Synthetic promoters, transcriptional units, vectors, and pharmaceutical compositions with enhanced ovarian cancer cell specificity and methods

Abstract

Disclosed are synthetic promoters, transcriptional units, vectors, pharmaceutical composition, and methods of use thereof which preferentially drive expression of an operatively linked nucleic acid in an ovarian cancer cell in vitro or in vivo, relative to a non-cancerous ovarian cell.

Description

[0001] SYNTHETIC PROMOTERS, TRANSCRIPTIONAL UNITS, VECTORS, AND PHARMACEUTICAL COMPOSITIONS WITH ENHANCED OVARIAN CANCER CELL SPECIFICITY AND METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003]

[0001] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 529,501, filed on July 28, 2023, the disclosure of which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005]

[0002] Cancer ranks as the second leading cause of death worldwide. However, most of the current treatments have serious side effects and off-target toxicities. Gene therapy, which involves the introduction of a gene encoding a therapeutic protein into a patient’s cells, represents a promising avenue as a cancer therapy. However, most cancer gene therapies have not exhibited high tumortargeting specificity. One reason is that most cancer-specific promoters used in cancer gene therapy are not substantively tumor specific (Nissim et al. Cell 171(5) A 138-1150 (2017)).

[0006]

[0003] Therefore, there is a critical need for improved cancer-specific treatments for cancer gene therapy.

[0007] SUMMARY

[0008]

[0004] The synthetic promoters, transcription units, vectors, compositions, and methods provided herein are expected to address the above need.

[0009]

[0005] In an aspect, there is provided a synthetic promoter containing 3 to 15 transcription factor binding sites (TFBSs), wherein each TFBS has any one of the following nucleic acid sequences: CACGTGNi (SEQ ID NO: 1), GCACGTG (SEQ ID NO: 2), AN2N3TGGCGGGAAAC (SEQ ID NO: 3), N4N5TTTTGGCGCCAAAAN6N7 (SEQ ID NO: 4), N8N9TTTTGGCGCCAAAATN10 (SEQ ID NO: 5), CCACGTGN11N12N13 (SEQ ID NO: 6), N14GCACGTGGN15 (SEQ ID NO: 7), N16N17TTCCCGCCN18AN19N20 (SEQ ID NO: 8), N21N22GGCGGGAA (SEQ ID NO: 9), GAAAATGGCGCCATTTTT (SEQ ID NO: 10), TN23TTCCCGCCAAAN24 (SEQ ID NO: 11), AGGCGGGAAN25N26 (SEQ ID NO: 12), N27TTN28GCGCN29AN30A (SEQ ID NO: 13), N31TTCCCGCCN32AA (SEQ ID NO: 14), N33N34TCGCGN35GA (SEQ ID NO: 15), TN36TCN37N38GAN39A (SEQ ID NO: 16), or AATATCGCGAGA (SEQ ID NO: 219), wherein each of Ni - N39 independently represents any nucleotide. Each of Ni - N39 may be the same or different for any of SEQ ID NOs: 1-16 and 219. Further, each of the 3 to 15 TFBSs in the synthetic promoter may be the same or different.

[0010]

[0006] As demonstrated in examples provided, the synthetic promoters may preferentially drive expression of an operatively linked nucleic acid coding sequence in an ovarian cancer cell, in vitro or in vivo, relative to a non-cancerous ovarian cell. And so, in another aspect, there is provided a transcriptional unit containing the synthetic promoter operatively linked to a nucleic acid coding sequence that encodes a therapeutic nucleic acid, peptide, polypeptide, or protein.

[0011]

[0007] In another aspect, a vector containing the transcriptional unit is provided.

[0012]

[0008] In another aspect, a pharmaceutical composition incorporating the vector is provided.

[0013]

[0009] In yet another aspect, a method of transcribing a nucleic acid coding sequence in an ovarian cancer cell is provided. The method entails contacting the ovarian cancer cell with the vector.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

[0010] FIG. l is a bar graph showing transcriptional units each containing a synthetic promoter and a nucleic acid coding sequence, wherein the synthetic promoter preferentially drives the expression of a nucleic acid coding sequence encoding a reporter in ovarian cancer cells relative to non-cancerous ovarian cells.

[0016] [OH] FIG. 2 is a bar graph showing the fold-difference of the expression of a nucleic acid coding sequence between ovarian cancer and non-cancerous ovarian cells for each synthetic promoter.

[0017]

[0012] FIG. 3 is a bar graph showing promoters identified by the screening platform that are active in murine ovarian cancer cell lines (BPPNM, KPCA, and PPNM); promoter names are listed on the X-axis.

[0018]

[0013] FIG. 4 is a bar graph showing radiance observed in mice harboring an ovarian cancer cell tumor (BPPNM) or without a tumor (control) infected with a lentivirus including luciferase under the control of a promoter of interest (MYC_v2, MYC_v4, ZBTB33_vl, or ZBTB33_v2).

[0019] DETAILED DESCRIPTION

[0020]

[0014] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used in the specification and the appended claims, unless specified to the contrary and / or otherwise clear from context, the following terms have the meaning indicated to facilitate the understanding of the present disclosure.

[0021]

[0015] The singular forms “a”, “an”, and “the” mean “one or more” and therefore include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, reference to “an inhibitor” includes mixtures of two or more such inhibitors, and the like.

[0022]

[0016] The term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. “About” can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term “about.”

[0017] The term “approximately” as used herein refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value (except where such number would exceed 100% of a possible value).

[0023]

[0018] The term "promoter" as used herein refers to a nucleic acid that facilitates, directly or indirectly, the transcription of a corresponding nucleic acid to which it is operatively linked. A promoter may function alone to facilitate transcription, or it may act in concert with one or more other regulatory units (e.g., enhancers and silencers that may be present in the nucleic acids or the vector). Promoters are located near the transcription start sites of nucleic acid coding sequences, on the same strand and upstream on the DNA (towards the 5' region of the sense strand).

[0024]

[0019] The term “synthetic” as used herein when referring to proteins or nucleic acids refers to a protein or nucleic acid that is not naturally found in an organism (i.e., non-naturally occurring).

[0025]

[0020] The term “non-cancerous” as used herein refers to a cell that is not dividing in an uncontrolled fashion, i.e., a cell that would arrest at a cell cycle checkpoint during mitosis.

