein consisting of WT mouse IL-12 and mouse IL-2, connected by a GS Linker), SEQ ID NO: 748 (encoding mIL12 fused by a GS linker to LAIR1 and then fused by a GS linker to mIL2), SEQ ID NO: 751 (encoding mIL12 fused by a GS linker to an RGD domain to enhance tumor retention by binding to Integrins fused by a GS linker to mIL2), SEQ ID NO: 754 (encoding mIL12 connected by a GS linker to Furin cleavage site and T2A self cleaving sequence then connected to amIL2 sequence), SEQ ID NO: 757 (encoding-mIL12 fused by a GS linker to an anti-PDLl antibody binding domain (scFv)), or no transfection (No Tnfx).

[0150] FIGs. 97A-97B show IL-2 or IL- 12 receptor functional data in reporter cells from supernatants of cancer cells transfected with cancer activated promoter constructs including SEQ ID NO: 733, SEQ ID NO: 736, SEQ ID NO: 739, SEQ ID NO: 742, SEQ ID NO: 745, SEQ ID NO: 748, SEQ ID NO: 751, SEQ ID NO: 754, SEQ ID NO: 757, SEQ ID NO: 779 (encoding an RGD integrin binding domain fused to mouse IL-2), SEQ ID NO: 781 (encoding an RGD integrin binding domain fused to mouse IL- 12, and NP1065 encoding mlLl connected by a GS linker to Furin cleavage site and T2A self-cleaving sequence then connected to a mIL12 sequence) or no transfection (No Transfx).

[0151] FIGs. 98A-98B show INFg production (pg / mL) from resting splenocytes (FIG. 98A) and in activated splenocytes (FIG. 98B), after transfection with SEQ ID NOs: 733, 736, 739, 742, 745, 748, 751, 754, 779, 782, 785, or a control.

[0152] FIG. 99 shows RLU of IL- 12 from the supernatant of Hl 299 cells transfected with SEQ ID NOs: 727, 757, 791, 788, or 794.

[0153] FIG. 100 shows RLU across concentrations of SEQ ID NOs: 788, 794, or 911, from the PD1 / PDL1 blockade reporter assay described herein.

[0154] FIG. 101A shows total tumor volume (mm3) at Day 10 in B16F10-tumor bearing mice after treatment with SEQ ID NOs: 733, 736, 739, 742, 745, 748, 751, 754, 757, or PBS.

[0155] FIGs. 101B-101C show total pharmacodynamic effects in tumor lymphocytes including CD44+CD62L- effector memory CD8 T cells (FIG. 101B) and granzyme B+ CD8 T cells (FIG. 101C), harvested from B16F10-tumor bearing mice 6 days after dosing with PBS, or SEQ ID NOs: 757, 754, 751, 748, 745, 742, 739, 736, or 733.

[0156] FIG. 102 shows human IL-2 levels (pg / mL) in the supernatant of Hl 299 cells transfected with SEQ ID NOs: 797, 806, 815, 818, 822, 825, 828, 849, 852, 855, 858, or no transfection (PBS). These data exhibit that multiple cancer activated promoter-cytokine constructs were expressed byAttorney Docket No. 53531-724602cancer cells and demonstrated a range of expression efficiencies across the various fusion proteins tested.

[0157] FIGs. 103A-103B show IL-2 (FIG. 103A) and IL-12 (FIG. 103B) expression levels (RLU) in the supernatant of IL-2 or IL-12 reporter cells transfected with SEQ ID NOs: 797, 806, 815, 818, 822, 825, 828, 849, 852, 855, or 858 for IL-2 expression analysis (FIG. 103A), or SEQ ID NOs: 800, 809, 812, 815, 818, 822, 825, 828, 849, 852, 855, or 858 or IL-12 expression analysis (FIG. 103B).

[0158] FIG. 103C shows IL-12 expression levels (RLU) in the supernatant of IL-12 reporter cells transfected with SEQ ID NOs 911, 788, or 794.

[0159] FIGs. 104A-104B show INFg concentrations (pg / mL) from the supernatants of human PBMCs (HD30106) (FIG. 104A) and human PBMCs (HD30198) (FIG. 104B) after transfection with SEQ ID NOs: 797, 800, 806, 809, 812, 815, 818, 822, 825, 828, or 849.

[0160] FIGs. 105A-105B show INFg concentrations (pg / mL) from the supernatants of activated human PBMCs (HD30106) (FIG. 105A) and activated human PBMCs (HD30198) (FIG. 105B) after transfection with SEQ ID NOs: 797, 800, 806, 809, 812, 815, 818, 822, 825, 828, or 849.

[0161] FIG. 106 shows binding, represented as absorbance optical density (OD) at 450 nm, of expression products of SEQ ID NOs: 797, 800, 809, 815, 818, 822, and 858 to collagen coated plates.

[0162] FIG. 107 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0163] FIG. 108 shows IL-12p70 expression levels (pg / mL) in the serum of healthy, tumor-free mice on Day 6 after treatment with SEQ ID NO: 728, 713, or 760, carried by LNPs described herein.

[0164] FIGs. 109A-109B show IL-12 concentrations (pg / mL) in bladder and tumor tissue (FIG. 109 A) or in urine (FIG. 109B) from MB49-FLuc-tumor bearing mice treated with LNPs carrying SEQ ID NO: 713, or PBS.

[0165] FIGs. 110A-110B show the concentration of IL-7 (FIG. 110A) or IL-21 (FIG. HOB) protein levels in the supernatant of H1299 cells transfected with SEQ ID NOs: 867, 873, or 876 (FIG. 110A) or with SEQ ID NOs: 870, 873, 876, 891, or 894 (FIG. HOB).

[0166] FIGs. 111A-111B show the fold change in cytokine expression over background for IL-7 reporter cells transfected with SEQ ID NOs: 867 or 873 (FIG. 111A) and for IL-21 reporter cells transfected with SEQ ID NOs: 761 or 873 (FIG. 111B).

[0167] FIG. 112 shows INFg concentration (pg / mL) in resting splenocytes after transfection with SEQ ID NOs: 713, 925, 761, 764, or 918.

[0168] FIGs. 113A-113B show IL-7 (FIG. 113A) or IL-21 (FIG. 113B) protein levels in the supernatant of H1299 cells transfected with SEQ ID NOs: 879, 885, 888, or 897.

[0169] FIGs. 114A-114B show IL-7 (FIG. 114A) or IL-21 (FIG. 114B) expression fold change over background in IL-7 reporter cells transfected with SEQ ID NOs: 879, 885, 888, or 891 (FIG. 114A) or IL-21 reporter cells transfected with SEQ ID NOs: 882, 885, or 888 (FIG. 114B).Attorney Docket No. 53531-724602

[0170] FIGs. 115A-115B show mouse IL-2 (FIG. 115A) or mouse IL- 12 (FIG. 115B) protein expression levels from H1299 cells transfected with SEQ ID NOs: 729, 733, 912, 773, 906 841, 846, or no transfection (FIG. 115A) or transfected with SEQ ID NOs: 713, 791, 912, 903, 773, 906, 861, 854, or no transfection.

[0171] FIGs. 116A-116B show IL-2 (FIG. 116A) or IL-12 (FIG. 116B) expression (RLUs) from IL-2 reporter cells transfected with SEQ ID NOs: 729, 733, 745, 912, 903, 770, 773, 906, 861, or 864 (FIG. 116A) or from IL-12 reporter cells transfected with SEQ ID NOs: 713, 791, 912, 903, 767, 770, 773, 906, 861, or 864 (FIG. 116B).

[0172] FIGs. 117A-117B show tumor volume (mm3) in MB49-tumor bearing mice after treatment with LNPs carrying SEQ ID NOs: 728, 761, 764, the combination of 728 and 713, the combination of 761 and 713, the combination of 764 and 713, 767, or PBS control (FIG. 117A) or with LNPs carrying the combination of SEQ ID NOs: 764 and 776, the combination of SEQ ID NOs: 728 and 774, the combination of SEQ ID NOs: 728 and 770, or the combination of SEQ ID NOs: 728 and 767 (FIG. 117B)

[0173] FIGs. 118A-118B show the percentage of Ki67+ cells out of total parent lineage cells for NK cells, CD4+ T cells, and CD8+ T cells in spleen after treatment with PBS, SEQ ID NO: 728, the combination of SEQ ID NO: 728 and SEQ ID NO: 713, or the combination of SEQ ID NO: 728 and SEQ ID NO: 767 (FIG. 118A) or in tumor after treatment with PBS, SEQ ID NO: 728, SEQ ID NO: 761, SEQ ID NO: 764, orthe combination of SEQ ID NO: 728 and SEQ ID NO: 713, the combination of SEQ ID NO: 761 and SEQ ID NO: 713, orthe combination of SEQ ID NO: 764 and SEQ ID NO: 713 (FIG. 118B).

[0174] FIGs. 119A-119B show median fluorescence intensity for MHC-I or MHC-II in Ly6C+ macrophages from healthy spleen (FIG. 119A) or MB49 tumors (FIG. 119B) harvested from mice administered PBS, or LNPs carrying SEQ ID NO: 728, the combination of SEQ ID NOs: 728 and 713, orthe combination of SEQ ID NOs: 728 and 767.

[0175] FIGs. 120A-120B show the proportion of CD44+ / CD62L- CD4+ T cells or CD8+ T cells of total parent lineage cells (FIG. 120A) or the proportion of CD44+ / CD62L+ CD4 T cells or CD8 T cells of total parent lineage cells (FIG. 120B) collected from the MB49 tumors in mice after administration with PBS, or LNPs carrying SEQ ID NO: 728, SEQ ID NO: 761, SEQ ID NO: 764, or the combination of SEQ ID NOs: 728 and 713, the combination of SEQ ID NOs: 761 and 713, or the combination of SEQ ID NOs: 764 and 713.

[0176] FIG. 121 A shows representative brightfield images of lungs from MB49-GFP / Luc-tumor bearing mice after administration with PBS, or LNPs (FRM177 or FRM638) carrying SEQ ID NO: 712 or SEQ ID NO: 713.Attomey Docket No. 53531-724602

[0177] FIG. 121B shows lung weight (mg) from MB49-GFP / Luc -tumor bearing mice after administration with PBS, or LNPs (FRM177 or FRM638) carrying SEQ ID NO: 712 or SEQ ID NO: 713.

[0178] FIG. 122 shows tumor volume (mm3) in MB49-tumor bearing mice across days post treatment with PBS, or LNPs (FRM177 or FRM638) carrying SEQ ID NO: 728 or SEQ ID NO: 713.

[0179] FIG. 123A shows representative brightfield images of lungs from MB49-GFP / Luc-tumor bearing mice after administration with PBS, or LNPs (FRM177 or FRM214) carrying SEQ ID NO: 712 or SEQ ID NO: 713.

[0180] FIG. 123B shows lung weight (mg) from MB49-GFP / Luc -tumor bearing mice after administration with PBS, or LNPs (FRM177 or FRM214) carrying SEQ ID NO: 712 or SEQ ID NO: 713.

[0181] FIGs. 124A-124C show serum levels (pg / mL) for IL-12p70 (FIG. 124A), INGg (FIG. 124B), IL-6 (FIG. 124C), and TNFa (FIG. 124D) from healthy mice administered PBS or LNPs carrying SEQ ID NO: 728, SEQ ID NO: 760, or SEQ ID NO: 713 (dashed line indicates lower level of quantification).

[0182] FIG. 125 shows IFN gamma (IFNg) concentrations in the supernatant of resting splenocytes which were treated with supernatants from Expi293™ cells transfected with CAP constructs SEQ ID NOs: 915, 918, 922, 925, 761, or 764.

[0183] FIGs. 126A-126D show the proportion of Granzyme B+ CD8 T cells (FIG. 126A), CD8 T cells (FIG. 126B), CD4 T cells (FIG. 126C), and CD4 T effector memory cells (FIG. 126D) of total parent lineage cells (%) from resting splenocytes after treatment with the supernatant of cancer cells transfected with SEQ ID NOs: 928, 931, 934, or 713.

[0184] FIGs. 127A-127D show the proportion of total T cells (FIG. 127A), CD8 T cells (FIG. 127B), CD8 T effector memory cells (FIG. 127C), and GranzymeB+ CD8 T cells (FIG. 127D) of total parent lineage cells (%) from resting splenocytes after treatment with the supernatant of cancer cells transfected with SEQ ID NOs: SEQ ID NOs: 939, 761, or 764.

[0185] FIGs. 128A-128L provide results of flow cytometry showing proportions of CD4+ / CD25+ cells (FIG. 128A), CD4+ Central Memory cells (FIG. 128B), CD8+ Effector Memory cells (FIG.128C), CD8+ Central Memory cells (FIG. 128D), CD4+ / CD25+ cells (FIG. 128E), CD4+ Central Memory cells (FIG. 128F), CD8+ Effector Memory cells (FIG. 128G), CD8+ Central Memory cells (FIG. 128H), CD8+ / CD25+ cells (FIG. 1281), CD8+ Granzyme B+ cells (FIG. 128J), CD25+ NK cells (FIG. 128K), and Granzyme B+ NK cells (FIG. 128L) as a percentage of total parent lineage cells from resting splenocytes treated with the supernatants of cancer cells transfected with SEQ ID NOs: 942, 945 or 948.Attorney Docket No. 53531-724602

[0186] FIGs. 129A-129B show tumor volume (mm3) in MB49-tumor bearing mice administered LNPs carrying SEQ ID NO: 713 at a dose of 7 ig or 3.5 ig of DNA, using LNP formulation FRM638 (FIG. 129A) or FRM640 (FIG. 129B).

[0187] FIG. 130 shows BLI (photons / s) of Hep55.1C-Luc tumors in mice across time post first dose of PBS or LNP FRM640 carrying SEQ ID NO: 713.

[0188] FIG. 131 shows tumor volume (mm3) of MC38-tumor bearing mice across days post first dose of PBS or SEQ ID NO: 713 carried by LNPs comprising formulations FRM177, FRM640, or FRM638, as described herein.

[0189] FIGs. 132A-132H provide schematic illustrations of various SRS nucleic acid sequences described herein, in some embodiments, including SEQ ID NO: 424 (FIG. 132A), SEQ ID NO: 426 (FIG. 132B), SEQ ID NO: 431 (FIG. 132C), SEQ ID NO: 456 (FIG. 132D), SEQ ID NO: 556 (FIG.132E), SEQ ID NO: 1005 (FIG. 132F), SEQ ID NO: 1009 (FIG. 132G), and SEQ ID NO: 1007 (FIG. 132H), illustrating promoter, SRE, and enhancer elements.

[0190] FIG. 133 shows the expression of the reporter gene (firefly luciferase) as percentage relative to the level generated by the strong constitutive promoter CAG that contains viral enhancer elements, across LU AD, LUSC, and large cell carcinoma patient cell samples (each bar represents a different cell line), under the transcriptional control of an endogenous BIRC promoter, or SRS011 (SEQ ID NO: 407).

[0191] FIG. 134 shows the fold change (log2) of gene expression between normal human lung and human lung tumor samples.

[0192] FIG. 135 shows expression (log2) of BIRC5, KIF20A, and FAM11 IB in benign lung disease tissue, healthy lung, lung adenocarcinoma, and lung squamous cell carcinoma.

[0193] FIG. 136A shows fold change, relative to a constitutive promoter, of luciferase expression using an endogenous (represented as E) or core promoter (represented as C) construct of BIRC5 promoter (BIRC), FAM11 IB (FAM) promoter, or KIF20A (KIF) promoter constructs in H1299 or LXFL430 cells.

[0194] FIG. 136B shows fold change, relative to constitutive promoters, of luciferase expression using a core (represented as C) or core promoter with a HIGH enhancer element (represented as H) construct of BIRC5 promoter (BIRC), FAM11 IB (FAM) promoter, or KIF20A (KIF) promoter in Hl 299, LXFL430 cells, or in normal lung fibroblasts.

[0195] FIG. 137 shows the variance (%) for Factors 1-13 in RNA, protein, and ptm.

[0196] FIG. 138 shows the factor value for Factors 1-5 in normal adjacent tissue (NAT) and tumor tissue.

[0197] FIG. 139 shows a heatmap of gene expression or protein expression in normal adjacent tissue (NAT) or tumor tissue, across cell types.Attorney Docket No. 53531-724602

[0198] FIGs. 140A-140B show log2 expression of FOSL RNA (FIG. 140A) and protein abundance of FOSL protein (FIG. 140B) in NAT and tumor samples.

[0199] FIG. 141 provides a schematic illustration of the consensus pathway landscape of LU AD TFs identified from MOFA as described herein.

[0200] FIG. 142 shows log2 fold change of luciferase expression from baseline for the tested SREs in A549, LXFA586, H1703, SKMES1, H1299, and LXFL430 cancer cells.

[0201] FIG. 143 shows heatmaps of log2 fold change of luciferase expression for SREs tested (Y -axis) comprising FOSL2, TCF7, TP53, or NFE2L2 binding elements.

[0202] FIG. 144 shows fold change, relative to a constitutive promoter, of FLUC expression from the coreBIRC5 construct, or SRE binding elements comprising FOS, TCF, or TP53, in H1299, LXFL430, or LXFA586 cells.

[0203] FIG. 145 shows log2 fold change of FLUC from tested SREs (Y-axis), with or without CREB3L3, in A549, LXFA586, H703, SKMES1, or LXFL430 cancer cells.

[0204] FIG. 146 shows fold change of reporter expression from SREs comprising TATA, FOS, or FOS / CREB, in H1299, LXFL430, or LXFA586 cancer cells.

[0205] FIG. 147 shows the expression of the engineered constructs in H1299 cancer cells transfected with constructs comprising a FAM11 IB core promoter and KIF20A 5’ UTR (SEQ ID NO: 573; PR166), a FAM11 IB core promoter and AGR25’ UTR (SEQ ID NO: 574; PR165), a KIF20a core promoter and FAM11 IB 5’ UTR (SEQ ID NO: 564; PR175), a KIF20a core promoter and AGR25’ UTR (SEQ ID NO: 563; PR176), a AGR2 core promoter and KIF20A 5’ UTR (SEQ ID NO: 568; PR171), and a AGR2 core promoter and FAM11 IB 5’ UTR (SEQ ID NO: 569; PR170).

[0206] FIG. 148 shows the fold change in expression relative to a constitutive promoter of the candidate promoters tested, as listed in Table 27, after transfection in LXFL430, LXFA586, H1299, fibroblast, or SAEC cells.

[0207] FIG. 149 shows expression strength of various SREs and core promoter combinations described herein, and as listed in Table 28, after transfection in various adenocarcinoma, squamous cell carcinomas, and large cell carcinomas, as compared to a constitutive CMV promoter.

[0208] FIG. 150 shows mean expression in cancer cells as a fold change to a constitutive active promoter, using the constructs listed in Table 28.

[0209] FIG. 151 shows IL-12 concentration (ng / mL) expressed from Expi293™ cells transfected with SEQ ID NOs: 728, 713, 727, 1001-1004, 1006, and 1008.

[0210] FIGs. 152A-152B show IFNg concentrations (pg / mL) expressed by resting human PBMCs (FIG. 152A) and activated human PBMCs (FIG. 152B) treated with the supernatants of cells transfected with SEQ ID NO: 958 or SEQ ID NO: 961, as compared to treatment with human IL-12 or human IL-2 protein.Attorney Docket No. 53531-724602DETAILED DESCRIPTION

[0211] The compositions and methods described herein contemplates a general strategy of identifying important elements of cancer-specific (or cancer-activated) promoters and designing and / or engineering cancer-specific promoters using elements of cancer-specific promoters identified. Cancer-specific promoters or cancer-activated promoters described herein can comprise promoters of genes that are preferentially expressed in cancer cells compared to non-cancer cells or expressed in higher level in cancer cells compared to non-cancer cells. Methods described herein can comprise identifying endogenous cancer-activated promoters by evaluating candidate promoter and / or enhancer sequences using bioinformatic analysis and designing / engineering a minimal cancer-activated promoter sequence (such as a core promoter described herein). For example, a candidate sequence (e.g., low -throughput or high-throughput screening) can be examined using a genome browser. The assessment range (e.g., sequence boundary) can be set based on the predicted transcriptional start site (TSS) of an endogenous promoter. For example, the assessment range can be from about -1000 bp to about +1000 bp relative to the predicted TSS. The assessment range can be adjusted based on chromatin immunoprecipitation (ChIP) data including, but not limited to, ChIP peaks of general transcription factors (TFs), indicators of active promoter regions, and TFs that may indicate cancer specificity by presence in cancer cells and absence in non-cancer cells; and abundance of predicted TF binding sequence (TFBS); and regions of high species conservation. In some embodiments, indicators of active promoter regions can include, but not limited to, RNA Polymerase II, DNAse I, H3K4mel, and H3K4me3. In some embodiments, TFBS abundance can be predicted using methods including, but not limited, to JASPAR or HOMER motif analysis. Methods described herein can also comprise testing highlight regulated TFs using Massively Parallel Reporter Assay (MPRA) to identify optimal sequences, optimal spacing between each sequence, and / or optimal combinations of different enhancer sequences to design synthetic tiled enhancers. Methods described herein can comprise a rationally designed (e.g., low-throughput) screening or a high-throughput screening to identify enhancer elements to increase transcription signal. In some embodiments, a synthetic tiled enhancer can comprise one or more copies of TFBS, or other highly conserved regulatory element repeats with spacing between repeats. One or more synthetic elements described herein can be placed upstream of core promoters. Synthetic elements described herein can also function as a promoter without a promoter or a core promoter.