[0026]

[0021] The term “nucleic acid” as used herein refers to a polymer of nucleotides that contain a nucleoside (a nucleobase and a five-carbon sugar) and a phosphate. The term nucleotide includes nucleosides that have a ribose sugar (i.e., a ribonucleotide that forms ribonucleic acid, RNA) or a 2’ -deoxyribose sugar i.e., a deoxyribonucleotide that forms deoxyribonucleic acid, DNA). Nucleotides serve as the monomeric units of nucleic acid polymers or polynucleotides. The four nucleobases in DNA are guanine (G), adenine (A), cytosine (C), and thymine (T). The four nucleobases in RNA are guanine (G), adenine (A), cytosine (C), and uracil (U). Nucleic acids are linear chains of nucleotides (e. , at least 3 nucleotides) chemically bonded by a series of ester linkages between the phosphoryl group of one nucleotide and the hydroxyl group of the sugar ( / '.<?., ribose or 2’ -deoxyribose) in the adjacent nucleotide. The term “nucleic acid coding sequence” as used herein refers to a segment of nucleic acids that encodes a therapeutic nucleic acid, peptide, polypeptide, or protein.

[0027]

[0022] The term “transcription factor binding site” or “TFBS” as used herein refers to a nucleic acid sequence contained in the synthetic promoters disclosed herein to which a transcription factor binds to facilitate expression of a nucleic acid coding sequence operatively linked to the promoter.

[0023] The terms “preferentially,” “preferentially driven” or “preferentially driven transcription” as used herein refer to the increased expression of a nucleic acid operatively linked to a synthetic promoter, i.e., a vector comprising the synthetic promoter, in an ovarian cancer cell relative to the level of expression of the same vector in a non-cancerous ovarian cell. Thus, a synthetic promoter that preferentially drives expression of an operatively linked nucleic acid in an ovarian cancer cell may drive expression of the nucleic acid in both an ovarian cancer cell and a non-cancerous ovarian cell (e.g., immortalized ovarian surface epithelium (IOSE) cells), but the level of expression of the nucleic acid will be higher in the ovarian cancer cell relative to the non-cancerous ovarian cell. Preferentially driven transcription in ovarian cancer cells may also be referred to herein in terms of a “fold-increase” in activity of the synthetic promoter in an ovarian cancer cell relative to the activity of the synthetic promoter in a non-cancerous ovarian cell.

[0028]

[0024] The term "operatively linked" as used herein is to be understood that a nucleic acid coding sequence disposed relative to the synthetic promoter such that the synthetic promoter facilitates the transcription of the nucleic acid coding sequence.

[0029]

[0025] The term “protein” as used herein refers to a polymer of amino acid residues, each of which are organic compounds consisting of an amino functional group and a carboxylic acid functional group, where the amino acid residues are bonded together by peptide bonds. Broadly, the therapeutic protein embraces any such protein that as an expression product, may exert an anticancer (e.g., immune stimulatory) effect, at least in the context of ovarian cancer.

[0030]

[0026] The term “derived from” as used herein when referring to proteins and nucleic acids refers to a protein or nucleic acid that originates from another, parental protein, or nucleic acid. The derived protein or nucleic acid has a sequence that may be identical to the parental sequence, may be a portion of the parent sequence, or may be a variant of the parent protein or nucleic acid in that it differs from the sequence of the parent. Variants may include amino acid and nucleotide substitutions, insertions, or deletions. Thus, for example, an amino acid sequence derived from a parent sequence may be identical for a specific range of amino acids of the parent but does not include amino acids outside that specific region.

[0031]

[0027] The term “cytokine” refers to low molecular weight extracellular polypeptides / gly coproteins that promote, modulate, and regulate the immune response (i.e., increase or decrease activity, differentiation, or proliferation).

[0032]

[0028] The term “tumor suppressor” as used herein refers to a gene product that reduces cancer cell growth.

[0033]

[0029] The term “signal peptide” as used herein refers to a short (e.g., 5-30 or 10-100 amino acids long) stretch of amino acids that directs the transport of the therapeutic protein during translation.

[0034]

[0030] As used herein, the term “lentiviral vector” refers to an infectious lentiviral particle. Lentivirinae (lentiviruses) is a subfamily of enveloped retroviridae (retroviruses), that is distinguishable from other viruses by virion structure, host range, and pathological effects. An infectious lentiviral particle is capable of invading a target host cell, including infecting, and transducing non-dividing cells and immune cells.

[0035]

[0031] The term “effective amount” as used herein refers to a sufficient amount of vector to provide the desired effect, e.g., the amount of a vector to introduce the transcriptional unit into a ovarian cancer cell, in vitro or in vivo, to express the nucleic acid coding sequence.

[0036]

[0032] The term “subject” (or “patient”) as used herein includes all members of the animal kingdom prone (or disposed) to or suffering from ovarian cancer. In some embodiments, the subject is a human. Therefore, a subject “having a cancer” or “in need of’ treatment according to the present disclosure broadly embraces subjects who have been positively diagnosed, including subjects having active disease who may have been previously treated with one or more rounds of therapy, and subjects who are not currently being treated (e.g., in remission) but who might still be at risk of relapse, and subjects who have not been positively diagnosed but who are predisposed to ovarian cancer (e.g., on account of the basis of prior medical history and / or family medical history, or who otherwise present with a one or more risk factors such that a medical professional might reasonably suspect that the subject was predisposed to ovarian cancer).

[0033] The terms “treat”, “treating”, and “treatment” as used herein refer to any type of intervention, process performed on, or the administration of an active agent to the subject in need thereof with the therapeutic objective (“therapeutic effect”) of reversing, alleviating, ameliorating, inhibiting, diminishing, slowing down, arresting, stabilizing, or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with ovarian cancer.

[0037]

[0034] The term “ovarian cancer” as used herein encompasses cancers that develop from cells that form the ovaries and associated tissues, including ovarian, fallopian, and peritoneal tissues. Ovarian cancers include ovarian carcinomas (e.g, epithelial ovarian carcinomas, low-grade serous ovarian carcinoma (LGSOC), high-grade serous ovarian carcinoma (HGSOC), serous adenocarcinomas, ovarian clear cell carcinoma (OCCC), primary squamous cell carcinoma (SCC) of the ovary, endometrioid carcinoma, and endometrioid adenocarcinomas), ovarian germ cell tumors, ovarian stromal cell tumors, and leiomyosarcomas.

[0038]

[0035] There is provided a synthetic promoter. The synthetic promoter contains a nucleic acid containing 3-15 transcription factor binding sites, wherein i) each TFBSs has any one of SEQ ID NOs: 1-16 or 219, ii) each N of SEQ ID NOs: 1-16 and 219 is independently any nucleotide and iii) each TFBS may be the same or different. Such synthetic promoters have been surprisingly discovered to exhibit increased in activity in an ovarian cancer cell relative to the activity of the synthetic promoter in a non-cancerous ovarian cancer cell. Each TFBS is about 27-314 nucleotides in length and is connected to an adjacent TFBS via a linker containing at least one nucleotide, wherein the linkers may be the same or different.

[0039]

[0036] A transcriptional unit is also provided and contains the synthetic promoter operatively linked to a nucleic acid coding sequence. In some embodiments, the transcription unit further contains a spacer. In some embodiments, the nucleic acid coding sequence encodes a therapeutic protein, e.g., a transcription factor, a surface T cell engager (STE), a cytokine, a tumor suppressor, or a checkpoint inhibitor. In some embodiments, the nucleic acid coding sequence further encodes a signal protein. In some embodiments, the nucleic acid coding sequence encodes a miRNA. In some embodiments, the transcriptional unit further comprises a regulatory unit.