[0212] A cancer-specific promoter described herein can comprise a non-naturally occurring polynucleotide comprising a core promoter sequence comprising a transcription start site (TSS). In some embodiments, a core promoter can be obtained from a gene and can be operably linked to an open reading frame (ORF). In some embodiments, a gene can comprise a human gene. In some embodiments, a core promoter can comprise a plurality of binding sites for a plurality of transcriptionAttorney Docket No. 53531-724602factors (TFs) that are expressed in higher levels in cancer cells compared to non-cancer cells. In some embodiments, a core promoter can comprise a plurality of binding sites for a plurality of transcription factors (TFs) that are more active in cancer cells compared to non-cancer cells. In some embodiments, a core promoter can comprise a plurality of enhancers obtained from two or more human genes. In one embodiment, each of the plurality of enhancers can comprise a transcription regulatory element with at least 80% sequence homology to the enhancer consensus sequence of the two or more human genes. In another embodiment, each of the plurality of enhancers can comprise a sequence capable of binding a transcription associated protein as assessed by ChlP. In some embodiments, measuring the higher expression of the ORF operably linked to a core promoter comprising a TSS in a cancer or tumor cell as compared to a corresponding healthy cell, as described herein, can be performed using RNA sequencing, multi-omic analysis including, for example, transcriptomic analysis, proteomic analysis, phosphor-proteomic analysis, ATAC-sequencing (ATAC-seq), or any combination thereof.I. COMPOSITIONSA. Nucleic Acid Molecules

[0213] Provided herein are nucleic acid molecules comprising a nucleic acid sequence comprising a core promoter sequence and a transcription start site (TSS). In some embodiments, the nucleic acid molecule comprises a non-naturally occurring nucleic acid sequence (e.g, a recombinant polynucleotide), a synthetic nucleic acid sequence (e.g., a synthetic polynucleotide), or both. In some embodiments a nucleic acid sequence described herein comprises a synthetic response sensor (SRS), wherein the SRS comprises one or more synthetic response elements and one or more core promoters described herein. In some embodiments, a nucleic acid molecule described herein comprises a linear nucleic acid sequence structure, a circular nucleic acid sequence structure, a hairpin nucleic acid sequence structure, a loop nucleic acid sequence structure, or any combination thereof. In some embodiments, a nucleic acid molecule described comprises a double -stranded nucleic acid sequence, a single stranded nucleic acid sequence, or both. In some embodiments, a nucleic acid molecule described herein is a non-naturally occurring polynucleotide. In some embodiments, the non-naturally occurring polynucleotide comprises a recombinant polynucleotide, a synthetic polynucleotide, or both.

[0214] In some aspects, provided herein is a non-naturally occurring polynucleotide comprising: (a) a core promoter operably linked to an open reading frame (ORF) comprising a gene encoding a peptide therapeutic agent, wherein said core promoter induces expression of said peptide therapeutic agent at a higher level in a cancer cell compared to a non-cancer cell, (b) one or more synthetic response elements comprising one or more enhancers and one or more transcription factor binding sites, and (c) a transcription start site (TSS) upstream of said ORF.Attorney Docket No. 53531-724602

[0215] In some embodiments, the disclosure provides for a nucleic acid, such as a non-naturally occurring polynucleotide described herein, comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of the sequences listed in Table 1A, or to reverse complements of any of the sequences listed in Table 1A. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of the sequences listed in Table 1A without spacer nucleotides, or to reverse complements of any of the sequences listed in Table 1A without spacer nucleotides. As described herein, spacer nucleotides can comprise one or more nucleotides which operably link a transcriptional element (e.g., a core promoter, a promoter, a transcription factor, a transcription factor binding site, an enhancer, or an open reading frame) to another transcriptional element of the non-naturally occurring polynucleotides described herein. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 1-343, or to reverse complements of any of SEQ ID NOs: 1-343. In some embodiments, the disclosure provides for a promoter comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 1-343, or to reverse complements of any of SEQ ID NOs: 1-343. In some embodiments, the nucleic acid can be a doublestranded nucleic acid. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, atAttomey Docket No. 53531-724602least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 1-343 without spacer nucleotides, or to reverse complements of any of SEQ ID NOs: 1-343 without spacer nucleotides.

[0216] In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of the sequences listed in Table IB, or to reverse complements of any of the sequences listed in Table IB. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of the sequences listed in Table IB without spacer nucleotides, or to reverse complements of any of the sequences listed in Table IB without spacer nucleotides. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 377-397, or 913, or to reverse complements of any of SEQ ID NOs: 377-397, or 913. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 377-397, or 913 without spacer nucleotides, or to reverse complements of any of SEQ ID NOs: 377-397, or 913 without spacer nucleotides. In some embodiments, the disclosure provides for a promoter comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 377-397, or 913, or to reverseAttomey Docket No. 53531-724602complements of any of SEQ ID NOs: 377-397, or 913. In some embodiments, the disclosure provides for an enhancer comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 377-397, or 913, or to reverse complements of any of SEQ ID NOs: 377-397, or 913. In some embodiments, the nucleic acid can be a double -stranded nucleic acid.

[0217] In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of the sequences listed in Table 1C, or to reverse complements of any of the sequences listed in Table 1C. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of the sequences listed in Table 1C without spacer nucleotides, or to reverse complements of any of the sequences listed in Table 1C without spacer nucleotides. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 398-486, 1007, or 1009, or to reverse complements to any of SEQ ID NOs: 398-486, 1007, or 1009. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 398-486, 1007, or 1009 without spacer nucleotides, or to reverse complements to any of SEQ ID NOs: 398-486, 1007, or 1009 without spacer nucleotides. In some embodiments, the disclosure provides forAttomey Docket No. 53531-724602a promoter having a sequence having at least 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of the sequences listed in Table 1C, or to reverse complements of any of the sequences listed in Table 1C. In some embodiments, the disclosure provides for a promoter comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any of SEQ ID NOs: 398-486, 1007, or 1009 and SEQ ID NOs: 556-557, or to reverse complements to any of SEQ ID NOs: 398-486, 1007, or 1009 and SEQ ID NOs: 556-557. In some embodiments, the nucleic acid can be a double -stranded nucleic acid.

[0218] In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table 1J, or to reverse complements of any one of the sequences listed in Table 1J. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table 1 J without spacer nucleotides, or to reverse complements of any one of the sequences listed in Table 1J without spacer nucleotides. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any SEQ ID NOs: 558-587, or 1012-1013, or to any reverse complements of any SEQ ID NOs: 558-587, or 1012-1013. In some embodiments, the disclosure provides for a core promoter comprising a sequence having at least about 80%, at least about 81%, at least about 82%, atAttorney Docket No. 53531-724602least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table 1J, or to reverse complements of any one of the sequences listed in Table 1 J. In some embodiments, the disclosure provides for the core promoter comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any SEQ ID NOs: 558-587, or 1012-1013, or to any reverse complements of any SEQ ID NOs: 558-587, or 1012-1013. In some embodiments, the nucleic acid can be a double -stranded nucleic acid.

[0219] In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 556, listed in Table 1C, or to a reverse complement thereof. In some embodiments, the nucleic acid can be a double-stranded nucleic acid. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 556 without spacer sequences, listed in Table 1C, or to a reverse complement thereof without spacer sequences. In some embodiments, the nucleic acid can be a double -stranded nucleic acid.

[0220] In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 557, listed in Table 1C, or to a reverse complement thereof. In some embodiments, the nucleic acid can be a double-stranded nucleic acid. In some embodiments, the disclosure provides for a nucleic acid comprising a sequence having at leastAttomey Docket No. 53531-724602about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 557 without spacer sequences, listed in Table 1C, or to a reverse complement thereof without spacer sequences. In some embodiments, the nucleic acid can be a double -stranded nucleic acid.

[0221] In some embodiments, any of the nucleic acids disclosed herein can have at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 220, at least about 240, at least about 260, at least about 280, at least about 300, at least about 320, at least about 340, at least about 360, at least about 380, at least about 400, at least about 420, at least about 440, at least about 460, at least about 480, at least about 500, at least about 520, at least about 540, at least about 560, at least about 580, at least about 600, at least about 620, at least about 640, at least about 680, at least about 700, at least about 720, at least about 740, at least about 760, at least about 780, at least about 800, at least about 820, at least about 840, at least about 860, at least about 880, at least about 900, at least about 920, at least about 940, at least about 960, at least about 980, at least about 1000, at least about 1020, at least about 1040, at least about 1060, at least about 1080, at least about 1100, at least about 1120, at least about 1140, at least about 1160, at least about 1180, at least about 1200, at least about 1220, at least about 1240, at least about 1260, at least about 1280, at least about 1300, at least about 1320, at least about 1340, at least about 1360, at least about 1380, at least about 1400, at least about 1420, at least about 1440, at least about 1460, at least about 1480, at least about 1500, at least about 1520, at least about 1540, at least about 1560, at least about 1580, at least about 1600, at least about 1620, at least about 1640, at least about 1660, at least about 1680, at least about 1700, at least about 1720, at least about 1740, at least about 1760, at least about 1780, at least about 1800, at least about 1820, at least about 1840, at least about 1860, at least about 1880, at least about 2000, at least about 2020, at least about 2040, at least about 2060, at least about 2080, at least about 2100, at least about 2120, at least about 2140, at least about 2160, at least about 2180, at least about 2200, at least about 2220, at least about 2240, at least about 2260, at least about 2280, at least about 2300, at least about 2320, at least about 2340, at least about 2360, at least about 2380, at least about 2400, at least about 2420, at least about 2440, at least about 2460, at least about 2480, at least about 2500, at least about 2520, at least about 2540, at least about 2560, at least about 2580, at least about 2600, at least about 2620, at least about 2640, at least about 2660, at least about 2680, at least about 2700, at least about 2720, at least about 2740, at least about 2760, at least about 2780, at least about 2800, at least about 2820, at least about 2840, at least about 2860, at least about 2880, at least about 2900, at least about 2920, at least about 2940, at least about 2960, at least about 2980, at least about 3000, at leastAttorney Docket No. 53531-724602about 3020, at least about 3040, at least about 3060, at least about 3080, at least about 3100, at least about 3120, at least about 3140, at least about 3160, at least about 3180, at least about 3200, at least about 3220, or at least about 3240 consecutive nucleotides of any of the nucleic acid sequences disclosed herein, or of any reverse complements of any of the nucleic acid sequences disclosed herein.

[0222] In some embodiments, a non-naturally occurring polynucleotide described herein can comprise a therapeutic nucleic acid molecule. In some embodiments, the therapeutic nucleic acid molecule comprises a short hairpin RNA (shRNA), a micro RNA (miRNA), an antisense or silencing RNA (e.g., siRNA), or any combination thereof.

[0223] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods, and materials are described below.B. Synthetic Promoters and Synthetic Promoter Design

[0224] Provided herein are synthetic promoters that can be activated in target cells with high sensitivity and specificity. These promoters can be modular and engineerable. In some embodiments, synthetic promoters described herein can be designed to drive specificity and sensitivity. For example, synthetic promoters can be designed to specifically respond to dysregulated pathways in cancer. In one embodiment, synthetic promoters described herein can comprise an endogenous promoter of a gene that is expressed specifically or preferentially in cancer cells compared to non-cancer cells. In another embodiment, synthetic promoters described herein can comprise a core promoter. A core promoter described herein can comprise a minimal promoter sequence of an endogenous promoter of a gene expressed specifically or preferentially in cancer cells compared to non-cancer cells. A minimal promoter can refer to a short DNA sequence that can allow for the formation of a transcription initiation complex or a DNA sequence comprising a minimal number of nucleotides sufficient to allow for the formation of a transcription initiation complex. In some embodiments, synthetic promoters described herein can comprise a structure comprising three major components (1) a cancer-specific promoter or core promoter, (2) cancer-activated response elements (e.g., binding sites of one or more transcription factors specific for cancer cells), and optionally (3) an enhancer to boost signal strength (e.g., see FIG. 1 or FIG. 72). In some embodiments, synthetic promoters described herein can comprise only (1) a cancer-specific promoter or core promoter. In some embodiments, synthetic promoters described herein can comprise only (1) a cancer-specific promoter or core promoter and (3) an enhancer to boost signal strength. In some embodiments, an enhancer or a transcription binding site can be referred to as a Synthetic Response Element (SRE). In some embodiments, a synthetic promoter comprising a promoter or core promoter and one or more SREs can be referred to as a Synthetic Response Sensor (SRS). In some embodiments, cancer-activated response elements can be designed and constructed to respond to specific dysregulated transcriptionAttomey Docket No. 53531-724602factors. In some embodiments, cancer-activated response elements described herein can demonstrate predictable activity based on transcriptomic and proteomic data when applied in new cancer models.

[0225] In some embodiments, bioinformatics can be used to identify endogenous cancer-activated core promoter sequences. In some embodiments, multi-omic approaches can be used to identify transcription factors (TFs) and their binding sites that are master-regulated. In some embodiments, such TF binding sites can be tiled and tested using high-throughput sequencing (HTS) to optimize promoter sequences, spacing, and combinations thereof. In some embodiments, one or more rationally designed enhancer elements that increase transcription and boost reporter signal can be used. An exemplary workflow and synthetic promoter are described in FIGs. 10-13. In some embodiments, transcriptional elements comprising a promoter, a core promoter, a transcription factor, a transcription factor binding site, an enhancer, or a transcriptional start site, as described herein, can be obtained from a corresponding endogenous transcriptional element with aid from an artificial intelligence (Al) or machine learning (ML) model or system. In some embodiments, the obtaining is based on a prediction of the Al or ML model or system, wherein the prediction is an output of the Al or ML model or system, and wherein the prediction is indicative of any one of the transcriptional elements having a capability of increasing expression of an open reading frame operably linked thereto in a target cell.

[0226] In some embodiments, candidate TF binding site sequences can be identified using Multi-Omics Factor Analysis (MOFA). In some embodiments, candidate TF binding site sequences can be highly dysregulated. In some embodiments, Multi-Omics Factor Analysis (MOFA) can be used to identify TFs specific for a cancer. In some embodiments, a cancer can comprise lung cancer, breast cancer, liver cancer, and / or colorectal cancer. In some embodiments, a lung cancer can comprise nonsmall cell lung cancer (NSCLC).

[0227] In some embodiments, a synthetic promoter can comprise a core promoter sequence. In some embodiments, a core promoter can be identified by analyzing one or more endogenous promoters that can drive cancer specific expression in vitro and / or in vivo, that is the one or more endogenous promoters can preferentially activate gene expression of a gene that is functionally or operatively linked to said one or more promotors in cancer cells (e.g., either in a subject or cancer cell lines) compared to corresponding healthy or normal cells. In some embodiments, one or more endogenous promoters can be analyzed and annotated using UCSC genome browser to build and test core promoters. In some embodiments, core promoters identified can be combined with other elements described herein. In some embodiments, a core promoter sequence can comprise a minimal cancer-activated core promoters. For example, a core promoter sequence can comprise a promoter sequence comprising a minimal number of nucleotides sufficient to drive expression (e.g., recruit transcription initiation complex) of a gene that is functionally or operatively linked to the core promoter in cancer cells. Examples of a minimal cancer-activated cores can include, but are not limited to, coreBIRC5,Attorney Docket No. 53531-724602coreCSTl, coreAGR2, coreFAMl 1 IB, CEACAM5, CEP55, UBE2C, FAM11 IB, KIF20A, F0XA1, MYC, or TP53 (e.g., FIGs. 2-5 and FIG. 11). In some embodiments, a core promoter sequence can provide specificity. In some embodiments, a synthetic promoter can comprise a response element. In some embodiments, a response element can comprise a binding site for a master regulated transcription factor (TF). Examples of a master regulated TF can include, but are not limited to, tiled TFBS for FOS, CREB, MYC, HOXCIO, TCF7, or combinations thereof. In some embodiments, a response element can provide specificity and / or sensitivity. In some embodiments, a synthetic promoter can comprise a signal strength enhancer. In some embodiments, a signal strength enhancer can comprise a synthetic enhancer (also referred herein as a Synthetic Response Element or SRE). Examples of a synthetic enhancer can include, but are not limited to enhancers of SP1, ETS, CEBP, NF-KB, or combinations thereof. In some embodiments, a synthetic enhancer can provide signal strength. Table A shows a table comparing different synthetic promoters. In some embodiments, synthetic promoters (FOS-AGR2, FOS-CST1, and HIGH-FAM11 IB) can drive high expression of the reporter gene and have improved signal -to-noise ratio (SNR) compared to BIRC5 variant promoters. In some embodiments, a non-naturally occurring polynucleotide described herein comprises one or more Internal Ribosome Entry Site (IRES) motifs and one or more ORFs operably linked to a response sensors described herein. In some embodiments, the non-naturally occurring polynucleotide, when expressed in a target cell, generates a fusion protein, as described elsewhere herein, encoding one or more peptides encoded by the one or more ORFs.

[0228] Table A. Exemplary Synthetic PromotersAttorney Docket No. 53531-724602

[0229] In some embodiments, synthetic promoters described herein that can drive expression in a broad range of cancer cells or cancer tissues including, but not limited to, lung cancer cells, can be identified using methods described herein. In one example, promoters identified using methods described herein can include promoters or binding sites / motifs of TCF7, one of TCFs that can be activated by Wnt / B-cat pathway, known for functioning in development pathways. In some embodiments, cancer cell lines based on Wnt / B-cat pathway can be used for further analysis. For example, a principal component analysis (PCA) of PDX database and CCLE focused on the B-cat / Wnt pathway can be used to choose cell lines for further analysis (e.g., 163 genes involved in Wnt / B-cat pathway, 50 CCLE lung cell lines, and 91 PDX lung cell lines). In some embodiments, a PCA including all lung-related PDXs from CRL as well as the CCLE transcriptome database can be used. Examples of cell lines include, but are not limited to, PC2, H520, LK2, or PDX430. In some embodiments, these cell lines can have similar level of expressions of Wnt7B, CCND1, FZD3, AXIN2 or NKD1. In another example, promoters identified using methods described herein can include promoters of TP53, a tumor suppressor that can activate or repress expression depending on location of the binding site. In some embodiments, TP53 binding sequence or motifs can be included in a promoter or a core promoter.

[0230] In some embodiments, synthetic promoters that can integrate multiple signaling can be engineered using methods described herein. For example, binding sequences or motifs of TCF, TP53, FOS, MNX1, HOXCIO, of CREB can be combined with core promoters described herein to engineer synthetic promoters. In some embodiments, synthetic promoters can comprise promoters or binding sequence s / motifs / sites TFs of genes in multiple regulatory pathways. In some embodiments, synthetic promoters comprising two or more endogenous or core promoters can result in gene expression with greater signal and coverage. Details of synthetic promoter design and construction are described inAttorney Docket No. 53531-724602Example 1 and Example 2. FIGs. 10-13 show the workflow of synthetic promoter design and construction, in some embodiments, as described in Examples 1-2.1. Synthetic Response Sensor (SRS or Synthetic Promoter) and Synthetic Response Elements (SREs)

[0231] In some aspects, provided herein is a non-naturally occurring polynucleotide comprising a Synthetic Response Sensor (SRS) that can drive expression of a gene or an ORF operatively linked to the SRS in tissue- or cell-specific manner. In some embodiments, an SRS described herein can drive cancer specific or cancer-activated expression of a gene or an ORF operatively linked to the SRS. For example, an SRS described herein can drive expression of a gene or an ORF operatively linked to the SRS preferentially or specifically in cancer cells or cancer tissues compared to non-cancer cells or non-cancer tissues. In some embodiments, the expression level of a gene or an ORF operatively linked to an SRS is higher in cancer cells or cancer tissues compared to non-cancer cells or non-cancer tissues. In some embodiments, an SRS can comprise a promoter or a core promoter and one or more Synthetic Response Elements (SREs). In some embodiments, the promoter or the core promoter can provide tissue- or cell-specificity for gene expression. In some embodiments, an SRE can provide tissue- or cell-specificity for gene expression and / or enhance the tissue- or cell-specificity of gene expression. In some embodiments, an SRE can comprise a plurality of binding sites for one or more transcription factors or a plurality of enhancers. For example, an SRE can comprise a plurality of binding sites for one or more transcription factors that are activated in cancer cells or cancer pathways or are dysregulated (e.g., expressed in aberrantly higher levels, etc) in cancer cells or cancer pathways. In some embodiments, an SRS can drive expression of an ORF operatively linked to the SRS in cancer cells or cancer tissues but not in normal cells or tissues (including normal tissues or cells adjacent to cancer cells or cancer tissues) and / or benign lesions. For example, FIGs. 132A-132H provide schematic illustrations of various SRS nucleic acid sequences described herein, in some embodiments, including SEQ ID NO: 424 (FIG. 132A), SEQ ID NO: 426 (FIG. 132B), SEQ ID NO: 431 (FIG. 132C), SEQ ID NO: 456 (FIG. 132D), SEQ ID NO: 556 (FIG. 132E), SEQ ID NO: 1005 (FIG. 132F), SEQ ID NO: 1009 (FIG. 132G), and SEQ ID NO: 1007 (FIG. 132H), illustrating promoter, SRE, and enhancer elements.

[0232] In some embodiments, an SRS can comprise a promoter and one or more SREs comprising a plurality of binding sites for one or more transcription factors and a plurality of enhancers. In some embodiments, an SRS can comprise a promoter and one or more SREs comprising a plurality of binding sites for one or more transcription factors. In some embodiments, an SRS can comprise a core promoter and one or more SREs comprising a plurality of binding sites for one or more transcription factors. In some embodiments, an SRS can comprise a promoter and one or more SREs comprising a plurality of enhancers. In some embodiments, an SRS can comprise a core promoter and one or moreAttomey Docket No. 53531-724602SREs comprising a plurality of enhancers. In some embodiments, an SRS can comprise a core promoter and one or more SREs comprising a plurality of binding sites for one or more transcription factors and a plurality of enhancers. An exemplary SRS is shown in FIG. 72. In one embodiment, an SRE can comprise a plurality of binding sites for one or more transcription factors, wherein each of the plurality of transcription binding sites can comprise the same binding site sequences or motifs (FIG. 72, left). In another embodiment, an SRE can comprise a plurality of binding sites for one or more transcription factors, wherein each of the plurality of transcription binding sites can comprise different binding site sequences or motifs. In yet another embodiment, an SRE can comprise a plurality of binding sites for one or more transcription factors, wherein the plurality of transcription binding sites can comprise a mixture of the same binding site sequences and different binding site sequences (FIG. 72, middle). In some embodiments, an SRS comprising an SRE that comprises a mixture of different transcription factor binding sequences or motifs can drive stronger or higher expression of an ORF operatively linked to the SRS in cancer cells or cancer tissues compared to a corresponding SRS comprising an SRE that that comprises a plurality of the same transcription binding sequences or motifs.

[0233] In some embodiments, an SRS can comprise one or more SREs comprising a plurality of binding sites for one or more transcription factors at the 5’ or upstream of a promoter or a core promoter. In some embodiments, an SRS can comprise one or more SREs comprising a plurality of enhancers at the 5 ’ or upstream of a promoter or a core promoter. In some embodiments, an SRS can comprise a plurality of enhancers at the 5’ or upstream of a plurality of binding sites for one or more transcription factors, wherein the plurality of binding sites for one or more transcription factors are at the 5’ or upstream of a promoter or a core promoter. For example, an SRS can comprise (i) a plurality of enhancers, (ii) a plurality of binding sites for one or more transcription factors, and (iii) a promoter or a core promotor in 5’ to 3’ direction. In some embodiments, an SRS can comprise a plurality of enhancers at the 5 ’ or upstream of a promoter or a core promoter and at the 3 ’ or downstream of a plurality of binding sites for one or more transcription factors. For example, an SRS can comprise (i) a plurality of binding sites for one or more transcription factors, (ii) a plurality of enhancers, and (ii) a promoter or a core promoter in 5’ to 3’ direction.

[0234] In some embodiments, an SRS described herein can drive the expression of an ORF operably linked to the SRS in one specific type of cancer cells. In some embodiments, an SRS described herein can drive the expression of an ORF operably linked to the SRS in two or more types of cancer cells.

[0235] In some embodiments, a non-naturally occurring polynucleotide comprising an SRS describe herein can drive the expression of an ORF operably linked to the SRS at a higher level compared to a corresponding non-naturally occurring polynucleotide comprising a constitutive promoter and an ORF operatively linked to the constitutive promoter. For example, a non-naturally occurring polynucleotide comprising an SRS describe herein can drive the expression of an ORF operably linked to the SRS atAttorney Docket No. 53531-724602a level that is at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% higher compared to a corresponding non-naturally occurring polynucleotide comprising a constitutive promoter and an ORF operatively linked to the constitutive promoter. In some embodiments, an ORF can comprise an ORF of a natural gene or a synthetic gene. In some embodiments, a natural gene or a synthetic can comprise a gene encoding a reporter protein, a biomarker protein, or a therapeutic protein.2. Promoters and Core Promoters

[0236] A core promoter described herein can comprise a minimal promoter that can comprise a transcription start site or a transcription start site sequence that is obtained from a promoter of one or more genes expressed in cancer cells or cancer tissues (also referred to as a cancer-responsive gene herein). In some embodiments, a core promoter described herein can comprise a minimal promoter that can comprise a transcription start site or a transcription start site sequence that is obtained from a promoter of one or more genes expressed at a higher level in cancer cells or cancer tissues compared to non-cancer cells or non-cancer tissues. For example, a core promoter described herein can comprise a minimal promoter that can comprise a transcription start site or a transcription start site sequence that is obtained from a promoter of one or more genes expressed at a level that is at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% higher in cancer cells or cancer tissues compared to non-cancer cells or non-cancer tissues.

[0237] In some embodiments, a core promoter can further comprise one or more promoter elements that are obtained from a promoter of one or more genes expressed in cancer cells or cancer tissues. In some embodiments, a core promoter can further comprise one or more promoter elements that are obtained from a promoter of one or more genes expressed at a level that is at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%;Attorney Docket No. 53531-724602400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% higher in cancer cells or cancer tissues compared to non-cancer cells or non-cancer tissues. In some embodiments, promoter elements can include, but are not limited to, elements specific for tissue, elements specific for development or development stage, elements specific for cancer (e.g., transcription factor binding sites specific for cancer or oncogenic transcription factor binding sites), elements important for transcription (e.g., general promoter elements). In some embodiments, a core promoter can comprise two or more promoter elements that are obtained from a promoter of two or more genes expressed in cancer cells or cancer tissues. For example, a core promoter can comprise two or more promoter elements that are obtained from a promoter of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, 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, or at least 20 genes expressed in cancer cells or cancer tissues. Nonlimiting examples of genes expressed in cancer cells or cancer tissues can include TCF7, MNX1, HOXC10, TP53, CEACAM5, CEP55, FAM111B, CST1, BIRC5, AGR2, FOXA1, cMYC, FOS, TWIST1, E2F2, UBE2C, KIF20A, or ETV4. FIGs. 1-5 describes, in some embodiments, various core promoter designs described herein.

[0238] In some embodiments, a core promoter can comprise a minimal promoter obtained from one or more genes expressed in cancer cells or cancer tissues. In one example, a core promoter can comprise a minimal promoter obtained from one or more genes comprising TCF7, MNX1, HOXC10, TP53, CEACAM5, CEP55, FAM11 IB, CST1, BIRC5, AGR2, FOXA1, cMYC, FOS, TWIST1, E2F2, UBE2C, KIF20A, or ETV4. In another example, a core promoter can comprise a hybrid minimal promoter obtained from two or more genes comprising TCF7, MNX1, HOXC10, TP53, CEACAM5, CEP55, FAM111B, CST1, BIRC5, AGR2, FOXA1, cMYC, FOS, TWIST1, E2F2, UBE2C, KIF20A, or ETV4. In some embodiments, a core promoter can comprise a minimal promoter and one or more promoter elements described herein obtained from two or more genes comprising TCF7, MNX1, HOXC10, TP53, CEACAM5, CEP55, FAM111B, CST1, BIRC5, AGR2, FOXA1, cMYC, FOS, TWIST1, E2F2, UBE2C, KIF20A, or ETV4. In some embodiments, a core promoter can comprise a minimal promoter and two or more promoter elements described herein obtained from TCF7 and HOXC10. In some embodiments, a core promoter can comprise a minimal promoter and two or more promoter elements described herein obtained from TP53 and CEP55. In some embodiments, a core promoter can comprise a minimal promoter and two or more promoter elements described herein obtained from FAM11 IB and KIF20A. In some embodiments, a core promoter can comprise a minimal promoter and two or more promoter elements described herein obtained fromAttorney Docket No. 53531-724602BIRC5 and E2F2. In some embodiments, a core promoter can comprise a minimal promoter and two or more promoter elements described herein obtained from CEACAM5 and TWIST1. In some embodiments, a core promoter can comprise a hybrid promoter comprising two or more promoter elements described herein obtained from two or more genes comprising TCF7, MNXI, HOXCIO, TP53, CEACAM5, CEP55, FAM11 IB, CST1, BIRC5, AGR2, FOXA1, cMYC, FOS, TWIST1, E2F2, UBE2C, KIF20A, or ETV4. In some embodiments, a core promoter can comprise a hybrid promoter comprising two or more promoter elements described herein obtained from TCF7 and HOXCIO. In some embodiments, a core promoter can comprise a hybrid promoter comprising two or more promoter elements described herein obtained from TP53 and CEP55. In some embodiments, a core promoter can comprise a hybrid promoter comprising two or more promoter elements described herein obtained from FAM11 IB and KIF20A. In some embodiments, a core promoter can comprise a hybrid promoter comprising two or more promoter elements described herein obtained from BIRC5 and E2F2. In some embodiments, a core promoter can comprise a hybrid promoter comprising two or more promoter elements described herein obtained from CEACAM5 and TWIST1. In some embodiments, a core promoter can comprise a hybrid promoter comprising a minimal promoter and two or more promoter elements described herein obtained from two or more genes comprising TCF7, MNXI, HOXCIO, TP53, CEACAM5, CEP55, FAM111B, CST1, BIRC5, AGR2, FOXA1, cMYC, FOS, TWIST1, E2F2, UBE2C, KIF20A, or ETV4. In some embodiments, a core promoter can comprise a hybrid promoter comprising a minimal promoter and two or more promoter elements described herein obtained from TCF7 and HOXCIO. In some embodiments, a core promoter can comprise a hybrid promoter comprising a minimal promoter and two or more promoter elements described herein obtained from TP53 and CEP55. In some embodiments, a core promoter can comprise a hybrid promoter comprising a minimal promoter and two or more promoter elements described herein obtained from FAM11 IB and KIF20A. In some embodiments, a core promoter can comprise a hybrid promoter comprising a minimal promoter and two or more promoter elements described herein obtained from BIRC5 and E2F2. In some embodiments, a core promoter can comprise a hybrid promoter comprising a minimal promoter and two or more promoter elements described herein obtained from CEACAM5 and TWIST1.

[0239] In some embodiments, a core promoter can comprise a hybrid promoter comprising a chimeric sequence of two or more promoter elements from two or more genes comprising TCF7, MNXI, HOXCIO, TP53, CEACAM5, CEP55, FAM111B, CST1, BIRC5, AGR2, FOXA1, cMYC, FOS, TWIST1, E2F2, UBE2C, KIF20A, or ETV4. In some embodiments, a core promoter can comprise a hybrid promoter comprising a chimeric sequence of two or more promoter elements obtained from TCF7 and HOXCIO. In some embodiments, a core promoter can comprise a hybrid promoter comprising a chimeric sequence of two or more promoter elements obtained from TP53 and CEP55. In some embodiments, a core promoter can comprise a hybrid promoter comprising aAttomey Docket No. 53531-724602chimeric sequence of two or more promoter elements obtained from FAM11 IB and KIF20A. In some embodiments, a core promoter can comprise a hybrid promoter comprising a chimeric sequence of two or more promoter elements obtained from BIRC5 and E2F2. In some embodiments, a core promoter can comprise a hybrid promoter comprising a chimeric sequence of two or more promoter elements obtained from CEACAM5 and TWIST 1.

[0240] In some embodiments, a core promoter described herein is obtained from an endogenous CEACAM5 promoter, an endogenous CEP55 promoter, an endogenous FAM11 IB promoter, an endogenous CST1 promoter, an endogenous BIRC5 promoter, an endogenous AGR2 promoter, an endogenous UBE2C promoter, or an endogenous KIF20A promoter. In some embodiments, a non-naturally occurring polynucleotide described herein comprises one or more core promoters, wherein each of the one or more core promoters are obtained from an endogenous CEACAM5 promoter, an endogenous CEP55 promoter, an endogenous FAM111B promoter, an endogenous CST1 promoter, an endogenous BIRC5 promoter, an endogenous AGR2 promoter, an endogenous UBE2C promoter, or an endogenous KIF20A promoter. In some embodiments, a non-naturally occurring polynucleotide described herein comprises two or more core promoters, wherein each of the two or more core promoters are obtained from an endogenous CEACAM5 promoter, an endogenous CEP55 promoter, an endogenous FAM11 IB promoter, an endogenous CST1 promoter, an endogenous BIRC5 promoter, an endogenous AGR2 promoter, an endogenous UBE2C promoter, or an endogenous KIF20A promoter. In some embodiments, a non-naturally occurring polynucleotide described herein comprises three or more core promoters, wherein each of the three or more core promoters are obtained from an endogenous CEACAM5 promoter, an endogenous CEP55 promoter, an endogenous FAM11 IB promoter, an endogenous CST1 promoter, an endogenous BIRC5 promoter, an endogenous AGR2 promoter, an endogenous UBE2C promoter, or an endogenous KIF20A promoter. In some embodiments, a non-naturally occurring polynucleotide described herein comprises four or more core promoters, wherein each of the four or more core promoters are obtained from an endogenous CEACAM5 promoter, an endogenous CEP55 promoter, an endogenous FAM11 IB promoter, an endogenous CST1 promoter, an endogenous BIRC5 promoter, an endogenous AGR2 promoter, an endogenous UBE2C promoter, or an endogenous KIF20A promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEACAM5 promoter and a core promoter obtained from an endogenous CEP55 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEACAM5 promoter and a core promoter obtained from an endogenous FAM11 IB promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEACAM5 promoter and a core promoter obtained from an endogenous CST1 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEACAM5 promoter and aAttorney Docket No. 53531-724602core promoter obtained from an endogenous BIRC5 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEACAM5 promoter and a core promoter obtained from an endogenous AGR2 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEACAM5 promoter and a core promoter obtained from an endogenous UBE2C promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEACAM5 promoter and a core promoter obtained from an endogenous KIF20A promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEP55 promoter and a core promoter obtained from an endogenous FAM11 IB promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEP55 promoter and a core promoter obtained from an endogenous CST1 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEP55 promoter and a core promoter obtained from an endogenous BIRC5 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEP55 promoter and a core promoter obtained from an endogenous AGR2 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEP55 promoter and a core promoter obtained from an endogenous UBE2C promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CEP55 promoter and a core promoter obtained from an endogenous KIF20A promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous FAM11 IB promoter and a core promoter obtained from an endogenous CST1 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous FAM11 IB promoter and a core promoter obtained from an endogenous BIRC5 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous FAM11 IB promoter and a core promoter obtained from an endogenous UBE2C promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous FAM11 IB promoter and a core promoter obtained from an endogenous KIF20A promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous FAM11 IB promoter and a core promoter obtained from an endogenous AGR2 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CST1 promoter and a core promoter obtained from an endogenous BIRC5 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CST1 promoter and a core promoter obtained from an endogenous UBE2C promoter. In some embodiments, the non-naturally occurring polynucleotideAttorney Docket No. 53531-724602comprises a core promoter obtained from an endogenous CST1 promoter and a core promoter obtained from an endogenous KIF20A promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous CST1 promoter and a core promoter obtained from an endogenous AGR2 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous BIRC5 promoter and a core promoter obtained from an endogenous AGR2 promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous BIRC5 promoter and a core promoter obtained from an endogenous UBE2C promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous BIRC5 promoter and a core promoter obtained from an endogenous KIF20A promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous AGR2 promoter and a core promoter obtained from an endogenous UBE2C promoter. In some embodiments, the non-naturally occurring polynucleotide comprises a core promoter obtained from an endogenous AGR2 promoter and a core promoter obtained from an endogenous KIF20A promoter.

[0241] In some embodiments, a core promoter can comprise a TATA box or a TATA box sequence. In some embodiments, a core promoter can comprise a sequence of a region from about -300 bp to about +100 bp, from about -250 bp to about +100 bp, from about -200 bp to about +100 bp, from about -150 bp to about +100 bp, from about -100 bp to about +100 bp, from about -90 bp to about +100 bp, from about -80 bp to about +100 bp, from about -70 bp to about +100 bp, from about -60 bp to about +100 bp, from about -50 bp to about +100 bp, from about -40 bp to about +100 bp, or from about -30 bp to about +100 bp relative to a transcription start site (TSS) of a gene. In some embodiments, a core promoter can comprise a sequence of a region from about 300 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 250 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 200 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 150 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 100 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 90 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 80 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 70 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 60 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 50 bp upstream of a TSS to about 100 bp downstream of a TSS, from about 40 bp upstream of a TSS to about 100 bp downstream of a TSS, or from about 30 bp upstream of a TSS to about 100 bp downstream of a TSS of a gene. In some embodiments, a gene can comprise a human gene.

[0242] In some embodiments, the sequence of a region from about -300 bp to about +100 bp relative to a TSS (or from about 300 bp upstream of a TSS to about 100 bp downstream of a TSS) can comprise elements that are important for transcription, elements that are tissue specific, elements thatAttorney Docket No. 53531-724602are specific for certain development stage, and / or one or more binding sites fortranscription factors specific for cancer (e.g., oncogenic transcription factors). In some embodiments, a promoter or a core promoter can comprise one or more elements or sequences binding to NKX2-1, NANOG, GATA3, TRPS1, SOX9, KSLF14, Sp5, ZEB1, ZEB2, TGIF, PITX, NKX6-1, THRb, ERRa, COUP-TFII, PR, Ascl2, Slug, E2A, PITX1, orNKX3.2.

[0243] In some embodiments, a promoter or a core promoter can be operably linked to an open reading frame (ORF) of a gene of interest. A gene of interest can be any gene for which expression is desired specifically in cancer cells. Non-limiting examples of a gene of interest can include a gene encoding a therapeutic protein, a gene encoding a synthetic protein, a gene encoding a marker protein (e.g. , biomarker for diagnostics, etc.), or a gene encoding a reporter protein, a gene encoding a tumor associated antigen, or a gene encoding an immune cell activator. In some embodiments, the therapeutic protein comprises a toxin. In some embodiments, the toxin is any one of the toxins listed in Table IK. In some embodiments, the therapeutic protein comprises a cytokine. In some embodiments, the cytokine is any one of the cytokines listed in Table IL. In some embodiments, the gene of interest encodes any one of the immune cell activators listed in Table IM. In some embodiments, the gene of interest encodes any one of the tumor associated antigens listed in Table IN. In some embodiments, the therapeutic protein comprises a protein binding element. In some embodiments, the protein binding element is directed against any one of the immune cell activators listed in Table IM. In some embodiments, the protein binding element is directed against any one of the tumor associated antigens listed in Table IN. In some embodiments, the protein binding element is any one of the protein binding elements listed in Table IO.

[0244] In some embodiments, the core promoter can be obtained from a promoter of one or more genes that are expressed at a higher level in cancer cells compared to non-cancer cells. For example, the core promoter can be obtained from a promoter of one or more genes that are expressed at a level that is at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% higher in cancer cells compared to non-cancer cells. In some embodiments, the core promoter can be obtained from a promoter of one or more genes that are more active in cancer cells compared to non-cancer cells. For example, the core promoter can be obtained from a promoter of one or more genes that are at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%,Attorney Docket No. 53531-724602380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% more active in cancer cells compared to non-cancer cells. In some embodiments, a phosphorylation assay can be used to measure activation or activity levels of genes described herein.

[0245] In some embodiments, the core promoter can be obtained from one or more genes that are expressed at a higher level in cancer cells compared to non-cancer cells. For example, the core promoter can be obtained from one or more genes that are either expressed at a level that is at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% higher in cancer cells compared to non-cancer cells. In some embodiments, the core promoter can be obtained from one or more genes that are more active in cancer cells compared to non-cancer cells. For example, the core promoter can be obtained from one or more genes that are at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% more active in cancer cells compared to non-cancer cells. In some embodiments, a phosphorylation assay can be used to measure activation or activity levels of genes described herein.3. Transcription Factors

[0246] In some embodiments, an SRS can comprise one or more SREs, wherein the one or more SREs can comprise a plurality of binding sites for one or more transcription factors. In some embodiments, a plurality of binding sites (e.g., binding site DNA sequence) for one or more transcription factors can be identified from a multi -omics approach, including but not limited to, transcriptomics, proteomics, and / or phospho-proteomics to be upregulated in cancer cells or tissues compared to normal (e.g., non-cancer) cells or tissues. In some embodiments, the one or more SREsAttomey Docket No. 53531-724602can comprise a plurality of binding sites for one or more transcription factors that are expressed in higher levels in cancer cells compared to non-cancer cells. In some embodiments, ChIP assay can be used to measure expression levels of transcription factors described herein. In some embodiments, the one or more SREs can comprise a plurality of binding sites for one or more transcription factors that are more active in cancer cells compared to non-cancer cells. For example, the one or more SREs can comprise a plurality of binding sites for one or more transcription factors that have higher level of phosphorylation in cancer cells compared to non-cancer cells. In some embodiments, a phosphorylation assay can be used to measure activation or activity levels of transcription factors described herein.