[0040]

[0037] A vector is also provided and contains an insertion site (z.e., a multiple cloning site) and the synthetic promoter. In other embodiments, the vector contains an insertion site and the transcriptional unit.

[0038] A pharmaceutical composition is also provided and contains effective amounts of the vector containing the synthetic promoter or transcriptional unit and a pharmaceutically acceptable carrier.

[0041] Synthetic Promoters

[0042]

[0039] In one aspect, there is provided a synthetic promoter that may preferentially drive expression of an operatively linked nucleic acid coding sequence in an ovarian cancer cell relative to a non-cancerous ovarian cell, providing a new means to treat the ovarian cancer cell. The synthetic promoter containing 3 to 15 transcription factor binding sites (TFBSs), wherein each TFBS has any one of the following nucleic acid sequences: CACGTGNi (SEQ ID NO: 1), GCACGTG (SEQ ID NO: 2), AN2N3TGGCGGGAAAC (SEQ ID NO: 3), N4N5TTTTGGCGCCAAAAN6N7 (SEQ ID NO: 4), N8N9TTTTGGCGCCAAAATN10 (SEQ ID NO: 5), CCACGTGN11N12N13 (SEQ ID NO: 6), N14GCACGTGGN15 (SEQ ID NO: 7), N16N17TTCCCGCCN18AN19N20 (SEQ ID NO: 8), N21N22GGCGGGAA (SEQ ID NO: 9), GAAAATGGCGCCATTTTT (SEQ ID NO: 10),

[0043] TN23TTCCCGCCAAAN24 (SEQ ID NO: 11), AGGCGGGAAN25N26 (SEQ ID NO: 12),

[0044] N27TTN28GCGCN29AN30A (SEQ ID NO: 13), N31TTCCCGCCN32AA (SEQ ID NO: 14),

[0045] N33N34TCGCGN35GA (SEQ ID NO: 15), TN36TCN37N38GAN39A (SEQ ID NO: 16), or

[0046] AATATCGCGAGA (SEQ ID NO: 219). Each of Ni - N39 may be the same or different for any of SEQ ID NOs: 1-16 and 219, e.g, one or more of N11-N13 of SEQ ID NO: 6 may be the same or different, and one or more of N36-N39 of SEQ ID NO: 16 may be the same or different. Further, each of the 3 to 15 TFBSs in the synthetic promoter may be the same or different, e.g., the synthetic promoter may include three of the same TFBSs nucleic acid sequences of SEQ ID NOs: 1-8, one of each of SEQ ID NOs: 1-16 or 219, or any combination therebetween.

[0047]

[0040] In some embodiments, the synthetic promoter exhibits at least about a 5-fold increase in activity in an ovarian cancer cell relative to the activity of the synthetic promoter in a non-cancerous ovarian cell. In some embodiments, the synthetic promoter exhibits a fold-increase of at least about 3-fold, about 5-fold, about 10-fold, about 20-fold, about 40-fold, about 50-fold, about 75-fold, about 100-fold, about 75-fold, or about 150-fold.

[0048]

[0041] The identity of the TFBSs in a synthetic promoter may vary. In some embodiments, all of the TFBSs in a synthetic promoter have the same nucleic acid sequence. In some embodiments, all of the TFBSs in a synthetic promoter have different nucleic acid sequences. In some embodiments, at least two TFBSs are the same. In some embodiments, at least three TFBSs are the same. In some embodiments, at least five TFBSs are the same. In some embodiments, at least seven TFBSs are the same. In some embodiments, at least nine TFBS are the same. In some embodiments, at least twelve TFBSs are the same. In some embodiments, at least fourteen TFBSs are the same. By way of a representative example, in a synthetic promoter that has 3 TFBSs, 2 TFBSs may have the same nucleic acid sequence and one TFBS may have a different nucleic acid sequence, such that this synthetic promoter has two different TFBSs. By way of another representative example, in a synthetic promoter that has 4 TFBSs, 2 TFBSs may have different nucleic acid sequences and two TFBSs may have the same nucleic acid sequence, such that this synthetic promoter has three different TFBSs. By way of a third representative example, in a synthetic promoter that has 5 TFBSs, all 5 TFBSs can be different, all 5 TFBSs can be the same, or 3 can be the same and 2 can be different.

[0049]

[0042] The order of the TFBSs may also vary. By way of a representative example, in a synthetic promoter that has, e.g., two TFBSs having the same nucleic acid sequence (TFBS1) and two TFBSs having different nucleic acid sequences (TFBS2 and TFBS3), the two TFBSls may be arranged contiguously (so that the TFBS sequence in the synthetic promoter (not including linkers interspersed between TFBSs) is TFBS1, TFBS1, TFBS2, TFBS3) or may be interspersed between TFBS2 and TFBS3 (so that the TFBS sequence in the synthetic promoter (not including linkers interspersed between TFBSs) is TFBS1, TFBS2, TFBS1, TFBS3). By way of another representative example, a synthetic promoter may have 3 TFBSs each having the same nucleic acid sequence, and 4 TFBSs each having the same nucleic acid sequence. In some of these embodiments, the first 3 TFBSs each having the same nucleic acid sequence is followed by the 4 TFBSs each having the same nucleic acid sequence. In some embodiments, the 3 TFBSs each having the same nucleic acid sequence is interspersed with the 4 TFBSs each having the same nucleic acid sequences. In some embodiments, the TFBSs alternate, from 5’ to 3’, repeating a TFBS with one sequence followed by a TFBS with a second, different sequence.

[0050]

[0043] In some embodiments, of SEQ ID NO: 1 (CACGTGNi), Ni is C or T. In such embodiments, at least one TFBS in the synthetic promoter has the nucleic acid sequence CACGTGC (SEQ ID NO: 17) or CACGTGT (SEQ ID NO: 18). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 17.

[0044] Tn some embodiments, at least one TFBS in the synthetic promoter has the sequence GCACGTG (SEQ ID NO: 2). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 2.

[0051]

[0045] In some embodiments, for SEQ ID NO: 3 (AN2N3TGGCGGGAAAC), N2 is A or T and N3 is A or T. In such embodiments, at least one TFBS has any one of the nucleic acid sequences ATATGGCGGGAAAC (SEQ ID NO: 19), ATTTGGCGGGAAAC (SEQ ID NO: 20), AAATGGCGGGAAAC (SEQ ID NO: 21), and AATTGGCGGGAAAC (SEQ ID NO: 22). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 19.