[0247] In some embodiments, an SRS comprising a promoter (or a core promoter) and a plurality of binding sites for one or more transcription factors can drive the expression of an ORF operably linked to the promoter (or the core promoter) at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, at least 3.7-fold, at least 3.8-fold, at least 3.9-fold, at least 4-fold, at least 4.1-fold, at least 4.2-fold, at least 4.3-fold, at least 4.4-fold, at least 4.5-fold, at least 4.6-fold, at least 4.7-fold, at least 4.8-fold, at least 4.9-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or at least 100-fold higher than the expression of a corresponding ORF driven by a promoter (or a core promoter) without the plurality of binding sites for one or more transcription factors.

[0248] In some embodiments, an SRS comprising a promoter described herein (or a core promoter described herein, e.g., a cancer-specific core promoter comprising a TATA-TSS and other elements in -300 bp to about +100 bp relative to a TSS) and a plurality of binding sites for one or more transcription factors can drive the expression of an ORF operably linked to the promoter (or the core promoter) at least 1.1-fold, at least 1.2-fold, at least 1.3-fold, at least 1.4-fold, at least 1.5-fold, at least 1.6-fold, at least 1.7-fold, at least 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, at least 2.2-fold, at least 2.3-fold, at least 2.4-fold, at least 2.5-fold, at least 2.6-fold, at least 2.7-fold, at least 2.8-fold, at least 2.9-fold, at least 3-fold, at least 3.1-fold, at least 3.2-fold, at least 3.3-fold, at least 3.4-fold, at least 3.5-fold, at least 3.6-fold, at least 3.7-fold, at least 3.8-fold, at least 3.9-fold, at least 4-fold, at least 4.1-fold, at least 4.2-fold, at least 4.3-fold, at least 4.4-fold, at least 4.5-fold, at least 4.6-fold, at least 4.7-fold, at least 4.8-fold, at least 4.9-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 11-fold, at least 12-fold, at least 13-fold, at least 14-fold, at least 15-fold, at least 16-fold, at least 17-fold, at least 18-fold, at least 19-fold, at least 20-fold, at least 21 -fold, at least 22-fold, at least 23 -fold, at least 24-fold, at least 25 -fold, at least 26-Attorney Docket No. 53531-724602fold, at least 27-fold, at least 28-fold, at least 29-fold, at least 30-fold, at least 31-fold, at least 32-fold, at least 33-fold, at least 34-fold, at least 35-fold, at least 36-fold, at least 37-fold, at least 38-fold, at least 39-fold, at least 40-fold, at least 41-fold, at least 42-fold, at least 43-fold, at least 44-fold, at least 45-fold, at least 46-fold, at least 47-fold, at least 48-fold, at least 49-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold, at least 80-fold, at least 85-fold, at least 90-fold, at least 95-fold, or at least 100-fold higher than the expression of a corresponding ORF driven by a non-cancer specific promoter (e.g., TATA-TSS promoter only) and the plurality of binding sites for one or more transcription factors.

[0249] Non-limiting examples of transcription factors can include TRPS1, MNX1, TWIST1, ETV4, FOSL2, NFIC, EN2, TFDP1, PITX2, TCF7L1, VENTX, H0XB9, DLX1, MYCN, SIX4, TP63, SOX11, E2F8, TFDP1, SURV, TOXE1, EN1, ZBTB7B, SP3, SIX2, XBP1, HIF-1A, CREB3L1, HSF-1, MTF1, NFE2L2, USF2, TP73, POU2F2, H0XA1, FOXO1, TFAP4, BACH1, E2F4, HOXCIO, KLF11, F0XM1, E2F2, E2F3, E2F1, GLIS3, GATA1, DLX3, LHX2, BARX1, HOXC9, FOXK1, RUNX2, RUNX1, SOX4, RREB1, HES6, ASCL1, FOXA3, HOXB2, DLX4, GRHL1, FOXA, HIF, E2F6, FOSL1, JUN, JUNB, FOSB, AP-1, NF-1, RFX6, EL4, TCF3, TCF12, SNAI2, REST, DMRTA2, RFX7, NRF1, ZNF148, ZNF652, PRDM1, HIF1A, TGIF1, STAT2, ESRRA, RELB, HSF1, MAFB, TFAP2C, YBX1, YY1, PITX1, SATB1, ARID3A, POU3F1, SP4, MGA, SALL4, AHR, MLXIP, PRDM4, NFIL3, TFAP2A, ZBTB17, ZFP91, ARID5A, IRF6, ZFX, POU2F1, NKX2-1, NKX2-8, FOXA1, NFKB1, HNF4G, ARID 1 A, NFATC2, SMAD2, ARID3B, TP53, FOS, FOS-CREB, ELK3, FOXO1::ELK3, TCF7, E2F2, CREB3L1, SHOX2, TCF7L1, H0XA1, MYBL2, NR2C2, MYCN, FOXN1, PITX2, EN2, NFIC, MYC, DLX4, SP3, FOXE1, VENTX, TP53, GLIS3, CUX1, MGA, DLX1, DLX6, GATA1, RUNX2, E2F7, GRHL1, ZBTB7B, HNF1A, FOXA3, NPAS2, TP63, RREB1, SOX4, ZIC2, TCF7, EN1, DMBX1, E2F8, FOSL2, PBX3, NKX3-2, DLX3, HOXB7, TRPS1, SOX11, PAX8, HES6, HOXCIO, MNX1, SIX2, ZNF281, ETV4, ZNF384, ASCL1, BARX1, PAX7, LHX2, OTX1, RUNX1, ETV6, FOXK1, HOXB9, E2F4, NR2F6, TWIST1 HOXC9, IRF6, NR2E1, RORB, E2F1, E2F3, TFDP1, FOXJ3, SIX4, MAX::MYC, ONECUT1, orNFKB.

[0250] In some embodiments, transcription factors enriched in lung adenocarcinoma (LU AD) can comprise E2F2, CREB3L1, SHOX2, TCF7L1, HOXA1, MYBL2, NR2C2, MYCN, FOXN1, PITX2, EN2, NFIC, MYC, DLX4, SP3, FOXE1, VENTX, TP53, GLIS3, CUX1, MGA, DLX1, DLX6, GATA1, RUNX2, E2F7, GRHL1, ZBTB7B, HNF1A, FOXA3, NPAS2, TP63, RREB1, SOX4, ZIC2, TCF7, EN1, DMBX1, E2F8, FOSL2, PBX3, NKX3-2, DLX3, HOXB7, TRPS1, SOX11, PAX8, HES6, HOXCIO, MNX1, SIX2, ZNF281, ETV4, ZNF384, ASCL1, BARX1, PAX7, LHX2, OTX1, RUNX1, ETV6, FOXK1, HOXB9, E2F4, NR2F6, TWIST1, HOXC9, IRF6, NR2E1, RORB, E2F1, E2F3, TFDP1, FOXJ3, SIX4, MAX::MYC, or ONECUT1.Attorney Docket No. 53531-724602

[0251] In some embodiments, transcription factors can comprise E2F4, E2F3, E2F1, GLIS3, GATA1, DLX1, DLX3, LHX2, BARX1, PBX3, HOXC9, FOXK1, FOXA3, TRPS1, RUNX2, H0XA1, NFE2L2, TCF3, TCF12, SNAI2, REST, DMRTA2, RFX7, NRF1, ZNF148, ZNF652, PRDM1, HIF1A, TGIF1, STAT2, ESRRA, RELB, HSF1, MAFB, TFAP2C, YBX1, YY1, PITX1, SATB1, ARID3A, USF2, POU3F1, SP4, MGA, SALL4, AHR, MLXIP, MTF1, PRDM4, ZBTB7B, NFIL3, TFAP2A, ZBTB17, ZFP91, BACH1, MLXIP, ARID5A, IRF6, ZFX, POU2F1, NKX2-1, NKX2-8, FOXA1, NFKB1, MGA, HNF4G, ARID 1 A, NFATC2, POU2F2, SMAD2, PRDM4, MLXIP, or ARID3B. In some embodiments, control TF tiles can comprise TCF7_v2, TCF7Ll_vl9, TP53_v5, TP53_v22, Control- l-FOSLl_vl, HOXC10_v24, HOXC10_vl4, CREB3Ll_v6, CREB3Ll_vl4, Control-Filler vl, Control-Filler_v2, Control-Filler_v3, Control-Filler_v4, or Control -Filler_v5. In some embodiments, TF tiles can comprise homotypic TF-tiles or heterotypic TF tiles. For examples, TF-tiles comprising mixed binding sequences / sites / motifs from the same TF can be referred to as homotypic TF-tiles. For example, TF-tiles comprising mixed binding sequences / sites / motifs from different TF can be referred to as heterotypic TF-tiles. In some embodiments, SREs can comprise binding sequences, sites, or motifs of TFs of dysregulated genes that are involved in the EGFR, KRAS or p53 pathways in NSCLC.

[0252] In some embodiments, a binding site for a transcription factor can comprise a known transcription factor binding site (TFBS) sequence element or DNA binding site sequence element. In some embodiments, a transcription factor can bind to TFBS sequence element or DNA binding site sequence element and can recruit additional transcriptional machinery and co-factors (e.g., RNA polymerase, etc.) to the promoter or the core promoter. In some embodiments, a transcription factor can comprise a transcription co-factor.

[0253] In one embodiment, transcription factors that bind to the plurality of transcription binding sites can drive the expression of an ORF operably linked to the promoter in one specific type of cancer cells. In another embodiment, transcription factors that bind to the plurality of transcription binding sites can drive the expression of an ORF operably linked to the promoter in two or more types of cancer cells.

[0254] In some embodiments, an SRE can comprise at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, or at least about ten binding sites for one or more transcription factors. In some embodiments, an SRE can comprise at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 binding sites for one or more transcription factors. In some embodiments, an SRE can comprise at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at mostAttorney Docket No. 53531-724602about 24, at most about 23, at most about 22, at most about 21, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, or at most about 5 binding sites for one or more transcription factors.

[0255] In some embodiments, an SRE can comprise a plurality of binding sites for at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, or at least about ten transcription factors. In some embodiments, an SRE can comprise a plurality of binding sites for at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 transcription factors. In some embodiments, an SRE can comprise a plurality of binding sites for at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at most about 24, at most about 23, at most about 22, at most about 21, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, or at most about 5 transcription factors.

[0256] In some embodiments, an SRE can comprise two or more transcription factor binding sites for one transcription factor, wherein each of the two or more transcription factor binding sites can be sequentially arranged or tiled in a sequential manner. For example, an SRE can comprise two or more transcription factor binding site sequences for one transcription factor and each of the two or more transcription factor binding sites can be sequentially arranged or tiled in a sequential manner (e.g., arranged side by side). In some embodiments, an SRE can comprise two or more transcription factor binding sites for one transcription factor, wherein each of two or more transcription factor binding sites can be sequentially arranged or tiled in a sequential manner at 5 ’ to a core promoter in the non-naturally occurring polynucleotide comprising the SRE and the core promoter.

[0257] In some embodiments, an SRE can comprise two or more transcription factor binding sites for two or more transcription factors, wherein each of two or more transcription factor binding sites can be non-sequentially arranged or tiled in a non-sequential manner. For example, an SRE can comprise two or more transcription factor binding site sequences for two or more transcription factors and the two or more transcription factor binding site sequences may be (i) the same, (ii) different, or (iii) a combination of (i) and (ii). In this example, the two or more transcription binding sites can comprise (ii) different transcription factor binding site sequences that are non-sequentially arranged or tiled in a non-sequential manner (e.g, shuffled) in the non-naturally occurring polynucleotide. In another example, the two or more transcription factor binding sites can comprise (iii) a combination of the same and different transcription factor binding site sequences, wherein all of the two or moreAttomey Docket No. 53531-724602transcription factor binding sites are non-sequentially arranged or tiled in a non-sequential manner in the non -naturally occurring polynucleotide. In yet another example, the two or more transcription factor binding sites can comprise (iii) a combination of the same and different transcription factor binding site sequences, wherein some of the two or more transcription factor binding sites are sequentially arranged or tiled in a sequential manner and the some of the two or more transcription factor binding sites are non-sequentially arranged or tiled in a non-sequential manner in the non-naturally occurring polynucleotide. In some embodiments, an SRE can comprise two or more transcription factor binding sites for two or more transcription factors, wherein each of two or more transcription factor binding sites can be non-sequentially arranged or tiled in a non-sequential manner at 5’ to a core promoter in the non-naturally occurring polynucleotide comprising the SRE and the core promoter.

[0258] In some embodiments, an SRE comprising a plurality of binding sites for one or more transcription factors can further comprise a spacer element between each of the plurality of binding sites for one or more transcription factors. In some embodiments, a spacer element can comprise a nucleotide sequence of from about 1 to about 10 nucleotides or base pairs. For example, a spacer element can comprise a nucleotide sequence of from about 1 to about 10 nucleotides, from about 2 to about 15 nucleotides, from about 3 to about 20 nucleotides, from about 4 to about 25 nucleotides, from about 4 to about 30 nucleotides, from about 5 to about 35 nucleotides, from about 6 to about 40 nucleotides, from about 7 to about 50 nucleotides, from about 8 to about 55 nucleotides, from about 9 to about 60 nucleotides, from about 10 to about 65 nucleotides, from about 15 to about 70 nucleotides, from about 20 to about 75 nucleotides, from about 25 to about 80 nucleotides, from about 30 to about 85 nucleotides, from about 35 to about 90 nucleotides, from about 40 to about 95 nucleotides, or from about 45 to about 100 nucleotides. In some embodiments, a spacer element can comprise a nucleotide sequence of at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 nucleotides. In some embodiments, a spacer element can comprise a nucleotide sequence of at most about 100, at most about 95, at most about 90, at most about 85, at most about 80, at most about 75, at most about 70, at most about 65, at most about 60, at most about 55, at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at most about 24, at most about 23, at most about 22, at most about 21, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most aboutAttorney Docket No. 53531-72460212, at most about 11, or at most about 10 nucleotides. In some embodiments, a spacer element can comprise a nucleotide sequence of 0, 3, 7, or 10 nucleotides or base pairs.

[0259] In some embodiments, an SRS can comprise a plurality of binding sites for one or more transcription factors (TFs), wherein said one or more TFs are expressed at higher levels in cancer cells compared to non-cancer cells. For example, the one or more TFs core promoter may be expressed at a level that is at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% higher in cancer cells compared to non-cancer cells.

[0260] In some embodiments, an SRS can comprise a plurality of binding sites for one or more transcription factors (TFs), wherein said one or more TFs are more active in cancer cells compared to non-cancer cells. For example, the one or more TFs may be at least 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 110%, 920%, 930%, 940%, 950%, 960%, 970%, 980%, 990%, or at least 1000% more active in cancer cells compared to non-cancer cells. In some embodiments, a phosphorylation assay can be used to measure activation or activity levels of TFs described herein.4. Enhancers

[0261] In some embodiments, an SRE can comprise a plurality of enhancers. For example, an SRE can comprise a plurality of any known enhancers that can increase the level of transcription of a gene. In some embodiments, an SRE can comprise a plurality of endogenous enhancer sequences. In some embodiments, an SRE can comprise a plurality of enhancers obtained from a gene described herein. In some embodiments, a gene can comprise a human gene. In some embodiments, an SRE can comprise at least about one, at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, or at least about ten enhancers obtained from a gene. In some embodiments, an SRE can comprise at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17,Attomey Docket No. 53531-724602at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, or at least about 50 enhancers obtained from a gene. In some embodiments, an SRE can comprise at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at most about 24, at most about 23, at most about 22, at most about 21, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, or at most about 5 enhancers obtained from a gene.

[0262] In some embodiments, an SRE can comprise a plurality of enhancers obtained from two or more genes described herein. In some embodiments, a gene can refer to a gene specifically or preferentially expressed in cancer cells or cancer tissues compared to non-cancer cells or non-cancer tissues. In some embodiments, a gene can comprise a human gene. In some embodiments, an SRE can comprise a plurality of enhancers obtained from at least about two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, or at least about ten genes. In some embodiments, an SRE can comprise a plurality of enhancers obtained from at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 genes. In some embodiments, an SRE can comprise a plurality of enhancers obtained from at most about 100, at most about 95, at most about 90, at most about 85, at most about 80, at most about 75, at most about 70, at most about 65, at most about 60, at most about 55, at most about 50, at most about 45, at most about 40, at most about 35, at most about 30, at most about 25, at most about 24, at most about 23, at most about 22, at most about 21, at most about 20, at most about 19, at most about 18, at most about 17, at most about 16, at most about 15, at most about 14, at most about 13, at most about 12, at most about 11, at most about 10, at most about 9, at most about 8, at most about 7, at most about 6, or at most about 5 genes.

[0263] In some embodiments, a plurality of enhancers described herein can comprise a transcription regulatory element (TRE). A TRE can refer to a region of DNA that can regulate transcription of a gene. In some embodiments, a TRE can increase the transcription of a gene. In some embodiments, a TRE can decrease the transcription of a gene. In some embodiments, a TRE can comprise a transcription binding site. In some embodiments, a plurality of enhancers can comprise a transcription regulatory element that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology to an enhancerAttorney Docket No. 53531-724602consensus sequence of two or more homologous genes. In some embodiments, a plurality of enhancers can comprise a transcription regulatory element that has 90% sequence homology to an enhancer consensus sequence of two or more homologous genes.

[0264] In some embodiments, a plurality of enhancers can comprise an enhancer consensus sequence of two or more homologous genes. In some embodiments, an enhancer consensus sequence of two or more homologous genes can comprise a consensus sequence of an enhancer sequence obtained from two or more genes that has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity between the two or more genes. In some embodiments, an enhancer consensus sequence of two or more homologous genes can comprise a consensus sequence of an enhancer sequence obtained from two or more genes that has at least 90% sequence identity between the two or more genes.

[0265] In some embodiments, an SRE can comprise a plurality of enhancers comprising at least two enhancer sequences, wherein each of the at least two enhancer sequences can comprise (i) the same enhancer sequences, (ii) different enhancer sequences, or (iii) a combination of (i) and (ii). In some embodiments, each of the at least two enhancer sequences can be sequentially arranged or tiled in a sequential manner in a non-naturally occurring polynucleotide. In some embodiments, each of the at least two enhancer sequences can be sequentially arranged or tiled in a sequential manner at 5 ’ to a core promoter in the non-naturally occurring polynucleotide comprising the core promoter and an SRE comprising the plurality of enhancers. In some embodiments, each of said at least two enhancer sequences can be sequentially arranged or tiled in a sequential manner at 5’ to a core promoter and / or at 3’ to a plurality of binding sites for one or more TFs, if present, in the non-naturally occurring polynucleotide comprising the core promoter, an SRE comprising the plurality of enhancers, and / or the plurality of transcription factor binding sites.

[0266] In some embodiments, an SRE can comprise a plurality of enhancers comprising at least two enhancer sequences, wherein each of the at least two enhancer sequences can comprise (ii) different enhancer sequences. In this embodiment, each of said plurality of enhancers comprising different enhancer sequences can be non-sequentially arranged or tiled in a non-sequential manner. In some embodiments, each of said plurality of enhancers comprising different enhancer sequences can be non-sequentially arranged or tiled in a non-sequential manner at 5’ to a core promoter in the non-naturally occurring polynucleotide comprising the core promoter and an SRE comprising the plurality of enhancers. In some embodiments, each of said plurality of enhancers comprising different enhancer sequences can be non-sequentially arranged or tiled in a non-sequential manner at 5’ to a core promoter and / or at 3’ to a plurality of binding sites for one or more TFs, if present, in the non-naturally occurring polynucleotide comprising the core promoter, an SRE comprising the plurality of enhancers, and / or the plurality of transcription factor binding sites.Attorney Docket No. 53531-724602

[0267] In some embodiments, an SRE can comprise a plurality of enhancers comprising at least two enhancer sequences, wherein each of the at least two enhancer sequences can comprise (iii) a combination of the same and different enhancer sequences. In this embodiment, each of said plurality of enhancers comprising a combination of the same and different enhancer sequences can be non-sequentially arranged or tiled in a non-sequential manner. In some embodiments, each of said plurality of enhancers comprising a combination of the same and different enhancer sequences can be non-sequentially arranged or tiled in a non-sequential manner at 5’ to a core promoter in the non-naturally occurring polynucleotide comprising the core promoter and an SRE comprising the plurality of enhancers. In some embodiments, each of said plurality of enhancers comprising a combination of the same and different enhancer sequences can be non-sequentially arranged or tiled in a nonsequential manner at 5’ to a core promoter and / or at 3’ to a plurality of binding sites for one or more TFs, if present, in the non-naturally occurring polynucleotide comprising the core promoter, an SRE comprising the plurality of enhancers, and / or the plurality of transcription factor binding sites.

[0268] In some embodiments, a plurality of enhancers described herein can comprise a sequence capable of binding to a transcription associated protein. A transcription associated protein as described herein can comprise any protein that is involved in transcription of a DNA sequence to an RNA sequence. In some embodiments, a transcription associated protein can bind to an enhancer sequence. In some embodiments, an assay can be used to determine if a transcription associated protein can bind to a sequence comprised in a plurality of enhancers. For example, chromatin immunoprecipitation (ChIP) assay, an in vitro transfection reporter assay, or any other suitable assays or methods can be used to determine if a transcription associated protein can bind to a sequence comprised in a plurality of enhancers. In some embodiments, a plurality of enhancers described herein can comprise a sequence capable of binding to a transcription associated protein determined by chromatin immunoprecipitation (ChIP) or an in vitro transfection reporter assay.

[0269] In some embodiments, a plurality of enhancers can comprise a CpG island. For example, at least one enhancer of the plurality of enhancers can comprise a CpG island. In some embodiments, a plurality of enhancers may not comprise a CpG island. For example, at least one enhancer of the plurality of enhancers may not comprise a CpG island.

[0270] In some embodiments, an SRS can comprise a core promoter and a plurality of binding sites for one or more transcription factors obtained from two or more genes, wherein the core promoter and the plurality of binding sites for one or more transcription factors are not obtained from the same gene. In some embodiments, an SRS can comprise a core promoter and a plurality of enhancers obtained from two or more genes, wherein the core promoter and the plurality of enhancers are not obtained from the same gene. In some embodiments, an SRS can comprise a core promoter, a plurality of binding sites for one or more transcription factors, and a plurality of enhancer obtained from two or more genes, wherein the core promoter, the plurality of binding sites for one or moreAttorney Docket No. 53531-724602transcription factors, and the plurality of enhancer are not obtained from the same gene. In some embodiments, a gene can comprise a human gene.

[0271] In some embodiments, a plurality of enhancers can comprise an enhancer sequence that can bind to SP1, ETS, CEBP, NF-KB, EBS, C / EBP, ARE, DRE, NFKB, GC-box, UN5CL, BOP1, RTN4RL2, ARNTL2, AGR2, LHX2, TRNP1, MU5AC, or D0K4. In some embodiments, a plurality of enhancers can comprise at least two, at least about three, at least about four, at least about five, at least about six, at least about seven, at least about eight, at least about nine, or at least about ten enhancer sequences. In some embodiments, a plurality of enhancers can comprise at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, or at least about 100 enhancer sequences. In some embodiments, a plurality of enhancers can comprise at least two SP1, ETS, CEBP, NF-KB, EBS, C / EBP, ARE, DRE, NFKB, GC-box, UN5CL, BOP1, RTN4RL2, ARNTL2, AGR2, LHX2, TRNP1, MU5AC, or DOK4 enhancer sequences.

[0272] In some embodiments, core promoter, plurality of binding sites for one or more transcription factors, or plurality of enhancers obtained from two or more genes can comprise a sequence listed in Table 1A, Table IB, or Table 1C. In some embodiments, an SRS described herein can comprise a sequence listed in Table 1A, Table IB, or Table 1C.

[0273] In some embodiments, an SRS can comprise a sequence comprising a human alphafetoprotein (AFP) promoter sequence comprising a plurality of HNF-1A transcription binding sites. AFP level is elevated in liver cancer including, but not limited to, hepatic carcinomas. In some embodiments, an HNF-1A transcription binding site can comprise a sequence of 5’-GTTAATTATTAAC-3’ (SEQ ID NO: 128).C. Therapeutic Proteins

[0274] Provided herein are nucleic acid molecules comprising an SRS described herein that can drive the expression of an open reading frame (ORF) operably linked to the SRS. In some embodiments, an ORF comprises a natural gene or a synthetic gene. In some embodiments, a natural gene or a synthetic gene comprises a gene encoding a tumor associated antigen, an immune cell activator, or any combination thereof. In some embodiments, a therapeutic protein comprises an antibody, a functional fragment of an antibody, a protein binding element, a toxin, a cytokine, or any combination thereof. In some embodiments, a therapeutic protein, expressed under the control of an SRS described herein, is differentially expressed in a cancer cell or a cancer tissue as compared to a corresponding healthy cell or healthy. In some embodiments, the therapeutic protein has a 1-fold, 2-fold, 3-fold, 4-fold, 5-fold,Attorney Docket No. 53531-72460210-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold, or higher fold change in expression in a cancer cell or a cancer tissue as compared to a corresponding healthy cell or healthy tissue. For example, FIGs. 82A-82B provide that expression of a non-naturally occurring polynucleotide comprising an SRS drove expression of an ORF operably linked to an SRS in tumor tissue (119 RNA copies of the ORF) compared to healthy tissues, which were below the limit of quantification of the RNA of the ORF.

[0275] In some embodiments, a therapeutic protein disclosed herein encoded in an ORF (e.g., operably linked to any of the promoters described herein) comprises a toxin, or derivative thereof. In some embodiments, the toxin has an amino acid sequence of any one of the toxins listed in Table IK.In some embodiments, the toxin, or derivative thereof, has an amino acid sequence of any one of SEQ ID NOs: 588-622 or a variant thereof. In some embodiments, the toxin, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IK. In some embodiments, the toxin, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 588- 622 or a variant thereof. In some embodiments, the toxin, or derivative thereof encoded in an ORF further comprises a secretion signal sequence. In some embodiments, the toxin, or derivative thereof encoded in an ORF does not comprise a secretion signal sequence. In some embodiments, the toxin, or derivative thereof encoded in an ORF comprises any of the sequences in Table IK, or a variant thereof, missing a secretion signal.

[0276] In some embodiments, the toxin, or derivative thereof encoded in an ORF (e.g., operably linked to any of the promoters described herein), comprises at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500 consecutive residues, or all any one of SEQ ID NOs: 588-622 or a variant thereof. In some embodiments, the toxin, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least aboutAttorney Docket No. 53531-72460288%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IK. In some embodiments, the toxin, or derivative thereof, comprises at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500 consecutive residues, or all any one of SEQ ID NOs: 588-622 or a variant thereof. In some embodiments, the toxin, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 588-622 or a variant thereof.

[0277] In some embodiments, a toxin, or derivative thereof encoded in an ORF (e.g., operably linked to any of the promoters described herein), comprises at most about 50, at most about 55, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 85, at most about 90, at most about 100, at most about 120, at most about 140, at most about 160, at most about 180, at most about 200, at most about 250, at most about 300, at most about 350, at most about 400, at most about 450, or at most about 500 consecutive residues, or all any one of SEQ ID NOs: 588-622 or a variant thereof. In some embodiments, the toxin, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IK. In some embodiments, the toxin, or derivative thereof, comprises at most about 50, at most about 55, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 85, at most about 90, at most about 100, at most about 120, at most about 140, at most about 160, at most about 180, at most about 200, at most about 250, at most about 300, at most about 350, at most about 400, at most about 450, or at most about 500 consecutive residues, or all any one of SEQ ID NOs: 588-621 or a variant thereof. In some embodiments, the toxin, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, atAttorney Docket No. 53531-724602least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 588-621 or a variant thereof.

[0278] Table IK: Toxin amino acid sequences

[0279] Cytokine therapies, including synthetic cytokine receptor agonist therapies, are promising for treating various conditions or diseases, such as cancer. However, cytokine therapies can also lead to significant toxicity, particularly when expressed non-specifically, rather than in a tissue-specific manner (e.g., under the control of a tissue-specific promoter). For example, non-specific cytokine therapies can cause a systematic inflammatory response comprising symptoms such as fever, hypotension, and organ dysfunction. In some cases, non-specific cytokine therapies can cause cytokine release syndrome (CRS). In some cases, non-specific cytokine therapies can cause neurotoxicity. In some cases, non-specific cytokine therapies can cause capillary leak syndrome. In some cases, non-specific cytokine therapies can cause cardiopulmonary toxicity. In some cases, non-Attorney Docket No. 53531-724602specific cytokine therapies can cause renal toxicity. In some cases, non-specific cytokine therapies can cause cardiopulmonary toxicity. In some cases, non-specific cytokine therapies can cause gastrointestinal toxicity. In some embodiments, expression of an ORF encoding for a cytokine or cytokine receptor agonist, or a derivative thereof, operably linked to any one of the promoters described herein, may reduce cytokine or cytokine receptor agonist toxicity by inducing increased expression of the cytokine or cytokine receptor agonist, or the derivative thereof, in a cancer cell compared to a non-cancer cell (e.g., a specific manner of expression). In some embodiments, the cytokine or cytokine receptor agonist, or the derivative thereof, is IL-2 or an agonist of the IL-2 receptor complexes (e.g., IL-2Ra / b / g, IL-2Rbg). In some embodiments, the cytokine or cytokine receptor agonist, or the derivative thereof, is IL-12 or an agonist of the IL-12 Receptor complexes (IL-12RB1 / 2). In some embodiments, the cytokine, or the derivative thereof, is IL-15 or an agonist of the IL-15 Receptor complexes (IL-15Ra-IL-2Rb / g). In some embodiments, the cytokine, or the derivative thereof, is IL-7 or an agonist of the IL-17 Receptor complexes (IL-7Ra-IL-2Rg). In some embodiments, the cytokine, or the derivative thereof, is IL-21 or an agonist of the IL-21 Receptor complexes (IL-21Ra-IL-2Rg). In some embodiments, the cytokine, or the derivative thereof, is IL-18 or an agonist of the IL- 18 Receptor complexes (IL-18R1-IL-18R2 / ILRAP). In some embodiments, inducing increased expression of the cytokine or cytokine receptor agonist, or the derivative thereof, in a cancer cell compared to a non-cancer cell can reduce the negative effects of cytokine toxicity, such as reducing one or more of a systematic inflammatory response, CRS, neurotoxicity, capillary leak syndrome, cardiopulmonary toxicity, gastrointestinal toxicity, or any combination thereof, compared to a non-specific cytokine therapy.

[0280] In some embodiments, a therapeutic protein disclosed herein encoded in an ORF (e.g., operably linked to any of the promoters described herein) comprises a cytokine or derivative thereof, a cytokine receptor agonist or derivative thereof, or both. In some embodiments, the cytokine has an amino acid sequence of any one of the cytokines listed in Table IL. In some embodiments, the cytokine, or derivative thereof, has an amino acid sequence of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the cytokine or cytokine receptor agonist stimulates any one of the following receptors: IL-2Ra, IL-2-Rb , IL-2Rg, IL-12Rbl, IL-12Rb2, IL-7Ra, IL-21Ra, IL-10R, IL-18R1, IL-8R2 (such as, IL-18RAP) or enhances the activity of these receptors by antagonizing negative regulators like IL-18BP. In some embodiments, the cytokine or cytokine receptor agonist stimulates any one of the following receptor multimers: IL-2Ra / b / g, IL-2Rb / g, IL-7Ra / IL-2Rg, IL-21Ra-IL-2Rg, IL-10R1-IL-10R2, IL-12RB1-IL-12RB2, IL-18Ra. In some embodiments, the cytokine, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, atAttorney Docket No. 53531-724602least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IL. In some embodiments, the cytokine, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the cytokine, or derivative thereof encoded in an ORF further comprises a secretion signal sequence. In some embodiments, the cytokine, or derivative thereof encoded in an ORF does not comprise a secretion signal sequence. In some embodiments, the cytokine, or derivative thereof encoded in an ORF comprises any of the sequences in Table IL, or a variant thereof, missing a secretion signal.

[0281] In some embodiments, the cytokine, or derivative thereof encoded in an ORF (e.g., operably linked to any of the promoters described herein), comprises at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500 consecutive residues, or all any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the cytokine, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IL. In some embodiments, the cytokine, or derivative thereof, comprises at least about 50, at least about 55, at least about 60, at least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, at least about 200, at least about 250, at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500 consecutive residues, or all any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the cytokine, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at leastAttorney Docket No. 53531-724602about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof.

[0282] In some embodiments, a cytokine, or derivative thereof encoded in an ORF (e.g., operably linked to any of the promoters described herein), comprises at most about 50, at most about 55, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 85, at most about 90, at most about 100, at most about 120, at most about 140, at most about 160, at most about 180, at most about 200, at most about 250, at most about 300, at most about 350, at most about 400, at most about 450, or at most about 500 consecutive residues, or all any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the cytokine, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IL. In some embodiments, the cytokine, or derivative thereof, comprises at most about 50, at most about 55, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 85, at most about 90, at most about 100, at most about 120, at most about 140, at most about 160, at most about 180, at most about 200, at most about 250, at most about 300, at most about 350, at most about 400, at most about 450, or at most about 500 consecutive residues, or all consecutive residues of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the cytokine, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof.

[0283] In some embodiments, a cytokine, or derivative thereof, encoded in an ORF (e.g., operably linked to any of the promoters described herein), comprises a fragment of the cytokine. In some embodiments, the fragment is a truncated peptide. In some embodiments, the fragment comprises at most about 50 to at most about 500 consecutive residues. In some embodiments, the fragment comprises at most about 50, at most about 55, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 85, at most about 90, at most about 100, at most about 120, at most about 140, at most about 160, at most about 180, at most about 200, at most about 250, at most about 300, at most about 350, at most about 400, at most about 450, or at most about 500Attomey Docket No. 53531-724602consecutive residues of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the fragment comprises all consecutive residues of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the fragment comprises at least one binding region. In some embodiments, the at least one binding region binds a cytokine receptor. In some embodiments, the cytokine receptor is an IL-2 receptor. In some embodiments, the cytokine receptor is an IL- 12 receptor. In some embodiments, the cytokine receptor is an IL- 15 receptor. In some embodiments, the fragment comprises at least one to at least four binding regions of the cytokine. In some embodiments, the fragment comprises at least one binding region of the cytokine. In some embodiments, the fragment comprises at least two binding regions of the cytokine. In some embodiments, the fragment comprises at least three binding regions of the cytokine. In some embodiments, the fragment comprises at least four binding regions of the cytokine.

[0284] In some embodiments, a therapeutic protein described herein encoded in an ORF (e.g., operably linked to any of the promoters described herein), comprises a fusion protein. In some embodiments, a peptide therapeutic agent described herein comprises a therapeutic protein described herein. In some embodiments, a therapeutic protein comprises a chimeric protein. In some embodiments, a chimeric protein comprises a first polypeptide or amino acid sequence, or a variant thereof, from a first species and a second polypeptide or amino acid sequence, or a variant thereof, from a second species. In some embodiments, a fusion protein described herein comprises a chimeric protein. In some embodiments, the fusion protein comprises a cleavable domain. In some embodiments, the cleavable domain comprises a protease cleavage site configured to allow for cleavage and separation of different subdomains of the fusion protein. In some embodiments, the fusion protein comprises a T2A self-cleavage sequence, a P2A self-cleavage sequence, or both. In some embodiments, a fusion protein described herein comprises a leader amino acid sequence. In some embodiments, the leader amino acid sequence comprises an amino acid sequence set forth in SEQ ID NO: 725. In some embodiments, the fusion protein comprises one or more to four or more cytokines. In some embodiments, the fusion protein comprises one or more cytokines, or derivatives thereof. In some embodiments, the fusion protein comprises two or more cytokines, or derivatives thereof. In some embodiments, the fusion protein comprises three or more cytokines, or derivatives thereof. In some embodiments, the fusion protein comprises four or more cytokines, or derivatives thereof. In some embodiments, the one or more cytokines, or derivatives thereof, the two or more cytokines, or derivatives thereof, the three or more cytokines, or derivatives thereof, or the four or more cytokines, or derivatives thereof, comprise at least two distinct cytokines, or derivatives thereof. In some embodiments, the one or more cytokines, or derivatives thereof, the two or more cytokines, or derivatives thereof, the three or more cytokines, or derivatives thereof, or the four or more cytokines, or derivatives thereof, comprise at least two of the same cytokine, or derivatives thereof. InAttorney Docket No. 53531-724602some embodiments, the fusion protein comprises at least one cytokine, or derivative thereof, having a sequence of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the at least one cytokine, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the fusion protein comprises two or more cytokines, or derivatives thereof, having a sequence of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the two or more cytokines, or derivative thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the fusion protein comprises three or more cytokines, or derivatives thereof, having a sequence of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the three or more cytokines, or derivatives thereof, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the fusion protein comprises four or more cytokines, or derivatives thereof, having a sequence of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the four or more cytokines, or derivatives thereof, comprise an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the fusion proteinAttomey Docket No. 53531-724602comprises one or more cytokines, or derivatives thereof, having a sequence of any one of the sequences listed in Table IL. In some embodiments, the fusion protein comprises two or more cytokines, or derivatives thereof, having a sequence of any one of the sequences listed in Table IL. In some embodiments, the fusion protein comprises three or more cytokines, or derivatives thereof, having a sequence of any one of the sequences listed in Table IL. In some embodiments, the fusion protein comprises four or more cytokines, or derivatives thereof, having a sequence of any one of the sequences listed in Table IL. In some embodiments, a fusion protein described herein is encoded by an ORF comprised by a non-naturally occurring polynucleotide described herein, wherein the ORF is expressed under the transcriptional control of an SRS described herein, and wherein the fusion protein is expressed from the non-naturally occurring polynucleotide as an intact protein. In some embodiments, a chimeric protein described herein is encoded by an ORF comprised by a non-naturally occurring polynucleotide described herein, wherein the ORF is expressed under the transcriptional control of an SRS described herein, and wherein the chimeric protein is expressed from the non-naturally occurring polynucleotide as an intact protein. In some embodiments, said fusion protein comprises one or more cleavable domains, wherein said fusion protein is configured to be cleaved into individual peptide subunits of said fusion protein. In some embodiments, said chimeric protein comprises one or more cleavable domains, wherein said fusion protein is configured to be cleaved into individual peptide subunits of said fusion protein. In some embodiments, the one or more cleavable domains of the fusion protein, the chimeric protein, or both comprises a proteolytic-sensitive cleavage amino acid sequence. In some embodiments, the one or more cleavable domains of the fusion protein, the chimeric protein, or both are cleaved via proteolysis.

[0285] In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 556, operably linked to a nucleic acid sequence encoding IL- 12, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-700, or variants thereof. In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least aboutAttorney Docket No. 53531-72460282%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 556, operably linked to a nucleic acid sequence encoding IL-2, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 702 or 709, or variants thereof. In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 556, operably linked to a nucleic acid sequence encoding IL-7, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NO: 1014, or variants thereof. In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 556, operably linked to a nucleic acid sequence encoding IL-21, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least aboutAttomey Docket No. 53531-72460294%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NO: 1015, or variants thereof.

[0286] In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 424, operably linked to a nucleic acid sequence encoding IL- 12, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 687-700, or variants thereof. In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 424, operably linked to a nucleic acid sequence encoding IL-2, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 702 or 709, or variants thereof. In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 424, operably linked to a nucleic acid sequence encoding IL-7, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acidAttomey Docket No. 53531-724602sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NO: 1014, or a variant thereof. In some embodiments, a non-naturally occurring polypeptide described herein comprises an SRS comprising a nucleic acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to SEQ ID NO: 424, operably linked to a nucleic acid sequence encoding IL-21, or a variant thereof, wherein the expression product of the nucleic acid sequence under the control of the SRS comprises an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NO: 1015, or a variant thereof.

[0287] In some embodiments, the fusion protein comprises a fragment of a cytokine. In some embodiments, the fusion protein comprises at least one to at least four fragments of a cytokine. In some embodiments, the fusion protein comprises at least one fragment of a cytokine. In some embodiments, the fusion protein comprises at least two fragments of a cytokine. In some embodiments, the fusion protein comprises at least three fragments of a cytokine. In some embodiments, the fusion protein comprises at least four fragments of a cytokine. In some embodiments, the fragment is a truncated polypeptide of the cytokine. In some embodiments, the truncated polypeptide is a binding region of the cytokine. In some embodiments, the fusion protein comprises at least one binding region of at least one cytokine. In some embodiments, the fusion protein comprises at least two binding regions of the at least one cytokine. In some embodiments, the fusion protein comprises at least three binding regions of at least one cytokine. In some embodiments, the fusion protein comprises at least four or more binding regions of the at least one cytokine. In some embodiments, the at least one cytokine comprises at most about 50 to at most about 500 consecutive residues of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the at least one cytokine comprises at most about 50, at most about 55, at most about 60, at most about 65, at most about 70, at most about 75, at most about 80, at most about 85, at most about 90, at most about 100, at most about 120, at most about 140, at most about 160, at most about 180, at most about 200, at most about 250, at most about 300, at most aboutAttomey Docket No. 53531-724602350, at most about 400, at most about 450, or at most about 500 consecutive residues, of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof. In some embodiments, the at least one cytokine comprises all consecutive residues of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof

[0288] In some embodiments, a polypeptide product is expressed from a non-naturally occurring polynucleotide described herein. In some embodiments, the polypeptide product comprises a fusion protein described herein. In some embodiments, the polypeptide product comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 715, 731, 734, 737, 740, 746, 752, 774, 777, 781, 784, 787, 805, 808, 811, 814, 817, 820, 830, 848, 851, 860, 863, 866, 869, 878, 893, 896, 905, 908, 917, 920, 924, 927, 930, 933, 935, 938, 941, 944, 947, 950, 953, 954, 957, 960, 969, 970, 977, 978, 985, 991, or 1000. In some embodiments, the polypeptide product is encoded by a nanoplasmid vector comprising a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 713, 278-279, 281-285, 289-293, 301-305, 310-312, 315-319, 324-326, 328-335, 342-343, 710-711, 727, 729, 730-731, 733, 736, 739, 742, 745, 748, 751, 757, 761, 764, 767, 770, 773, 776, 779, 782, 785, 788, 791, 794, 797, 800, 803, 806, 809, 812, 815, 818, 825, 828, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 876, 891, 894, 903, 906, 912, 915, 918, 921, 922, 925, 928, 931, 934, 936, 939, 942, 945, 948, 951, 955, 958, 961, 967, 975, 983, 989, 998, 1001-1004, 1006, or 1008.