[0052]

[0046] In some embodiments, for SEQ ID NO: 4 (N4N5TTTTGGCGCCAAAAN6N7), N4is A, C, or G, Ns is A, G, or T, Ne is A, C, or T, and N7 is any nucleotide. In such embodiments, at least one TFBS has any one of the nucleic acid sequences AATTTTGGCGCCAAAACG (SEQ ID NO: 23), AATTTTGGCGCCAAAATT (SEQ ID NO: 24), AGTTTTGGCGCCAAAATC (SEQ ID NO: 25), GTTTTTGGCGCCAAAATG (SEQ ID NO: 26), ATTTTTGGCGCCAAAATA (SEQ ID NO: 27), CTTTTTGGCGCCAAAATG (SEQ ID NO: 28), GATTTTGGCGCCAAAAAG (SEQ ID NO: 29), and AGTTTTGGCGCCAAAAAG (SEQ ID NO: 30). In some embodiments, at least two TFBSs in the synthetic promoter have any one of the nucleic acid sequences of SEQ ID NOs: 23- 26. In some embodiments, each TFBS in the synthetic promoter has any one of the nucleic acid sequences of SEQ ID NOs: 23-26, and each TFBS in the synthetic promoter is the same.

[0053]

[0047] In some embodiments, at least one TFBS in the synthetic promoter has the sequence N4N5TTTTGGCGCCAAAAAT (SEQ ID NO: 31), which corresponds to SEQ ID NO: 4 (N4N5TTTTGGCGCCAAAAN6N7), wherein N4is A, C, or G, Ns is A, G, or T, N6is A, and N7 is T. Taking into account all possible combinations of N4and Ns, SEQ ID NO: 31 includes the TFBSs set forth in Table 1.

[0054] Table 1: Examples of TFBSs Represented by SEQ ID NO: 31

[0055]

[0048] In some embodiments, for SEQ ID NO: 5 (NsNgTTTTGGCGCCAAAATNio), Ns is A or G, N9 is A, G, or T, and N10 is C or G. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequence AATTTTGGCGCCAAAATC (SEQ ID NO: 41), GGTTTTGGCGCCAAAATG (SEQ ID NO: 42), ATTTTTGGCGCCAAAATG (SEQ ID NO: 43), AGTTTTGGCGCCAAAATG (SEQ ID NO: 44), GATTTTGGCGCCAAAATC (SEQ ID NO: 45), GGTTTTGGCGCCAAAATG (SEQ ID NO: 46), ATTTTTGGCGCCAAAATC (SEQ ID NO: 47), GTTTTTGGCGCCAAAATC (SEQ ID NO: 48), and AATTTTGGCGCCAAAATG (SEQ ID NO: 49). In some embodiments, at least two TFBSs in the synthetic promoter have any one of the nucleic acid sequences of SEQ ID NOs: 41-43. In some embodiments, each TFBS in the synthetic promoter has any one of the nucleic acid sequences of SEQ ID NOs: 41-43, and each TFBS in the synthetic promoter is the same.

[0056]

[0049] In some embodiments, for SEQ ID NO: 6 (CCACGTGN11N12N13), N11 is C or G, N12 is C, or T, and N13 is C, G, or T. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences CCACGTGCTT (SEQ ID NO: 50), CCACGTGCTC (SEQ ID NO: 51), CCACGTGCCT (SEQ ID NO: 52), CCACGTGCCC (SEQ ID NO: 53), CCACGTGCTG (SEQ ID NO: 54), CCACGTGGCC (SEQ ID NO: 55), CCACGTGCCG (SEQ ID NO: 56), CCACGTGGCG (SEQ ID NO: 57), and CCACGTGGCT (SEQ ID NO: 58). In some embodiments, at least two TFBSs in the synthetic promoter have any one of the nucleic acid sequences of SEQ ID NOs: 50 and 51. In some embodiments, each TFBS in the synthetic promoter has any one of the nucleic acid sequences of SEQ ID NO: 50 and 51, and each TFBS in the synthetic promoter is the same.

[0057]

[0050] In some embodiments, for SEQ ID NO: 7 (N14GCACGTGGN15), Ni4 is A or C and N15 is C or T. In some embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences CGCACGTGGT (SEQ ID NO: 59), AGCACGTGGC (SEQ ID NO: 60), AGCACGTGGT (SEQ ID NO: 61), and CGCACGTGGC (SEQ ID NO: 62). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 59. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 59, wherein each TFBS in the synthetic promoter is the same.

[0058]

[0051] In some embodiments, for SEQ ID NO: 8 (N16N17TTCCCGCCN18AN19N20), Ni6 is any nucleotide, N17 is A, G, or T, Nis is A or T, N19 is A or T, and N20 is A or T. In such embodiments, one or more of the TFBSs in the synthetic promoter has any one of the nucleic acid sequences ATTTCCCGCCAATT (SEQ ID NO: 63), GTTTCCCGCCAATT (SEQ ID NO: 64),

[0059] CTTTCCCGCCAATA (SEQ ID NO: 65), TTTTCCCGCCTAAA (SEQ ID NO: 66),

[0060] AATTCCCGCCAAAT (SEQ ID NO: 67), GGTTCCCGCCAAAA (SEQ ID NO: 68), and AGTTCCCGCCAAAA (SEQ ID NO: 69). In some embodiments, at least two TFBSs in the synthetic promoter have any one of the nucleic acid sequences of SEQ ID NOs: 63-64. In some embodiments, each TFBS in the synthetic promoter has any one of the nucleic acid sequences of SEQ ID NOs: 63-64, and each TFBS in the synthetic promoter is the same.

[0061]

[0052] In some embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences N16ATTCCCGCCTAN19A (SEQ ID NO: 70), which correspond to SEQ ID NO: 8 (N16N17TTCCCGCCN18AN19N20), wherein Ni6 is any nucleotide, N17 is A, Nis is T, N19 is A or T, and N20 is A. Taking into account all possibilities of combinations of Ni6 and N19, SEQ ID NO: 70 includes the TFBSs set forth in Table 2.

[0062] Table 2

[0063]

[0053] In some embodiments, at least one TFBS in the synthetic promoter has the sequence N21N22GGCGGGAA (SEQ ID NO: 9), wherein N21 is A, G, or T, and N22 is A or T. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences TTGGCGGGAA (SEQ ID NO: 79), GAGGCGGGAA (SEQ ID NO: 80), ATGGCGGGAA (SEQ ID NO: 81), GTGGCGGGAA (SEQ ID NO: 82), AAGGCGGGAA (SEQ ID NO: 83), and TAGGCGGGAA (SEQ ID NO: 84). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 79. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 79.

[0064]

[0054] In some embodiments, at least one TFBS in the synthetic promoter has the nucleic acid sequence GAAAATGGCGCCATTTTT (SEQ ID NO: 10), In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 10.

[0065]

[0055] In some embodiments, for SEQ ID NO: 11 (TN23TTCCCGCCAAAN24), N23 is A or G, and N24 is A or T. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences TGTTCCCGCCAAAA (SEQ ID NO: 85), TATTCCCGCCAAAT (SEQ ID NO: 86), TATTCCCGCCAAAA (SEQ ID NO: 87), and TGTTCCCGCCAAAT (SEQ ID NO: 88). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 85. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 85.

[0066]

[0056] In some embodiments, for SEQ ID NO: 12 (AGGCGGGAAN25N26), N25 is A or G, and N26 is A, C, or G. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences AGGCGGGAAAA (SEQ ID NO: 89), AGGCGGGAAGA (SEQ ID NO: 90), AGGCGGGAAAG (SEQ ID NO: 91), AGGCGGGAAGC (SEQ ID NO: 92), and AGGCGGGAAAC (SEQ ID NO: 93). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 89. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 89.

[0067]

[0057] In some embodiments, for SEQ ID NO: 13 (N27TTN28GCGCN29AN30A), N27 is A, G, or T, N28 is C or G, N29 is C or G, and N30 is A, C, or T. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences TTTCGCGCCAAA (SEQ ID NO: 94), ATTGGCGCCATA (SEQ ID NO: 95), TTTGGCGCCACA (SEQ ID NO: 96), GTTGGCGCCAAA (SEQ ID NO: 97), ATTGGCGCGAAA (SEQ ID NO: 98). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 94. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 94.

[0058] In some embodiments, at least one TFBS in the synthetic promoter has the sequence N27TTN28GCGCGAN30A (SEQ ID NO: 99), which corresponds to SEQ ID NO: 13 (N27TTN28GCGCN29AN30A), wherein N27 is A, G, or T, N28 is C or G, N29 is G, and N30 is A or T. Taking into account all possible combinations of N27, N28, and N30, SEQ ID NO: 99 includes the TFBSs set forth in Table 3.

[0068] Table 3

[0069]

[0059] In some embodiments, for SEQ ID NO: 14 (N31TTCCCGCCN32AA), N31 is G or T and N32 is A or T. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences TTTCCCGCCTAA (SEQ ID NO: 111), GTTCCCGCCAAA (SEQ ID NO: 112), TTTCCCGCCAAA (SEQ ID NO: 113), and GTTCCCGCCTAA (SEQ ID NO: 114). In some embodiments, at least two TFBS have the nucleic acid sequence of SEQ ID NO: 111 or 112. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 111 or 112, and each TFBS in the synthetic promoter is the same.

[0070]

[0060] In some embodiments, for SEQ ID NO: 15 (N33N34TCGCGN35GA), N33 is any nucleotide, N34 is A, C, or G, and N35 is A or G. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences TGTCGCGAGA (SEQ ID NO: 115), TATCGCGGGA (SEQ ID NO: 116), TATCGCGAGA (SEQ ID NO: 117), GATCGCGAGA (SEQ ID NO: 118), CATCGCGAGA (SEQ ID NO: 119), TCTCGCGAGA (SEQ ID NO: 120), ACTCGCGAGA (SEQ ID NO: 121), AATCGCGAGA (SEQ ID NO: 122), AGTCGCGAGA (SEQ ID NO: 123), CGTCGCGAGA (SEQ ID NO: 124), and GGTCGCGAGA (SEQ ID NO: 125). In some embodiments, at least two TFBS in the synthetic promoter have any one of the nucleic acid sequences of SEQ ID NOs: 115-117. In some embodiments, each TFBS in the synthetic promoter has any one of the nucleic acid sequences of SEQ ID NO: 115-117, and each TFBS in the synthetic promoter is the same.

[0071]

[0061] In some embodiments, at least one TFBS in the synthetic promoter has the sequence N33N34TCGCGGGA (SEQ ID NO: 126), which corresponds to SEQ ID NO: 15 (N33N34TCGCGN35GA), wherein N33 is any nucleotide, N34 is A, C, or G, and N35 is G. Taking into account all possible combinations among N33 and N34, SEQ ID NO: 126 includes the TFBSs set forth in Table 4.

[0072] Table 4

[0073]

[0062] In some embodiments, for SEQ ID NO: 16 (TN40TCN41N42GAN43A), N36 is C or G, N37 is A or G, N38 is C or T, and N39 is C, G, or T. In such embodiments, at least one TFBS in the synthetic promoter has any one of the nucleic acid sequences TCTCGCGACA (SEQ ID NO: 137), TGTCGCGACA (SEQ ID NO: 138), TGTCGCGATA (SEQ ID NO: 139), TCTCACGAGA (SEQ ID NO: 140), TCTCGCGATA (SEQ ID NO: 141), and TCTCGTGAGA (SEQ ID NO: 142). In some embodiments, at least two TFBSs in the synthetic promoter have the nucleic acid sequence of SEQ ID NO: 137. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NOs: 137, where each TFBS in the synthetic promoter is the same.

[0074]

[0063] In some embodiments, at least one TFBS in the synthetic promoter has the sequence TN36TCN37N38GACA (SEQ ID NO: 143), which corresponds to SEQ ID NO: 16 (TN36TCN37N38GAN39A), wherein N36 is C or G, N37 is A or G, N38 is C or T, and N39 is C. Taking into account all possible combinations of N36-N38, SEQ ID NO: 143 includes the TFBSs set forth in Table 5.

[0075] Table 5

[0076]

[0064] In some embodiments, at least one TFBS in the synthetic promoter has the nucleic acid sequence AATATCGCGAGA (SEQ ID NO: 219). In some embodiments, at least two TFBS have the nucleic acid sequence of SEQ ID NO: 219. In some embodiments, each TFBS in the synthetic promoter has the nucleic acid sequence of SEQ ID NO: 219.

[0077]

[0065] A person skilled in the art would immediately recognize that there are additional TFBSs within the scope of SEQ ID NOs: 1-16 and 219, and would be able to elucidate their sequences using standard techniques and methodologies.

[0078]

[0066] The synthetic promoters contain 3 to 15 TFBSs. Each TFBS may be the same or different. In some embodiments, the synthetic promoter contains 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 TFBSs. In some embodiments, the synthetic promoter contains 3 TFBSs. In some embodiments, the synthetic promoter contains 5 TFBSs. In some embodiments, the synthetic promoter contains 6 TFBSs. In some embodiments, the synthetic promoter contains 8 TFBSs. In some embodiments, the synthetic promoter contains 9 TFBSs. In some embodiments, the synthetic promoter contains 12 TFBSs. In some embodiments, the synthetic promoter contains 15 TFBSs.

[0079]

[0067] In some embodiments, each TFBS in the synthetic promoter is connected to an adjacent TFBS via a linker containing at least one nucleotide, where each linker has the same or different nucleic acid sequence. Without being bound by theory, linkers provide steric space between adjacent TFBSs, enabling more efficient or more transcription factor (TF) proteins binding to each TFBS in the synthetic promoter. The nucleotide sequences of the linkers are not critical, provided that they do not interfere with the function of the promoter. Table 26 includes the sequences of synthetic promoters having undefined trinucleotide linkers.