[0289] In some embodiments, a therapeutic protein described herein comprises a fusion protein. In some embodiments, the fusion protein comprises a collagen binding domain. In some embodiments, the collagen binding domain is a leukocyte-associated immunoglobulin-like receptor- 1 (LAIR) domain. In some embodiments, a fusion protein described herein comprises a cleavable domain. In some embodiments, the cleavable domain is a furin cleavable domain. In some embodiments, the cleavable domain is a Thosea asigna virus 2A (T2A) domain. In some embodiments, the furin cleavable domain comprises a T2A domain (FurT2A). In some embodiments, the fusion protein comprises a PDL1 antigen binding moiety. In some embodiments, the PDL1 antigen binding moiety is an anti-PDLl antibody or functional fragment thereof. In some embodiments, the fusion protein comprises an Arg-Gly-Asp (RGD) peptide motif. In some embodiments, the fusion protein comprisesAttomey Docket No. 53531-724602an CD8 antigen binding domain. In some embodiments, the CD8 antigen binding domain is an anti-CD8 antibody or functional fragment thereof. In some embodiments, the fusion protein comprises an albumin domain or variant thereof. In some embodiments, the albumin domain is human serum albumin (HSA) or a variant thereof. In some embodiments, the fusion protein comprises a PD-1 antigen binding domain. In some embodiments, the PD-1 antigen binding domain is an anti -PD-1 antibody or fragment thereof.

[0290] In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-7 or a variant thereof, and IL-21 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof, and a LAIR domain or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL- 12 or a variant thereof, a LAIR domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminal, IL-12 or a variant thereof, a first LAIR domain or a variant thereof, a T2A domain or variant thereof, a second LAIR domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof, and an anti-PDL 1 antibody or functional fragment thereof or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a first IL-12 or a variant thereof, and a second IL- 12 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a first IL-12 or a variant thereof, a first LAIR domain or a variant thereof, a second LAIR domain or a variant thereof, and a second IL- 12 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a LAIR domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof, a first LAIR domain or a variant thereof, a second LAIR domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-21 or a variant thereof, and IL-7 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-7 or a variant thereof, a FurT2A domain or a variant thereof, and IL-21 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-21 or a variant thereof, a FurT2A domain or a variant thereof, and IL-7 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, an RGD motif and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof and an RGD motif. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof, an RGD motif, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus,Attorney Docket No. 53531-724602IL- 12 or a variant thereof, a FurT2A or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-2 or a variant thereof, a FurT2A or a variant thereof, and IL- 12 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a LAIR domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N-to C-terminus, an RGD domain and IL- 12 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof, IL-7 or a variant thereof, and IL-21 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-7 or a variant thereof, IL-21 or a variant thereof, and IL-12 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-7 or a variant thereof, IL-21 or a variant thereof, IL- 12 or a variant thereof, and a LAIR domain or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, IL-12 or a variant thereof, a human serum albumin (HSA) domain or variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, an anti-CD8 antibody or fragment thereof, IL-12 or a variant thereof, HSA domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, an anti-PD-1 antibody or a fragment thereof, IL- 12 or a variant thereof, an HSA domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a first anti-PD-1 antibody or a fragment thereof, IL- 12 or a variant thereof, a second anti-PD-1 antibody or a fragment thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a first anti-PD-1 antibody or a fragment thereof, IL-12 or a variant thereof, a T2A domain or a variant thereof, a second anti-PD-1 antibody or a fragment thereof, and IL-21 or a fragment thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, an anti-PD-1 antibody or a fragment thereof, IL-12 or a variant thereof, an HSA domain or a variant thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a first anti-PD-1 antibody or fragment thereof, IL- 12 or a variant thereof, a second anti-PD-1 antibody or fragment thereof, and IL-21 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a first anti-PD-1 antibody or fragment thereof, IL- 12 or a variant thereof, a T2A domain, a second anti-PD-1 antibody or fragment thereof, and IL-2 or a variant thereof. In some embodiments, a fusion protein described herein comprises, from N- to C-terminus, a first anti-PD-1 antibody or fragment thereof, IL- 12 or a variant thereof, a T2A domain, a second anti-PD-1 antibody or fragment thereof, and IL-21 or a variant thereof. In some embodiments, a therapeutic protein described herein encoded in an ORF (e.g., operably linked to any of the promoters described herein) is one or moreAttorney Docket No. 53531-724602cytokine or cytokine receptor agonists fused to one or more antibody domains, or antibody fragment domains.

[0291] In some embodiments, a therapeutic protein disclosed herein encoded in an ORF (e.g., operably linked to any of the promoters described herein) comprises an antibody or a fragment thereof. In some embodiments, the antibody or the fragment of the antibody comprises an scFv, an Fc, a F(ab’)2, a minibody, a diabody, a heavy chain only antibody, a heavy chain variable region, a light chain only antibody, a light chain variable region, a VHH, a fynomer, a anticalin, or a DARPin. In some embodiments, a fusion protein described herein can comprise an antibody or a fragment thereof. In some embodiments, the antibody, or a fragment thereof, is a bi-specific antibody comprising two antibody or antibody fragment domains. In some embodiments, the antibody, or fragment thereof, is a tri-specific antibody comprising three antibody or antibody fragment domains. In some embodiments, the antibody, or a fragment thereof, is a T cell engager. In some embodiments, the antibody, or fragment thereof, is a bi-specific T cell engager (BiTE) comprising two antibody or antibody fragment domains. In some embodiments, the antibody, or fragment thereof, is a tri-specific T cell engager (TriTE) comprising three antibody or antibody fragment domains. In some embodiments the antibody or fragment thereof is a T cell engager with 4 domains. In some embodiments, the antibody or fragment thereof is a T cell engager with 5 antibody or antibody fragment domains. In some embodiments, the bi-specific antibody binds a check point receptor and a tumor associated antigen. In some embodiments, the tri-specific antibody binds one or more check point receptors and one or more tumor associated antigens. In some embodiments, the BiTE binds a T cell receptor and a tumor associated antigen. In some embodiments, the TriTE binds a T cell receptor, a check point receptor, a tumor associated antigen, or any combination thereof. In some embodiments, a fusion protein described herein can be expressed as a BiTE or a TriTE. In some embodiments, a BiTE or a TriTE described herein can bind, for example, one or more tumor-associated antigens comprising of a BCMA antigen, a GPRC5D antigen, a DLL3 antigen, a CD 19 antigen, a CD20 antigen, and a T cell receptor comprising, for example, one or more of CD3, CD8, or PD-1. In some embodiments, a BiTE described herein comprises blinatumomab, teclistamab, talquetamab, epcoritamab, glofitamab, mosunetuzumab, or tarlatamab. In some embodiments, a TriTE described herein comprises SIM0500.

[0292] Table IL: Cytokine amino acid sequencesAtorney Docket No. 53531-724602

[0293] In some embodiments, a therapeutic protein encoded in an ORF (e.g., operably linked to any of the promoters described herein) is an immune cell activator. In some embodiments, the immune cell activator is a T cell activator and / or a natural killer (NK) cell activator. In some embodiments, the immune cell activator comprises an antibody or antigen-binding fragment(s) thereof configured to bind any one of the tumor associated antigens listed in Table IM. In some embodiments, the immune cell activator is configured to bind an amino acid sequence of any one of SEQ ID NOs: 622-636 or a variant thereof. In some embodiments, the immune cell activator is configured to bind an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IM. In some embodiments, the immune cell activator is configured to bind an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, atAttorney Docket No. 53531-724602least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 622-636 or a variant thereof.

[0294] Table IM: Targets of Immune cell activators

[0295] In some embodiments, a therapeutic protein encoded in an ORF (e.g. , operably linked to any of the promoters described herein) comprises an antibody or antigen-binding fragment(s) thereof configured to bind tumor associated antigen. In some embodiments, the tumor associated antigen has an amino acid sequence of any one of the tumor associated antigens listed in Table IN. In some embodiments, the antibody or a functional fragment or derivative thereof is configured to bind an amino acid sequence of any one of SEQ ID NOs: 637-675 or a variant thereof. In some embodiments, the tumor associated antigen, comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IN. In some embodiments, the antibody or antigen-binding fragment(s) thereof configured to bind is an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, atAttorney Docket No. 53531-724602least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 637-675 or a variant thereof.

[0296] Table IN: Targets of Tumor associated antigen bindersAttorney Docket No. 53531-724602

[0297] In some embodiments, a therapeutic protein disclosed herein encoded in an ORF (e.g., operably linked to any of the promoters described herein) comprises an antibody or antigen-binding fragment(s) thereof configured to bind a tumor associated antigen and / or an immune cell activator. In some embodiments, a therapeutic protein disclosed herein comprises antibody or antigen-binding fragment(s) thereof configured to bind a tumor associated antigen and immune cell activator. In some embodiments antibody or antigen-binding fragment(s) thereof configured to bind a tumor associated antigen and immune cell activator is a multispecific antibody (e.g., a bi-, tri-, or quad-specific antibody). In some embodiments, the therapeutic protein comprises: (i) an antibody or antigenbinding fragment(s) thereof configured to bind an immune cell activator in Table IM or any one of SEQ ID NOs: 622-636 or a variant thereof; and (ii) an antibody or antigen-binding fragment(s) thereof configured to bind a tumor associated antigen in Table IN or any one of SEQ ID NOs: 637-666 or a variant thereof.

[0298] In some embodiments, disclosed herein is a protein binding element, or derivative thereof, directed against any of the targets listed in Table IM and / or Table IN.

[0299] In some embodiments, a therapeutic protein disclosed herein encoded in an ORF (e.g., operably linked to any of the promoters described herein) comprises an antibody or antigen-binding fragment(s) thereof comprising any of the sequences listed in Table IO. In some embodiments, the protein binding element comprises any one of SEQ ID NOs: 667-686 or a variant thereof. In some embodiments, the protein binding element comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of the of the sequences listed in Table IO. In some embodiments, the protein binding element comprises an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% sequence identity to any one of SEQ ID NOs: 667-686 or a variant thereof.Attorney Docket No. 53531-724602

[0300] Table IO: Protein binding element amino acid sequencesAtorney Docket No. 53531-724602Attorney Docket No. 53531-724602D. Cancer or tumor cells described herein

[0301] Described herein is a method of selectively expressing a protein in a target cell or target tissue. The target cell or target tissue can be a diseased cell or a diseased tissue. In some embodiments, the target cell is a cancer or tumor cell. In some embodiments, the method can comprise contacting cancer or tumor cells with a non-naturally occurring polynucleotide comprising any SRS described herein that comprises a promoter or a core promoter, one or more SREs, and an open reading frame (ORF) encoding a protein. In some embodiments, the ORF can be operatively linked to the SRS or the promoter (or the core promoter) in the SRS. In some embodiments, cancer or tumor cells described herein can comprise malignant cancer cells. Examples of cancer or tumor cells include, but are not limited to, colorectal cancer (CRC) cells, hepatocellular carcinoma cells, breast cancer cells, or lung cancer cells. In some embodiments, cancer or tumor cells can comprise cancer or tumor cells associated with colorectal cancer (CRC), hepatocellular carcinoma, lung cancer, liver cancer, breast cancer, prostate cancer, cervix cancer, uterus cancer, pancreas cancer, kidney cancer, stomach cancer, bladder cancer, ovary cancer, brain cancer, head and neck cancer, eye cancer, mouth cancer, throat cancer, esophagus cancer, chest cancer, bone cancer, rectum or other gastrointestinal tract organ cancer, spleen cancer, skeletal muscle cancer, subcutaneous tissue cancer, testicles or other reproductive organ cancer, skin cancer, thyroid cancer, blood cancer, lymph nodes cancer, or any combination of one or more thereof. In some embodiments, adenocarcinoma (LU AD) cells can comprise LXFA586, LXFA629, LXFA2184, or A549. In some embodiments, large cell carcinoma cells can comprise H1299, LXFL430, LXFL1121, or LXFL529. In some embodiments, squamous cell carcinoma (LUSC) cells can comprise LK2, H520, H1703, SK-MES-1, or Calu-1. In some embodiments, hepatocellular carcinoma (HCC) cells can comprise HUH7.Attorney Docket No. 53531-724602

[0302] In some embodiments, the target cell is a metastatic cancer cell or tumor cell. In some embodiments, the metastatic cancer cell or tumor cell is metastatic from a primary cancer cell or tumor. In some embodiments, the primary cancer cell or tumor comprises cancer or tumor cells associated with colorectal cancer (CRC), hepatocellular carcinoma, lung cancer, liver cancer, breast cancer, prostate cancer, cervix cancer, uterus cancer, pancreas cancer, kidney cancer, stomach cancer, bladder cancer, ovary cancer, brain cancer, head and neck cancer, eye cancer, mouth cancer, throat cancer, esophagus cancer, chest cancer, bone cancer, rectum or other gastrointestinal tract organ cancer, spleen cancer, skeletal muscle cancer, subcutaneous tissue cancer, testicles or other reproductive organ cancer, skin cancer, thyroid cancer, blood cancer, lymph nodes cancer, or any combination of one or more thereof. In some embodiments, the metastatic cancer cell or tumor cell is a lung metastatic cancer cell or tumor cell.

[0303] In some embodiments, promoters active in LXFA586 cell lines can comprise promoters of TP53, HES6, FOS, FOS-CREB, FOXO1::ELK3, or MTF1. In some embodiments, promoters active in LXFA629 cell lines can comprise promoters of FOS, CREB3L1, or HES6. In some embodiments, promoters active in LXFA2184 cell lines can comprise promoters of FOS or MNX. In some embodiments, promoters active in H1299 cell lines can comprise promoters of FOS, CREB3L1, HES6, FOS-CREB, NFE2L2, FOXO1::ELK3, or XBP1. In some embodiments, promoters active in LXFL430 cell lines can comprise promoters of TCF7, ETV4, HOXCIO, FOS-CREB, FOXO1::ELK3, or XBP1. In some embodiments, promoters active in LXFL1121 cell lines can comprise promoters of FOS, CREB3L1, or ETV4. In some embodiments, promoters active in LXFL529 cell lines can comprise promoters of FOS.

[0304] In some embodiments, expression of the protein encoded by the ORF may be increased in cancer cells compared to non-cancer cells. In some embodiments, expression of the protein encoded by the ORF may be increased when the non-naturally occurring polynucleotide comprising the SRS and the ORF is introduced to cancer cells compared to non-cancer cells. For example, expression of the protein encoded by the ORF may be increased at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, at least about 160%, at least about 170%, at least about 180%, at least about 190%, at least about 200%, or at least about 250% in cancer cells compared to non-cancer cells. In some embodiments, the ORF can comprise a sequence encoding a therapeutic protein, marker protein (e.g., for diagnostic imaging,Attorney Docket No. 53531-724602etc.), or a reporter protein (e.g., luciferase). In some embodiments, the ORF can comprise a sequence encoding a recombinant, synthetic, or engineered protein.

[0305] In some embodiments, expression of the protein encoded by the ORF may be increased in a first plurality of cancer cells when said non-naturally occurring polynucleotide is introduced to the first plurality of cancer cells compared to a second plurality of cancer cells, wherein the first plurality of cancer cells and the second plurality of cancer cells are different types of cancer cells. In some embodiments, expression of the protein encoded by the ORF may be increased in a first plurality of cancer cells when the non-naturally occurring polynucleotide comprising the SRS and the ORF is introduced to the first plurality of cancer cells compared to a second plurality of cancer cells, wherein the first plurality of cancer cells and the second plurality of cancer cells are different types of cancer cells. For example, expression of the protein encoded by the ORF operatively linked to a first type of SRS in the non-naturally occurring polynucleotide may be increased in cells of one type of cancer in which the first type of SRS can drive expression of the ORF compared to in cells of another type of cancer in which the first type of SRS cannot drive expression of the ORF. For example, expression of the protein encoded by the ORF operatively linked to an SRS that is specific for lung cancer may be increased in lung cancer cells compared to in liver cancer cells.

[0306] In some embodiments, expression of the protein encoded by the ORF may be increased in a first plurality of cancer cells comprising two or more types of cancer cells when the non-naturally occurring polynucleotide comprising the SRS and the ORF is introduced to the first plurality of cancer cells compared to a second plurality of cancer cells. For example, expression of the protein encoded by the ORF operatively linked to a first type of SRS in the non-naturally occurring polynucleotide may be increased in cells of two or more types of cancer in which the first type of SRS can drive expression of the ORF compared to in cells of another type of cancer in which the first type of SRS cannot drive expression of the ORF. For example, expression of the protein encoded by the ORF operatively linked to an SRS that is specific for lung and liver cancer may be increased in lung cancer cells and liver cancer cells compared to in non-lung cancer cells and non-liver cancer cells (e.g., breast cancer cells, etc.). In some embodiments, the first plurality of cancer cells comprising two or more types of cancer cells can comprise cells associated with two or more cancers comprising colorectal cancer, hepatocellular carcinoma, lung cancer, liver cancer, breast cancer, prostate cancer, cervix cancer, uterus cancer, pancreas cancer, kidney cancer, stomach cancer, bladder cancer, ovary cancer, brain cancer, head and neck cancer, eye cancer, mouth cancer, throat cancer, esophagus cancer, chest cancer, bone cancer, rectum or other gastrointestinal tract organ cancer, spleen cancer, skeletal muscle cancer, subcutaneous tissue cancer, testicles or other reproductive organ cancer, skin cancer, thyroid cancer, blood cancer, lymph nodes cancer, or any combination of one or more thereof.Attorney Docket No. 53531-724602E. Pharmaceutical Formulations

[0307] Provided herein are pharmaceutical compositions comprising any non-naturally occurring polynucleotide described herein or any vector comprising the non-naturally occurring polynucleotide described herein and a pharmaceutically acceptable excipient, carrier, diluents, or any combination thereof. A pharmaceutical composition can denote a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients to be administered to a subject in need thereof. The term “pharmaceutically acceptable” can denote an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use. The term “Pharmaceutically acceptable” can refer to a material, such as an excipient, carrier, diluents, or any combination thereof, which does not abrogate the biological activity or properties of the non-naturally occurring polynucleotide or the compound, and is relatively nontoxic, i. e. , the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. A pharmaceutically acceptable excipient can denote any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fdlers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives, or lubricants used in formulating pharmaceutical products. Pharmaceutical compositions can facilitate administration of a non-naturally occurring polynucleotide, a vector comprising a non-naturally occurring polynucleotide, or a compound to an organism and can be formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. A proper formulation is dependent upon the route of administration chosen and a summary of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins 1999), herein incorporated by reference. In some embodiments, pharmaceutical compositions can be formulated by dissolving active substances (e.g., non-naturally occurring polynucleotides or vectors comprising the non-naturally occurring polynucleotides described herein) in aqueous solution for administration into a cell, a tissue, or a subject (e.g., a disease cell, disease tissue, or a subject in need thereof). In some embodiments, pharmaceutical compositions can be formulated by dissolving active substances (e.g., non-naturally occurringAttorney Docket No. 53531-724602polynucleotides or vectors comprising the non-naturally occurring polynucleotides described herein) in aqueous solution for administration into a cell, a tissue, or a subject (e.g, a disease cell, disease tissue, or a subject in need thereof).

[0308] Provided herein are non-naturally occurring polynucleotides (or any vector, pharmaceutical composition, or lipid nanoparticle comprising any non-naturally occurring polynucleotides described herein) useful for the diagnosis or the treatment of a disease or condition. In some aspects, non-naturally occurring polynucleotides described herein (or any vector, pharmaceutical composition, or lipid nanoparticle comprising any non-naturally occurring polynucleotides described herein) are present or administered in an amount for sufficient expression of a protein (e.g. , a reporter protein or a biomarker) useful for a diagnosis of a disease or condition. In some embodiments, the disease or condition comprise a cancer. In some aspects, provided herein is a method of selectively expressing a reporter protein or a biomarker in a cancer or tumor cell. In some aspects, the method comprises contacting a tumor cell with any of non-naturally occurring polynucleotides described herein, any of vectors comprising non-naturally occurring polynucleotide described herein, any of pharmaceutical composition comprising non-naturally occurring polynucleotide described herein, or any of lipid nanoparticle (LNP) comprising the non-naturally occurring polynucleotide, the vector, or the pharmaceutical composition described herein, wherein non-naturally occurring polynucleotides can comprise an open reading frame (ORF) encoding the reporter protein or the biomarker operatively linked to a synthetic promoter described herein (e.g., a synthetic promoter that can drive expression of the ORF preferentially or specifically in cancer cells).