[0080]

[0068] In some embodiments, the linkers (L) are from two to eight nucleotides in length and may be represented by the formula N40N41N42N43N44N45N46N47, where each of N40-N47 may be any one of A, C, T, or G and may be the same or different, and further wherein any one or more of N42-N47 may be absent. In some embodiments, the linkers are two nucleotides in length. In such embodiments, the linkers may have any one of the following sequences: AA, AC, AG, AT, CA, CC, CG, CT, GA, GC, GG, GT, TA, TC, TG, and TT. In some embodiments, the linkers are three nucleotides in length. In such embodiments, the linkers may have any one of the following sequences: AGA, TCG, GAC, ATA, ACT, TAC, TAG, CAT, ATC, GAT, CGT, AGT, CAC, CTG, TAC, TAT, ACA, TAC, CGA, and ACG. In some embodiments, the linkers are from two to four nucleotides in length. In some embodiments, the linkers are from four to eight nucleotides in length. In such embodiments, the linkers have any one of the following sequences: AGAT, TCGG, GACAT, ACTTA, TACTAG, TAGCAT, CATATCG, ATCGATC, and GATCGTAGT. In some embodiments, each linker in the synthetic promoter has a different nucleotide sequence.

[0081]

[0069] In some embodiments, the synthetic promoter may be represented by the formula TFBSi- L1-TFBS2-L2-TFBS3-L3-TFBS4-L4-TFBS5-L5-TFBS6-L6-TFBS7-L7-TFBS8-L8-TFBS9-L9-TFBS10- Lio-TFBSii-Lii-TFBSi2-Li2-TFBSi3-Li3-TFBSi4-Li4-TFBSi5, wherein each of TFBS1-TFBS15 is independently any one of SEQ ID NOs: 1-149 and 219 ( / . ., each TFBS may be the same or different), and wherein any one or more of TFBS4-15 (and the adjacent L4-14) may be absent, and wherein each of L1-L14 independently represents a linker from two to eight nucleotides in length, and where each linker independently represents N40N41N42N43N44N45N46N47, where each of N40-N47 may be any one of A, C, T, or G and may be the same or different, and further wherein any one or more of N42-N47 may be absent.

[0082]

[0070] In some embodiments, the synthetic promoter is about 27 to about 370 nucleotides in length. In some embodiments, the synthetic promoter is about 33 to about 342 nucleotides in length. In some embodiments, the synthetic promoter is about 37 to about 314 nucleotides in length.

[0083]

[0071] In some embodiments, the synthetic promoter has 11 TFBSs, each having the sequence of SEQ ID NO: 17, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, TAG, CAT, ATC, and GAT, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 150):

[0084] 1 cacgtgcaga cacgtgctcg cacgtgcgac cacgtgcata cacgtgcact cacgtgctac 61 cacgtgctag cacgtgccat cacgtgcatc cacgtgcgat cacgtgc

[0085]

[0072] In some embodiments, the synthetic promoter has 11 TFBSs, each having the sequence of SEQ ID NO: 2, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence s AGA, TCG, GAC, ATA, ACT, TAC, TAG, CAT, ATC, and GAT, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 151):

[0086] 1 gcacgtgaga gcacgtgtcg gcacgtggac gcacgtgata gcacgtgact gcacgtgtac 61 gcacgtgtag gcacgtgcat gcacgtgatc gcacgtggat gcacgtg

[0087]

[0073] In some embodiments, the synthetic promoter has 6 TFBSs, each having the sequence of SEQ ID NO: 19, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, and ACT, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 152):

[0088] 1 atatggcggg aaacagaata tggcgggaaa ctcgatatgg cgggaaacga catatggcgg 61 gaaacataat atggcgggaa acactatatg gcgggaaac

[0089]

[0074] In some embodiments, the synthetic promoter has 4 TFBSs, each having the sequence of SEQ ID NO: 23, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, and GAC, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 153):

[0090] 1 aattttggcg ccaaaacgag aaattttggc gccaaaacgt cgaattttgg cgccaaaacg 61 gacaattttg gcgccaaaac g

[0091]

[0075] In some embodiments, the synthetic promoter has 4 TFBSs, each having the sequence of SEQ ID NO: 41 , wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, and GAC, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 154):

[0092] 1 aattttggcg ccaaaatcag aaattttggc gccaaaatct cgaattttgg cgccaaaatc 61 gacaattttg gcgccaaaat c

[0093]

[0076] In some embodiments, the synthetic promoter has 8 TFBSs, each having the sequence of SEQ ID NO: 50, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, and TAG, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO:

[0094] 155):

[0095] 1 ccacgtgctt agaccacgtg ctttcgccac gtgcttgacc cacgtgctta taccacgtgc 61 ttactccacg tgctttaccc acgtgcttta gccacgtgct t

[0096]

[0077] In some embodiments, the synthetic promoter has 8 TFBSs, each having the sequence of SEQ ID NO: 59, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, and TAG, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO:

[0097] 156):

[0098] 1 cgcacgtggt agacgcacgt ggttcgcgca cgtggtgacc gcacgtggta tacgcacgtg 61 gtactcgcac gtggttaccg cacgtggtta gcgcacgtgg t

[0099]

[0078] In some embodiments, the synthetic promoter has 6 TFBSs, each having the sequence of SEQ ID NO: 63, separated by linkers containing three nucleotides, the linkers having the nucleic acid sequences AGA, TCG, GAC, ATA, and ACT, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 157):

[0100] 1 atttcccgcc aattagaatt tcccgccaat ttcgatttcc cgccaattga catttcccgc 61 caattataat ttcccgccaa ttactatttc ccgccaattt

[0101]

[0079] In some embodiments, the synthetic promoter has 8 TFBSs, each having the sequence of SEQ ID NO: 79, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, and TAG, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 158):

[0102] 1 ttggcgggaa agattggcgg gaatcgttgg cgggaagact tggcgggaaa tattggcggg 61 aaactttggc gggaatactt ggcgggaata gttggcggga

[0103]

[0080] In some embodiments, the synthetic promoter has 7 TFBSs, each having the sequence of SEQ ID NO: 64, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, and TAC, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 159):

[0104] 1 gtttcccgcc aattagagtt tcccgccaat ttcggtttcc cgccaattga cgtttcccgc 61 caattatagt ttcccgccaa ttactgtttc ccgccaattt acgtttcccg ccaatt

[0105]

[0081] In some embodiments, the synthetic promoter has 4 TFBSs, each having the sequence of SEQ ID NO: 10, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, and GAC, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 160):

[0106] 1 gaaaatggcg ccatttttag agaaaatggc gccatttttt cggaaaatgg cgccattttt 61 gacgaaaatg gcgccatttt t

[0107]