[0309] In some aspects, provided herein is a method for diagnosing a disease or a condition. In some embodiments, the method can comprise administering to any of non-naturally occurring polynucleotide described herein, a vector comprising the non-naturally occurring polynucleotide described herein, the pharmaceutical composition comprising the non-naturally occurring polynucleotide described herein, or a lipid nanoparticle (LNP) comprising the non-naturally occurring polynucleotide, the vector, or the pharmaceutical composition described herein to a subject. In some embodiments, the non-naturally occurring polynucleotide can further comprise an open reading frame (ORF) encoding a reporter protein or a biomarker, wherein the ORF is operatively linked to a synthetic promoter in the non-naturally occurring polynucleotide that can drive expression of the ORF selectively, preferentially, or specifically in diseased cells compared to non-disease cells. In some embodiments, the method can further comprise detecting the reporter protein or a biomarker of which expression can be induced by a synthetic promoter in the non-naturally occurring polynucleotide described herein selectively, preferentially, or specifically in diseased cells compared to non-disease cells. In some embodiments, a relative ratio of the reporter protein or the biomarker expressed in the diseased cells over the non-diseased cells can be greater than 1.0. For example, a relative ratio of the reporter protein or the biomarker expressed in the diseased cells over the non-diseased cells can beAttorney Docket No. 53531-724602greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, or about 100.0. In some embodiments, the disease or condition can comprise a cancer.

[0310] In some aspects, non-naturally occurring polynucleotides (or any vector, pharmaceutical composition, or lipid nanoparticle comprising any non-naturally occurring polynucleotides described herein) are present or administered in an amount sufficient to treat or prevent a disease or condition. In some aspects, provided herein, is a method of treating a disease or condition comprising administering to a subject in need thereof the non-naturally occurring polynucleotide described herein, a vector comprising the non-naturally occurring polynucleotide described herein, a pharmaceutical composition comprising the non-naturally occurring polynucleotide described herein, or a lipid nanoparticle (LNP) comprising the vector, the pharmaceutical composition or the non-naturally occurring polynucleotide described herein. In some aspects, provided herein, is non-naturally occurring polynucleotide described herein, a vector comprising the non-naturally occurring polynucleotide described herein, the pharmaceutical composition comprising the non-naturally occurring polynucleotide described herein, or a lipid nanoparticle (LNP) comprising the non-naturally occurring polynucleotide, the vector, or the pharmaceutical composition described herein for use in a method of treating a disease or a condition in a subject in need thereof. In some aspects, provided herein, is the use of non-naturally occurring polynucleotide described herein, a vector comprising the non-naturally occurring polynucleotide described herein, the pharmaceutical composition comprising the non-naturally occurring polynucleotide described herein, or a lipid nanoparticle (LNP) comprising the non-naturally occurring polynucleotide, the vector, or the pharmaceutical composition described herein for the manufacture of a medicament for treating a disease or a condition in a subject in need thereof.II. KITS

[0311] Disclosed herein, are kits useful for to detection of cancer or tumor cells described herein using any of the non-naturally occurring polypeptides described herein. In some embodiments, the kits disclosed herein may be used to diagnose and / or treat a disease or condition in a subject; or select a patient for treatment and / or monitor a treatment disclosed herein. In some embodiments, the kit comprises the compositions described herein, which can be used to perform the methods described herein. Kits comprise an assemblage of materials or components, including at least one of the compositions. Thus, in some embodiments the kit contains a composition including of theAttorney Docket No. 53531-724602pharmaceutical composition, for the treatment of a cancer. In other embodiments, the kits contain all of the components necessary and / or sufficient to perform an assay for detecting and measuring cancer or tumor cells, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.

[0312] In some instances, the kits described herein comprise components for detecting the presence, absence, and / or quantity of a cancer or tumor cells described herein. In some embodiments, the kit comprises the compositions (e.g., primers, probes, antibodies) described herein. The disclosure provides kits suitable for assays such as enzyme-linked immunosorbent assay (ELISA), single-molecular array (Simoa), PCR, and qPCR. The exact nature of the components configured in the kit depends on its intended purpose.

[0313] In some embodiments, the kits described herein are configured for the purpose of treating and / or characterizing a disease or condition (e.g., cancer) in a subject. In some embodiments, the kit is configured particularly for the purpose of treating mammalian subjects. In some embodiments, the kit is configured particularly for the purpose of treating human subjects. In further embodiments, the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals. In some embodiments, the kit is configured to select a subject for a therapeutic agent, such as those disclosed herein. In some embodiments, the kit is configured to select a subject for treatment with a therapeutic agent disclosed herein.

[0314] Instructions for use may be included in the kit. Optionally, the kit also contains other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, bandaging materials, or other useful paraphernalia. The materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility. For example, the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated or frozen temperatures. The components are typically contained in suitable packaging material(s). As employed herein, the phrase “packaging material” refers to one or more physical structures used to house the contents of the kit, such as compositions and the like. The packaging material is constructed by well-known methods, preferably to provide a sterile, contaminant-free environment. The packaging materials employed in the kit are those customarily utilized in gene expression assays and in the administration of treatments. As used herein, the term “package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components. Thus, for example, a package can be a glass vial, or prefilled syringes used to contain suitable quantities of the pharmaceutical composition. The packaging material has an external label which indicates the contents and / or purpose of the kit and its components.Attorney Docket No. 53531-724602III. METHODS1. Therapeutic or Diagnostic Applications

[0315] In some aspects, provided herein is a method for treating a subject having or suspected of having a disease or a condition. In some embodiments, the method can comprise administering any of non-naturally occurring polynucleotide described herein, a vector comprising the non-naturally occurring polynucleotide described herein, the pharmaceutical composition comprising the non-naturally occurring polynucleotide described herein, or a lipid nanoparticle (LNP) comprising the non-naturally occurring polynucleotide, the vector, or the pharmaceutical composition described herein to a subject. In some embodiments, the non-naturally occurring polynucleotide can further comprise an open reading frame (ORF) encoding a therapeutic protein, wherein the ORF is operatively linked to a synthetic promoter in the non-naturally occurring polynucleotide that can drive expression of the ORF selectively, preferentially, or specifically in diseased cells compared to nondisease cells. In some embodiments, a relative ratio of the therapeutic protein expressed in the diseased cells over the non-diseased cells can be greater than 1.0. For example, a relative ratio of the therapeutic protein expressed in the diseased cells over the non-diseased cells can be greater than about 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, or about 15.0.

[0316] In some embodiments, the disease or disorder can comprise a cancer. Examples of cancer can include, but are not limited to, colorectal cancer (CRC), hepatocellular carcinoma, breast cancer, lung cancer, liver cancer, prostate cancer, cervix cancer, uterus cancer, pancreas cancer, kidney cancer, stomach cancer, bladder cancer, ovary cancer, brain cancer, head and neck cancer, eye cancer, mouth cancer, throat cancer, esophagus cancer, chest cancer, bone cancer, rectum or other gastrointestinal tract organ cancer, spleen cancer, skeletal muscle cancer, subcutaneous tissue cancer, testicles or other reproductive organ cancer, skin cancer, thyroid cancer, blood cancer, lymph nodes cancer, or any combination of one or more thereof. In some embodiments, the cancer can comprise a metastatic cancer. In some embodiments, the metastatic cancer is metastatic from a primary cancer cell or tumor. In some embodiments, the primary cancer cell or tumor comprises cancer or tumor cells associated with colorectal cancer (CRC), hepatocellular carcinoma, lung cancer, liver cancer, breast cancer, prostate cancer, cervix cancer, uterus cancer, pancreas cancer, kidney cancer, stomach cancer, bladder cancer, ovary cancer, brain cancer, head and neck cancer, eye cancer, mouth cancer, throat cancer, esophagus cancer, chest cancer, bone cancer, rectum or other gastrointestinal tract organ cancer, spleen cancer, skeletal muscle cancer, subcutaneous tissue cancer, testicles or other reproductiveAttomey Docket No. 53531-724602organ cancer, skin cancer, thyroid cancer, blood cancer, lymph nodes cancer, or any combination of one or more thereof. In some embodiments, the metastatic cancer is a lung metastatic cancer.

[0317] Also provided herein are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any non-naturally occurring polynucleotide described herein, any vector comprising the non-naturally occurring polynucleotide described herein, or pharmaceutical compositions described herein. The terms “effective amount” or “therapeutically effective amount,” as used herein, can refer to a sufficient amount of an agent, a compound, any non-naturally occurring polynucleotide described herein, any vector comprising non-naturally occurring polynucleotide described herein, or pharmaceutical compositions described herein being administered which will relieve to some extent one or more of the symptoms of the disease or the condition being treated; for example a reduction and / or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses can be an amount of an agent that provides a clinically significant decrease in one or more disease symptoms. An appropriate “effective” amount may be determined using techniques, such as a dose escalation study, in individual cases. In some embodiments, an “effective amount” can comprise an amount for sufficient expression of a protein (e.g., a reporter protein or a biomarker) useful for diagnosing a disease or condition in a subject.

[0318] The terms “treat,” “treating” or “treatment,” as used herein, can include alleviating, abating or ameliorating at least one symptom of a disease or a condition, preventing additional symptoms, inhibiting the disease or the condition, e.g., arresting the development of the disease or the condition, relieving the disease or the condition, causing regression of the disease or the condition, relieving a condition caused by the disease or the condition, or stopping the symptoms of the disease or the condition either prophylactically and / or therapeutically. In some embodiments, treating a disease or condition comprises reducing the size of disease tissues or diseased cells. In some embodiments, treating a disease or a condition in a subject comprises increasing the survival of a subject. In some embodiments, treating a disease or condition comprises reducing or ameliorating the severity of a disease, delaying onset of a disease, inhibiting the progression of a disease, reducing hospitalization of or hospitalization length for a subject, improving the quality of life of a subject, reducing the number of symptoms associated with a disease, reducing or ameliorating the severity of a symptom associated with a disease, reducing the duration of a symptom associated with a disease, preventing the recurrence of a symptom associated with a disease, inhibiting the development or onset of a symptom of a disease, inhibiting of the progression of a symptom associated with a disease, or obtaining beneficial or desired results in the recipient.

[0319] In some cases, a subject can encompass mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees,Attomey Docket No. 53531-724602and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In some cases, the mammal is a human mammal. In some cases, the subject may be a non-human mammal. In some cases, the subject may be an animal. In some cases, an animal may comprise human beings and non-human animals. In one embodiment, a non-human animal may be a mammal, for example a rodent such as rat or a mouse. In another embodiment, a non-human animal may be a mouse. In some instances, the subject is a mammal. In some instances, the subject is a human. In some instances, the subject is an adult, a child, or an infant. In some instances, the subject is a companion animal. In some instances, the subject is a feline, a canine, or a rodent. In some instances, the subject is a dog or a cat.

[0320] Non-naturally occurring polynucleotides, such as the non-naturally occurring polynucleotides described herein, vectors, or pharmaceutical compositions described herein can be administered to a subject using any suitable methods known in the art. Suitable formulations for use in the present invention and methods of delivery are generally well known in the art. For example, compositions described herein can be administered to the subject in a variety of ways, including parenterally, intravenously, intradermally, intramuscularly, colonically, rectally, or intraperitoneally. In some embodiments, compositions described herein is administered by intraperitoneal injection, intramuscular injection, subcutaneous injection, or intravenous injection of the subject. In some embodiments, compositions described herein can be administered parenterally, intravenously, intramuscularly, or orally. In some embodiments, compositions described herein can be administered via injection into disease tissues or cells.

[0321] Provided herein are methods of delivering the nucleic acids, vectors, or pharmaceutical compositions, as described herein, to an individual in need thereof. In some embodiments, compositions or pharmaceutical compositions comprising any non-naturally occurring polynucleotide described herein can be delivered to a cell via direct DNA transfer (Wolff et al. (1990) Science 247, 1465-1468). In some embodiments, non-naturally occurring polynucleotides can be delivered to cells following mild mechanical disruption of the cell membrane, temporarily permeabilizing the cells. Such a mild mechanical disruption of the membrane can be accomplished by gently forcing cells through a small aperture (Sharei et al. PLOS ONE (2015) 10(4), eOl 18803). In another embodiment, compositions or pharmaceutical compositions comprising any non-naturally occurring polynucleotide described herein can be delivered to via liposome or lipid nanoparticle (LNP) (e.g., Gao & Huang (1991) Biochem. Ciophys. Res. Comm. 179, 280-285, Crystal (1995) Nature Med. 1, 15-17, Caplen et al. (1995) Nature Med. 3, 39-46)). A liposome or LNP can encompass a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Non-naturally occurring polynucleotides can be encapsulated in the aqueous interior of a liposome or LNP, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule thatAttorney Docket No. 53531-724602is associated with both the liposome and the oligonucleotide, entrapped in a liposome, or complexed with a liposome. In some embodiments, liposomes described herein comprise micelles, solid lipid nanoparticles, or both. In some embodiments, the nucleic acids described herein can be delivered in a vector, wherein the vector comprises an adeno-associated viral vector, a lentiviral vector, a retroviral vector, or a non-viral vector. In some embodiments, the non-viral vector is a plasmid vector. In some embodiments, the plasmid vector is a nanoplasmid vector. In some embodiments, the nucleic acids described herein can be delivered to a target cell or a tissue of the individual by a host cell, wherein the nucleic acids are carried by the host cell and delivered to the target cell or the tissue of the individual. In some embodiments, a peptide product of the nucleic acids described herein can be expressed in the host cell and delivered to the target cell or the tissue of the individual. In some embodiments, the host cell can deliver the nucleic acids or the peptide product of the nucleic acids to the target cell or the tissue of the individual. In some embodiments, the delivery of the nucleic acids or the peptide product of the nucleic acids to the target cell or the tissue of the individual is performed by delivering an exosome or vesicle of the host cell to the target cell or tissue of the individual. In some embodiments, the target cell, or a cell of the tissue endocytosis the exosome or vesicle of the host cell. In some embodiments, the host cell is an engineered cell or a natural cell. In some embodiments, the host cell is a eukaryotic cell or a prokaryotic cell. In some embodiments, the eukaryotic cell is a yeast cell or a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the prokaryotic cell is a bacterial cell. In some embodiments, the host cell is nucleated or enucleated.

[0322] In some aspects, provided herein is a method comprising: (a) administering to a subject any of the pharmaceutical composition described herein; or a composition any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, or any of the LNP described herein; wherein the non-naturally occurring polynucleotide further comprises an open reading frame (ORF) encoding a reporter protein, wherein said ORF is operatively linked to a synthetic promoter in said non-naturally occurring polynucleotide, and (b) localizing a tumor or an absence thereof in a body of said subject via expression of said reporter protein using an imaging technique performed on said body of said subject. In some embodiments, the imaging technique comprises photoacoustic imaging, Magnetic resonance imaging (MRI) imaging, positron emission tomography (PET) imaging, or single-photon emission computed tomography (SPECT) imaging.

[0323] In some aspects, provided herein is a method comprising: (a) administering to a subject any of the pharmaceutical composition described herein; or a composition any of the non-naturally occurring polynucleotide described herein, any of the vector described herein, or any of the LNP described herein; wherein the non-naturally occurring polynucleotide further comprises an open reading frame (ORF) encoding a protein, wherein said ORF is operatively linked to a synthetic promoter in said non-naturally occurring polynucleotide, (b) localizing a tumor or an absence thereof in a body of saidAttorney Docket No. 53531-724602subject via expression of said protein, and (c) killing a cancer or tumor cell of the tumor in the body of said subject via expression of said protein.Combination Therapies

[0324] Also provided herein are methods of treating a disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of any non-naturally occurring polynucleotide described herein, any vector comprising a non-naturally occurring polynucleotide described herein, or pharmaceutical compositions described herein in combination with an additional therapeutic agent or therapy. In some embodiments, the additional therapeutic agent or therapy comprises a small molecule. In some embodiments, the additional therapeutic agent or therapy comprises a pharmaceutical drug. In some embodiments, the additional therapeutic agent or therapy comprises a non-steroidal anti-inflammatory drug (NSAID). In some embodiments, nonlimiting examples of an NSAID can include acetylsalicylic acid, ibuprofen, naproxen, celecoxib, ketoprofen, indomethacin, diclofenac, meclofenamate, etodolac, or any combination thereof. In some embodiments, the additional therapeutic agent or therapy is an anti -histamine drug. In some embodiments, non-limiting examples of an anti-histamine drug can include diphenhydramine, cetirizine, loratadine, fexofenadine, or any combination thereof. In some embodiments, the additional therapeutic agent or therapy is a corticosteroid. In some embodiments, non-limiting examples of a corticosteroid can include prednisone, prednisolone, methyl prednisone, dexamethasone, hydrocortisone, betamethasone, or any combination thereof. In some embodiments, the additional therapeutic agent or therapy is a complement inhibitor. In some embodiments, non-limiting examples of a complement inhibitor can include a C5 inhibitor, a C3 inhibitor, a Cl inhibitor, a Factor B inhibitor, or any combination thereof. In some embodiments, non-limiting examples of a complement inhibitor can include eculizumab, ravulizumab, avacopan, or any combination thereof. In some embodiments, the additional therapeutic agent comprises a checkpoint inhibitor. In some embodiments, non-limiting examples of a checkpoint inhibitor can include nivolumab, pembrolizumab, cemiplimab, atezolizumab, durvalumab, avelumab, ipilimumab, or any combination thereof. In some embodiments, the checkpoint inhibitor comprises an anti-PD-1 antibody or fragment thereof.IV. SYSTEMS

[0325] Disclosed herein are systems for identifying or obtaining one or more transcriptional elements, such as core promoters, transcription factors, transcription factor binding sites, or enhancers as described herein, useful for enhancing the expression of a protein product encoded by an open reading frame operably linked to the one or more transcriptional elements in a diseased cell as compared to a corresponding healthy cell. In some embodiments, the systems described herein comprise kits and compositions for detecting or treating diseased cells, such as cancer cells or tumorAttorney Docket No. 53531-724602cells, in an individual. The system may comprise a computer system for implementing one or more methods of the disclosure, such as for example, receiving genomic sequence data comprising the one or more transcriptional elements, inputting the genomic sequence data into an algorithm, such as an Al or ML model or system described herein, to output a prediction of whether one or more transcriptional elements of the genomic sequence data may enhance expression in a diseased cell as compared to a corresponding healthy cell. The system may comprise imaging systems for detecting the expression of a protein encoded by a non-naturally occurring polypeptide described herein, in a target cell. The system may comprise bioinformatic workflows for obtaining the one or more transcriptional elements as described herein.Computer Systems

[0326] Disclosed herein, in some embodiments, are methods and systems of the present disclosure utilizing one or more computer systems. Referring to FIG. 107, a block diagram is shown depicting an exemplary machine that includes a computer system 10701 (e.g., a processing or computing system) within which a set of instructions can execute for causing a device to perform or execute any one or more of the aspects and / or methodologies for generating a non-naturally occurring polynucleotide described herein, or for detecting an expressed product of a non-naturally occurring polynucleotide described herein in a target cell . The components in FIG. 107 are examples only and do not limit the scope of use or functionality of any hardware, software, embedded logic component, or a combination of two or more such components implementing particular embodiments.

[0327] FIG. 107 shows a computer system 10701 that is programmed or otherwise configured generating a non-naturally occurring polynucleotide described herein, or for detecting an expressed product of a non-naturally occurring polynucleotide described herein in a target cell in a subject. The computer system 10701 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device, such as a mobile electronic device belonging to a physician. The computer system 10701 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 10705, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 10701 also includes memory or memory location 10710 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 10715 (e.g., hard disk), communication interface 10720 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 10725, such as cache, other memory, data storage and / or electronic display adapters. The memory 10710, storage unit 10715, interface 10720 and peripheral devices 10725 are in communication with the CPU 10705 through a communication bus (solid lines), such as a motherboard. The storage unit 10715 can be a data storage unit (or data repository) for storing data. The computer system 10701 can be operatively coupled to a computer network (“network”) 10730 with the aid of the communication interface 10720. The network 10730 can be the Internet, an internetAttorney Docket No. 53531-724602and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 10730 in some cases is a telecommunication and / or data network. The network 10730 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 10730, in some cases with the aid of the computer system 10701, can implement a peer-to-peer network, which may enable devices coupled to the computer system 10701 to behave as a client or a server.

[0328] The CPU 10705 can execute a sequence of machine -readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 10710. The instructions can be directed to the CPU 10705, which can subsequently program or otherwise configure the CPU 10705 to implement methods of the present disclosure. Examples of operations performed by the CPU 10705 can include fetch, decode, execute, and writeback.

[0329] The CPU 10705 can be part of a circuit, such as an integrated circuit. One or more other components of the system 10701 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0330] The storage unit 10715 can store files, such as drivers, libraries and saved programs. The storage unit 10715 can store user data, e.g., user preferences and user programs. The computer system 10701 in some cases can include one or more additional data storage units that are external to the computer system 10701, such as located on a remote server that is in communication with the computer system 10701 through an intranet or the Internet.

[0331] The computer system 10701 can communicate with one or more remote computer systems through the network 10730. For instance, the computer system 10701 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e.g., portable PC), slate or tablet PC's (e.g., Apple® Wad, Samsung® Galaxy Tab), telephones, Smart phones (e.g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 10701 via the network 10730.