[0082] In some embodiments, the synthetic promoter has 6 TFBSs, each having the sequence of SEQ ID NO: 85, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, and ACT, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 161):

[0108] 1 tgttcccgcc aaaaagatgt tcccgccaaa atcgtgttcc cgccaaaaga ctgttcccgc 61 caaaaatatg ttcccgccaa aaacttgttc ccgccaaaa

[0109]

[0083] In some embodiments, the synthetic promoter has 7 TFBSs, each having the sequence of SEQ ID NO: 89, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, and TAC, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 162):

[0110] 1 aggcgggaaa aagaaggcgg gaaaatcgag gcgggaaaag acaggcggga aaaataaggc 61 gggaaaaact aggcgggaaa atacaggcgg gaaaa

[0111]

[0084] In some embodiments, the synthetic promoter has 7 TFBSs, each having the sequence of SEQ ID NO: 94, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, and TAC, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 163):

[0112] 1 tttcgcgcca aaagatttcg cgccaaatcg tttcgcgcca aagactttcg cgccaaaata 61 tttcgcgcca aaacttttcg cgccaaatac tttcgcgcca aatagtttcg cgccaaa

[0113]

[0085] In some embodiments, the synthetic promoter has 7 TFBSs, each having the sequence of SEQ ID NO: 111, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, and TAC, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO: 164):

[0114] 1 tttcccgcct aaagatttcc cgcctaatcg tttcccgcct aagactttcc cgcctaaata 61 tttcccgcct aaacttttcc cgcctaatac tttcccgcct aatagtttcc cgcctaa

[0115]

[0086] In some embodiments, the synthetic promoter has 8 TFBSs, each having the sequence of SEQ ID NO: 51, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, and TAG, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO:

[0116] 165):

[0117] 1 ccacgtgctc agaccacgtg ctctcgccac gtgctcgacc cacgtgctca taccacgtgc 61 tcactccacg tgctctaccc acgtgctcta gccacgtgct

[0118]

[0087] In some embodiments, the synthetic promoter has 8 TFBSs, each having the sequence of SEQ ID NO: 115, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, and TAG, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO:

[0119] 166):

[0120] 1 tgtcgcgaga agatgtcgcg agatcgtgtc gcgagagact gtcgcgagaa tatgtcgcga 61 gaacttgtcg cgagatactg tcgcgagata gtgtcgcgag a

[0121]

[0088] In some embodiments, the synthetic promoter has 8 TFBSs, each having the sequence of SEQ ID NO: 137, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, and TAG, respectively. The nucleic acid sequence of the promoter is set forth below (SEQ ID NO:

[0122] 167):

[0123] 1 tctcgcgaca agatctcgcg acatcgtctc gcgacagact ctcgcgacaa tatctcgcga 61 caacttctcg cgacatactc tcgcgacata gtctcgcgac a

[0124]

[0089] In some embodiments, the synthetic promoter (referred to as MYC_v2) has 9 TFBSs, each having the sequence of SEQ ID NO: 50, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, TAG, and CAT, respectively; has the nucleic acid sequence set forth below (SEQ ID NO: 220):

[0125] CCACGTGCTTAGACCACGTGCTTTCGCCACGTGCTTGACCCACGTGCTTATACCACGTG CTTACTCCACGTGCTTTACCCACGTGCTTTAGCCACGTGCTTCATCCACGTGCTT

[0126]

[0090] In some embodiments, the synthetic promoter (referred to as MYC_v4) has 12 TFBSs, each having the sequence of SEQ ID NO: 17, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, TAG, ATC, GAT, and CGT respectively; has the nucleic acid sequence set forth below (SEQ ID NO: 221): CACGTGCAGACACGTGCTCGCACGTGCGACCACGTGCATACACGTGCACTCACGTGCTA

[0127] CCACGTGCTAGCACGTGCCATCACGTGCATCCACGTGCGATCACGTGCCGTCACGTGC

[0128]

[0091] In some embodiments, the synthetic promoter (referred to as ZBTB33_vl) has 8 TFBSs, each having the sequence of SEQ ID NO: 219, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, and TAG, respectively; has the nucleic acid sequence set forth below (SEQ ID NO: 222):

[0129] AATATCGCGAGAAGAAATATCGCGAGATCGAATATCGCGAGAGACAATATCGCGAGAAT AAATATCGCGAGAACTAATATCGCGAGATACAATATCGCGAGATAGAATATCGCGAGA

[0130]

[0092] In some embodiments, the synthetic promoter (referred to as ZBTB33_v2) has 9 TFBSs, each having the sequence of SEQ ID NO: 137, wherein each TFBS is separated from an adjacent TFBS by a linker containing three nucleotides, and which has the nucleic acid sequence AGA, TCG, GAC, ATA, ACT, TAC, TAG, and CAT, respectively; has the nucleic acid sequence set forth below (SEQ ID NO: 223):

[0131] TCTCGCGACAAGATCTCGCGACATCGTCTCGCGACAGACTCTCGCGACAATATCTCGCG ACAACTTCTCGCGACATACTCTCGCGACATAGTCTCGCGACACATTCTCGCGACA

[0132]

[0093] The sequences of additional synthetic promoters are listed below in Table 25.

[0133] Transcriptional Units

[0134]

[0094] In one aspect, the disclosure provides a transcriptional unit containing a synthetic promoter operatively linked to a nucleic acid (also referred to herein as a “nucleic acid coding sequence”) that encodes a therapeutic nucleic acid (e.g. , a miRNA), peptide, polypeptide, or protein. In some embodiments, the nucleic acid coding sequence encodes a therapeutic nucleic acid, a reporter, or a therapeutic protein. The synthetic promoter preferentially drives expression of the nucleic acid coding sequence in an ovarian cancer cell, in vitro or in vivo.

[0135]

[0095] In some embodiments, the transcriptional unit further contains a regulatory unit. In some embodiments, the transcriptional unit contains a spacer containing additional nucleotides disposed between the synthetic promoter and the nucleic acid coding sequence. In some embodiments, the spacer contains 30 nucleotides or less disposed toward the 3’ end of the transcriptional unit, downstream of the synthetic promoter (i.e., between the synthetic promoter and the nucleic acid coding sequence). In some embodiments, the spacer contains 30 nucleotides or less disposed toward the 5’ end of the transcriptional unit, upstream of the synthetic promoter.

[0136]

[0096] Methods of determining the therapeutic effect of a therapeutic protein or a therapeutic nucleic acid in ovarian cancer cells are known in the art. See, e.g., Nissim et al., Cell 171(5)'.1138- 1150 (2017).

[0137] Nucleic Acid Coding Sequences

[0138]

[0097] The nucleic acid coding sequence encodes a therapeutic nucleic acid, peptide, polypeptide, or protein. In some embodiments, the nucleic acid coding sequence is a reporter. In some embodiments, the nucleic acid coding sequence encodes a therapeutic protein or a therapeutic nucleic acid. In some embodiments, the therapeutic protein is a transcription factor, a surface T cell engager (STE), a cytokine, a tumor suppressor, or a checkpoint inhibitor.