[0332] Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 10701, such as, for example, on the memory 10710 or electronic storage unit 10715. The machine executable or machine readable code can be provided in the form of software. During use, the code can be executed by the processor 10705. In some cases, the code can be retrieved from the storage unit 10715 and stored on the memory 10710 for ready access by the processor 10705. In some situations, the electronic storage unit 10715 can be precluded, and machine-executable instructions are stored on memory 10710.

[0333] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in aAttomey Docket No. 53531-724602programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.

[0334] Aspects of the systems and methods provided herein, such as the computer system 10701, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machineexecutable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the s...

Claims

Attorney Docket No. 53531-724602CLAIMS WHAT IS CLAIMED IS:

1. A non-naturally occurring polynucleotide comprising:a) a core promoter operably linked to an open reading frame (ORF) comprising a gene encoding a peptide therapeutic agent, wherein said core promoter induces expression of said peptide therapeutic agent at a higher level in a cancer cell compared to a non-cancer cell;b) one or more synthetic response elements comprising one or more enhancers and one or more transcription factor binding sites; andc) a transcription start site (TSS) upstream of said ORF.

2. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter further comprises one or more promoter elements obtained from one or more genes that are either expressed at a higher level or are more active in cancer cells compared to non-cancer cells.

3. The non-naturally occurring polynucleotide of claim 2, wherein said one or more genes are obtained from a human subject.

4. The non-naturally occurring polynucleotide of claim 1, wherein said higher levels of expression or activity of said one or more genes in said cancer cell compared to said non-cancer cell is determined by chromatin immunoprecipitation (ChIP).

5. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises one or more synthetic promoter elements.

6. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises one or more promoter elements that are endogenous to said subject.

7. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises one or more promoter elements that are non-endogenous to said subject.

8. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises two or more promoter elements, wherein at least two promoter elements of said two or more promoter elements are obtained from different genes.

9. The non-naturally occurring polynucleotide of claim 1, wherein said one or more enhancers comprise a CpG island.Attorney Docket No. 53531-72460210. The non-naturally occurring polynucleotide of claim 1, wherein said one or more enhancers does not comprise a CpG island.

11. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises a fusion protein.

12. The non-naturally occurring polynucleotide of claim 11, wherein said fusion protein comprises a cytokine, a cytokine receptor agonist or fragment thereof, or a derivative thereof.

13. The non-naturally occurring polynucleotide of claim 12, wherein said cytokine or derivative thereof comprises a sequence of any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof.

14. The non-naturally occurring polynucleotide of claim 12, wherein said cytokine, said cytokine receptor agonist or fragment thereof, or derivative thereof comprises a sequence in Table IL.

15. The non-naturally occurring polynucleotide of claim 11, wherein said fusion protein comprises:a cytokine or fragment thereof, wherein said fragment of said cytokine comprises a receptor binding domain of said cytokine; orsaid cytokine and said fragment thereof.

16. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises a toxin, a cytokine, an antibody or antigen binding fragment thereof, or any combination thereof.

17. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises a cytokine.

18. The non-naturally occurring polynucleotide of claim 17, wherein said peptide therapeutic agent further comprises an antibody or antigen-binding fragment thereof.

19. The non-naturally occurring polynucleotide of claim 17, wherein said cytokine is IL-2 or a fragment thereof, IL- 12 or a fragment thereof, IL-7 or a fragment thereof, IL-21 or a fragment thereof, or any combination thereof.

20. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises a cytokine or fragment thereof, a cytokine receptor agonist orAttorney Docket No. 53531-724602fragment thereof, an immune checkpoint inhibitor or fragment thereof, an immune cell activator agonist or fragment thereof, a tumor associated antigen binding moiety, a collagen binding domain or a fragment thereof, or an integrin binding domain or a fragment thereof, or any combination thereof.

21. The non-naturally occurring polynucleotide of claim 20, wherein:a) said cytokine comprises IL-2, IL-12, IL-7, IL-21, or any combination thereof; b) said immune checkpoint inhibitor comprises an anti-Programmed Death-Ligand 1 (PD-L1) antibody or an anti- Programmed cell death protein 1 (PD-1) antibody, or any combination thereof;c) said immune cell activator agonist comprises an anti-CD3 antibody, an anti 4-1BB (CD137) antibody, an anti-OX40 (CD134) antibody, an anti-CD28 antibody, an antiinducible T cell co-stimulator (ICOS) antibody, an anti -Glucocorticoid-Induced TNFR Family Related gene (GITR), an anti-CD2 antibody, an anti-NKG2D antibody, an anti-T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3) antibody, an anti- Lymphocyte Activation Gene-3 (LAG3) antibody, an anti-CD27 antibody, an anti- Killer-cell Immunoglobulin-like Receptor (KIR) antibody, an anti- V-domain Ig Suppressor of T-cell Activation (VISTA) antibody, an anti-CD7 antibody, an anti-CD44 antibody, or any combination thereof; andd) said tumor associated antigen binding moiety bind to a tumor associated antigen comprising mesothelin, CD19, NY-ESO-1, Fibroblast Activation Protein (FAP), Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1), Melanoma Antigen Gene A3 (MAGE-A3), (Disialoganglioside)Cer-Glc-Gal(NeuAc-NeuAc)-GalNAc (GD2), GM2 / GD2 synthase (B4GALNT1), B1-H3 (CD276), CD20, Cancer Antigen 125 (CA125), Carbonic Anhydrase IX (CAIX), Tumor Endothelial Marker 8 (TEM8), Interleukin-3 Receptor Alpha Chain (CD123), Preferentially Expressed Antigen in Melanoma (PRAME), Epithelial Cell Adhesion Molecule (EPCAM), Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), AXL, Interleukin- 13 Receptor Alpha 2 (IL12Ra2), Signaling Lymphocytic Activation Molecule Family 7 (SLAMF7), or any combination thereof.

22. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises a cleavable domain.Attorney Docket No. 53531-72460223. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises at least two cytokines or fragments thereof.

24. The non-naturally occurring polynucleotide of claim 23, wherein said at least two cytokines comprise IL-2, IL-12, IL-7, IL-21, or any combination thereof.

25. The non-naturally occurring polynucleotide of claim 23, wherein said at least two cytokines thereof comprise IL-2 and IL- 12.

26. The non-naturally occurring polynucleotide of claim 23, wherein said at least two cytokines thereof comprise IL-7 and IL-21.

27. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises at least three cytokines or fragments thereof.

28. The non-naturally occurring polynucleotide of claim 27, wherein said at least three cytokines comprise IL-2, IL-12, IL-7, IL-21, or any combination thereof.

29. The non-naturally occurring polynucleotide of claim 27, wherein said at least three cytokines comprise IL-2, IL-21, IL-7, or any combination thereof.

30. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises an amino acid sequence of any one of SEQ ID NOs: 588-621, or a variant thereof.

31. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 687-702, SEQ ID NO: 709, SEQ ID NOs: 1014-1015, or a variant thereof.

32. The non-naturally occurring polynucleotide of claim 1, wherein said peptide therapeutic agent comprises an antibody or antigen binding fragment thereof comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 667-686, or a variant thereof.

33. The non-naturally occurring polynucleotide of claim 1, further comprising a spacer element comprising 1-20 nucleotides at a position comprising:a) between at least two of said one or more synthetic response elements;b) between at least two of said one or more enhancers;c) between at least two of said one or more transcription factor binding sites;Attorney Docket No. 53531-724602d) between said one or more transcription factor binding sites and said one or more enhancers; ore) any combination thereof.

34. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises two or more promoter elements obtained from two or more of CEACAM5, CEP55, FAM111B, CST1, BIRC5, AGR2, UBE2C, KIF20A.

35. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises two or more promoter elements obtained from FAM11 IB and KIF20A.

36. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises a region of said non-naturally occurring polynucleotide that is from about -300 bp to about +100 bp relative to said TSS.

37. The non-naturally occurring polynucleotide of claim 1, wherein a synthetic response element of said one or more synthetic response elements is 5’ to said core promoter in said non-naturally occurring polynucleotide.

38. The non-naturally occurring polynucleotide of claim 1, wherein said cancer cell is a colorectal cancer cell, a hepatocellular carcinoma cell, a lung cancer cell, a liver cancer cell, a breast cancer cell, a prostate cancer cell, a cervix cancer cell, a uterus cancer cell, a pancreas cancer cell, a kidney cancer cell, a stomach cancer cell, a bladder cancer cell, an ovary cancer cell, a brain cancer cell, a head and neck cancer cell, an eye cancer cell, a mouth cancer cell, a throat cancer cell, an esophagus cancer cell, a chest cancer cell, a bone cancer cell, a rectum or other gastrointestinal tract organ cancer cell, a spleen cancer cell, a skeletal muscle cancer cell, a subcutaneous tissue cancer cell, a testicles or other reproductive organ cancer cell, a skin cancer cell, thyroid cancer cell, a blood cancer cell, a lymph nodes cancer cell, or any combination of one or more thereof.

39. The non-naturally occurring polynucleotide of claim 1, wherein said one or more synthetic response elements comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 377-397, or 913, or a reverse complement thereof.Attorney Docket No. 53531-72460240. The non-naturally occurring polynucleotide of claim 1, wherein said one or more synthetic response elements comprises a sequence having at least 80% sequence identity to at least one of SEQ ID NOs: 386, 388, or 384, or a reverse complement thereof.

41. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises at least 35 consecutive nucleotides having at least 80% sequence identity to any one of SEQ ID NOs: 558-587, or 1010-1012, or a reverse complement thereof.

42. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises at least 35 consecutive nucleotides having at least 80% sequence identity to any one of SEQ ID NO: 560 or SEQ ID NO: 570.

43. The non-naturally occurring polynucleotide of claim 1, wherein said core promoter comprises a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 584-587.

44. The non-naturally occurring polynucleotide of claim 1, further comprising a nucleic acid sequence comprising any one of SEQ ID NOs: 713, 278-279, 281-285, 289-293, 301-305, 310-312, 315-319, 324-326, 328-335, 342-343, 710-711, 727, 729, 730- 731, 733, 736, 739, 742, 745, 748, 751, 757, 761, 764, 767, 770, 773, 776, 779, 782, 785, 788, 791, 794, 797, 800, 803, 806, 809, 812, 815, 818, 825, 828, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 876, 891, 894, 900, 903, 906, 912, 915, 918, 921, 922, 925, 928, 931, 934, 936, 939, 942, 945, 948, 951, 955, 958, 961, 964, 967, 971, 975, 979, 983, 986, 989, 992, 995, 998, 1001-1004, 1006, or 1008.

45. The non-naturally occurring polynucleotide of claim 1, wherein said non-naturally occurring polynucleotide is double-stranded.

46. The non-naturally occurring polynucleotide of claim 1, wherein said non-naturally occurring polynucleotide is single-stranded.

47. The non-naturally occurring polynucleotide of claim 1, wherein said non-naturally occurring polynucleotide is a linear polynucleotide.

48. The non-naturally occurring polynucleotide of claim 1, wherein said non-naturally occurring polynucleotide is a circular polynucleotide.Attorney Docket No. 53531-72460249. The non-naturally occurring polynucleotide of claim 1, wherein said non-naturally occurring polynucleotide is synthetic, a recombinant polynucleotide, or a combination thereof.

50. The non-naturally occurring polynucleotide of claim 1, wherein said non-naturally occurring polynucleotide comprises a ribonucleic acid (RNA) polynucleotide.

51. The non-naturally occurring polynucleotide of claim 1, wherein said non-naturally occurring polynucleotide comprises a deoxyribonucleic acid (DNA) polynucleotide.

52. The non-naturally occurring polynucleotide of claim 1, wherein said ORF comprises two or more genes encoding two or more peptide therapeutic agents.

53. The non-naturally occurring polynucleotide of claim 1, further comprising a second ORF comprising a second gene encoding a second peptide therapeutic agent.

54. The non-naturally occurring polynucleotide of claim 1, wherein said higher level of expression of said peptide therapeutic agent in said cancer cell is at least a 10% increase in expression as compared to said non-cancer cell.

55. A pharmaceutical formulation, comprising:a) the non-naturally occurring polynucleotide of claim 1; andb) a pharmaceutically acceptable: excipient, carrier, diluent, or any combination thereof.

56. The pharmaceutical formulation of claim 55, wherein said carrier is a liposome, a micelle, a lipid nanoparticle, a cell, an exosome, or a vesicle.

57. The pharmaceutical formulation of claim 56, wherein said cell is an enucleated cell.

58. The pharmaceutical formulation of claim 56 or 57, wherein said cell is a mammalian cell.

59. The pharmaceutical formulation of claim 58, wherein said mammalian cell is a human cell.

60. The pharmaceutical formulation of claim 59, wherein said human cell is an immune cell.

61. The pharmaceutical formulation of claim 58, wherein said cell is allogenic.

62. The pharmaceutical formulation of claim 58, wherein said cell is autologous.

63. A vector comprising said non-naturally occurring polynucleotide of claim 1.Attorney Docket No. 53531-72460264. The vector of claim 63, wherein said vector comprises an adeno-associated viral vector, a lentiviral vector, a retroviral vector, or any combination thereof.

65. The vector of claim 63, wherein said vector is non-viral.

66. The vector of claim 63, wherein said vector comprises a bacterial plasmid, a minicircle plasmid, or a nanoplasmid.

67. The vector of claim 63, wherein said vector is single stranded.

68. The vector of claim 63, wherein said vector is double stranded.

69. The vector of claim 63, wherein said vector is linear.

70. A method, comprising: administering said non-naturally occurring polynucleotide of any one of claims 1-54, said pharmaceutical formulation of any one of claims 55-62, or said vector of any one of claims 63-69 to a subject with cancer, thereby increasing expression of said peptide therapeutic agent in said cancer cell of said subject compared to a non-cancer cell.

71. The method of claim 70, further comprising administering to said subject an additional therapeutic agent.

72. The method of claim 71, wherein said additional therapeutic agent is a non-steroidal anti-inflammatory drug (NSAID).

73. The method of claim 71, wherein said additional therapeutic agent is an anti-histamine drug.

74. The method of claim 71, wherein said additional therapeutic agent is a corticosteroid.

75. The method of claim 74, wherein said corticosteroid is methyl prednisone.

76. The method of claim 71, wherein said additional therapeutic agent is a complement inhibitor.

77. The method of claim 71, wherein said additional therapeutic agent is a check-point inhibitor.

78. The method of claim 70, wherein said administering is performed by intratumoral injection, intravesicular injection, hepatic artery injection, or any combination thereof.

79. The method of claim 70, wherein said non-naturally occurring polynucleotide is administered systemically.Attorney Docket No. 53531-72460280. The method of claim 70, wherein said non-naturally occurring polynucleotide is administered in a tissue-specific manner.

81. The method of claim 70, wherein said administering is performed locoregionally.

82. The method of claim 70, wherein said peptide therapeutic agent comprises a toxin, a cytokine, an antibody or antigen binding fragment thereof, or any combination thereof.

83. The method of claim 70, wherein said peptide therapeutic agent comprises a cytokine.

84. The method of claim 70, wherein said subject has a tumor comprising said cancer cell.

85. The method of claim 84, further comprising reducing a size of said tumor by at least two-fold as compared to a control subject with a comparable tumor that has not been administered said non-naturally occurring polynucleotide.

86. The method of claim 84, further comprising reducing a size of said tumor by at least two-fold as compared to said size of said tumor prior to said administering.

87. The method of claim 70, wherein said expression of said peptide therapeutic agent is increased in said cancer cell by two-fold to 200-fold.

88. The method of claim 70, wherein said expression of said peptide therapeutic agent is increased in said cancer cell by two-fold to 200-fold compared to a reference expression level in said subject prior to said administering.

89. The method of claim 70, wherein said expression of said peptide therapeutic agent is increased in said cancer cell by two-fold to 200-fold compared to a reference expression level in a control subject with said cancer that has not been administered said non- naturally occurring polynucleotide.

90. The method of claim 70, wherein said non-naturally occurring polynucleotide is provided in a liposome.

91. The method of claim 90, wherein said liposome comprises one or more of a micelle, a solid lipid nanoparticle, or a combination thereof.

92. The method of claim 70, wherein said non-naturally occurring polynucleotide is provided in a vector.

93. The method of claim 92, wherein said vector comprises an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a non-viral vector, or any combination thereof.Attorney Docket No. 53531-72460294. The method of claim 93, wherein said non-viral vector comprises a plasmid vector.

95. The method of claim 70, wherein said non-naturally occurring polynucleotide is provided by a host cell.

96. The method of claim 95, wherein said non-naturally occurring polynucleotide is provided by an exosome or a vesicle of said host cell.

97. The method of claim 95, wherein said host cell comprises an engineered cell, a natural cell, or a combination thereof.

98. The method of claim 95, wherein said host cell comprises a eukaryotic cell, a prokaryotic cell, or a combination thereof.

99. The method of claim 98, wherein said eukaryotic cell comprises a yeast cell, a mammalian cell, or a combination thereof.

100. The method of claim 99, wherein said mammalian cell is a human cell.

101. The method of claim 98, wherein said prokaryotic cell is a bacterial cell.

102. The method of claim 95, wherein said host cell is a nucleated host cell, an enucleated host cell, or a combination thereof.

103. A kit comprising:a) said non-naturally occurring polynucleotide of any one of claims 1-54, said pharmaceutical formulation of any one of claims 55-62, or said vector of any one of claims 63-69; andb) instructions for providing said non-naturally occurring polynucleotide to subject with cancer.

104. The kit of claim 103, wherein said instructions comprise the method of any one of claims 70-102.

105. A lipid nanoparticle (LNP) comprising said non-naturally occurring polynucleotide of any one of claims 1-54 or said vector of any one of claims 63-70.

106. An engineered nucleic acid molecule comprising a nucleic acid sequence comprising SEQ ID NOs: 713, 278-279, 281-285, 289-293, 301-305, 310-312, 315-319, 324-326, 328-335, 342-343, 710-711, 727, 729, 730-731, 733, 736, 739, 742, 745, 748, 751, 757, 761, 764, 767, 770, 773, 776, 779, 782, 785, 788, 791, 794, 797, 800, 803, 806, 809, 812,Attorney Docket No. 53531-724602815, 818, 825, 828, 846, 849, 852, 855, 858, 861, 864, 867, 870, 873, 876, 891, 894, 900, 903, 906, 912, 915, 918, 921, 922, 925, 928, 931, 934, 936, 939, 942, 945, 948, 951, 955, 958, 961, 964, 967, 971, 975, 979, 983, 986, 989, 992, 995, 998, 1001-1004, 1006, or 1008.

107. A composition comprising an engineered peptide, wherein said engineered peptide comprises:a. a cytokine or a fragment thereof;b. an antibody or a fragment thereof; andc. a collagen binding domain or a fragment thereof, an integrin binding domain or a fragment thereof, a cleavable domain or a fragment thereof, or any combination thereof.

108. The composition of claim 107, wherein said engineered peptide is a chimeric peptide or a synthetic peptide.

109. The composition of claim 107, wherein said engineered peptide is a peptide therapeutic agent.

110. The composition of claim 107, wherein said engineered peptide is a fusion protein.

111. A nucleic acid molecule encoding said composition of any one of claims 107-110.

112. A method of detecting an expression product in situ, said method comprising:a) administering a polynucleotide to a subject, wherein said polynucleotide comprises:i. one or more synthetic response elements comprising one or more enhancers and a plurality of transcription factor binding sites; ii. a core promoter operably linked to an open reading frame (ORF) comprising a gene encoding said expression product; and iii. a transcription start site (TSS) upstream of said ORF, wherein said one or more synthetic response elements and said core promoter increase expression of said expression product in a target cell of said subject as compared with a non-target cell; andb) detecting said expression product in situ.Attorney Docket No. 53531-724602113. The method of claim 112, wherein said expression product is detected in said target cell ex vivo.

114. The method of claim 112, wherein said expression product is detected in said subject in vivo.

115. The method of claim 112, wherein said expression product is secretable from said target cell.

116. The method of claim 112, wherein said detecting comprises using magnetic resonance imaging (MRI) imaging, positron emission tomography (PET) imaging, singlephoton emission computed tomography (SPECT) imaging, photoacoustic imaging, fluorescence or luminescence imaging, or any combination thereof.

117. The method of claim 116, wherein said detecting comprises said luminescence imaging, and wherein said expression product comprises a luciferase, an Enhanced Green Fluorescent Protein (EGFP), a Green Fluorescent Protein (GFP), a Red Fluorescent Protein (RFP), or any combination thereof.

118. The method of claim 112, wherein said expression product comprises a polypeptide contrast agent, a lanthanide-binding protein, or an engineered fusion thereof.

119. The method of claim 112, wherein said expression product comprises P-galactosidase, GFP, mCherry or derivatives thereof, aeCP597, cjBlue or derivatives thereof, IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP713 / 256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry, GAF-FP, BphPl-FP / C20S, AphB variants, Dronpa, Dronpa-M159T, BphPl, or variants thereof.

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