[0139]

[0098] In embodiments wherein the therapeutic protein is a transcription factor, representative examples of transcription factors include a fusion protein containing a galactose-responsive transcription factor GAL4 fused to VP 16 (GAL4-VP16), GAL4 fused to VP64 (GAL4-VP64), and GAL4 fused to VP64, p65, and Rta (VPR) domains (GAL4-VPR).

[0140]

[0099] In some embodiments, the therapeutic protein is a STE. In some embodiments, the STE is a fusion protein containing an antibody fragment, e.g., a single-chain variable antibody fragment (scFv), that binds CD3 and a membrane anchor domain. The membrane anchor domain localizes the STE to the cell plasma membrane and may be any transmembrane domain. In some embodiments, the antibody fragment of the STE fusion protein is derived from a commercially available anti-CD3 antibody, antibody fragment, or derivative thereof. The names of anti-CD3 antibodies from which the therapeutic protein may be derived are provided at the NCBI and FDA Global Substance Registration System (GSRS), with accession numbers set forth herein, include, e.g., blinatumomab (available under the tradename BLINCYTO; NCBI 6AL5), catumaxomab (available under the tradename REMOVAB; NCBI 4DZ8), flotetuzumab (MGD006; GSRS 0AHT0IC02G), muromonab-CD3 (available under the tradename ORTHOCLONE OKT3; NCBI 8F0L), otelixizumab (ChAglyC D3, TRX4; GSRS I5HF2X04PB), tebentafusp (available under the tradename KIMMTRAK; GSRS N658GY6L3E), teplizumab (available under the tradename TZIELD; GSRS S4M959U2IJ), and visilizumab (available under the tradename NUV10N; GSRS H18SKU3289). In some embodiments, the membrane anchor domain of the STE fusion protein is derived from the Duffy antigen / receptor for chemokines (DARC). DARC is also known as Atypical Chemokine Receptor 1 (Duffy blood group) (ACKR1; NCBI gene ID 2532).

[0141]

[0100] In some embodiments, the therapeutic protein is a cytokine or a non-naturally occurring variant or fragment thereof that binds to the cytokine’s cognate target. Representative examples of cytokines include chemokines, interferons (IFNs), interleukins (Ils), lymphokines and tumor necrosis factors (TNFs). Cytokine variants are non-naturally occurring proteins capable of binding to a cytokine receptor on an immune cell and initiating signal transduction through that receptor to achieve substantially the same effect as the naturally occurring cytokine.

[0142]

[0101] In embodiments wherein the therapeutic protein is a cytokine, or a non-naturally occurring variant or fragment thereof, the cytokine may be derived from IL-la, IL-10, IL-2, IL-5, IL- 12, IL- 17, CCL21, TNF-a, IFN-a, IFN-0, or IFN-y. The amino acid sequences of additional representative cytokines from which the therapeutic protein may be derived are provided at the NCBI Accession numbers set forth in Table 6.

[0143] Table 6: Examples of Therapeutic Proteins of Interest

[0144]

[0145]

[0102] In those embodiments wherein the therapeutic protein is or is derived from a cytokine, the cytokine may be CCL21 (NCBI gene ID 6366) or single-chain IL- 12 (derived from IL-12A; NCBI gene ID 3592 and IL-12B; NCBI gene ID 3593).

[0146]

[0103] In some embodiments, the therapeutic protein is a tumor suppressor. In those embodiments wherein the therapeutic protein is or is derived from a tumor suppressor, the tumor suppressor may be derived from GSDMD (NCBI gene ID 79792), CDKN2A (NCBI gene ID 1029), CDK4 (NCBI gene ID 1019), p53 (NCBI gene ID 7157), PTEN (NCBI gene ID 5728), INK4 (NCBI gene ID 1029), RBI (NCBI gene ID 5925), or APC (NCBI gene ID 324).

[0147]

[0104] In some embodiments, the therapeutic protein is a checkpoint inhibitor. Checkpoint inhibitors are inhibitors that target key regulators of the immune system. When the key regulators, or immune checkpoints are stimulated they can dampen the immune response to an immunologic stimulus. In those embodiments wherein the therapeutic protein is or is derived from a checkpoint inhibitor, the checkpoint inhibitor may be an antibody fragment, e.g.. a scFv, that binds EGFR, NKG2A, PD-1, or PD-Ll.

[0148]

[0105] In those embodiments wherein the therapeutic protein is or is derived from a checkpoint inhibitor, the checkpoint inhibitor may be derived from a commercially available anti-EGFR antibody, antibody fragment, or derivative thereof. Additional information relating to the anti-CD3 antibodies from which the therapeutic protein may be derived are provided at the NCBI and FDA GSRS Accession numbers set forth herein, including, e.g., amivantamab (available under the tradename RYBREVANT; NCBI 6WVZ), cetuximab (available under the tradename ERBITUX; GSRS PQX0D8J21J), duligotuzumab (GSRS 8PMF8YQX2T), imgatuzumab (GSRS V77J4WJF9Z), modotuximab (GSRS 1W7BD1M08N), necitumumab (available under the tradename PORTRAZZA; NCBI 6B3S), panitumumab (available under the tradename VECTIBIX; GSRS 6A901E312A), petosemtamab (GSRS Z6Z6IVE7W4), serclutamab (GSRS RY62FD82YX), and zalutumumab (GSRS DA709Q5020).

[0149]

[0106] In those embodiments wherein the therapeutic protein is or is derived from a checkpoint inhibitor, the checkpoint inhibitor may be derived from a commercially available anti-NKG2A antibody, antibody fragment, or derivative thereof, e.g., monalizumab (GSRS 3ZXZ2V0588).

[0150]

[0107] In those embodiments wherein the therapeutic protein is or is derived from a checkpoint inhibitor, the checkpoint inhibitor may be derived from a commercially available anti-PD-1 antibody, antibody fragment, or derivative thereof. Additional information relating to the anti-PD- 1 antibodies from which the therapeutic protein may be derived is provided at the FDA GSRS Accession numbers set forth herein, including, e.g., balstilimab (GSRS 1Q2QT5M7EO), budigalimab (GSRS 6VDO4TY3OO), cadonilimab (GSRS 6FYG1DS4NW), cemiplimab (available under the tradename LIBTAYO; GSRS 6QVL057INT), cetrelimab (GSRS LYK98WP91F), dostarlimab (available under the tradename IEMPERLI; GSRS P0GVQ9A4S5), izuralimab (GSRS 9D2R4T39AY), nivolumab (available under the tradename OPDIVO; GSRS nivolumab), pacmilimab (GSRS 31YO63LBSN), pembrolizumab (available under the tradename KEYTRUDA; GSRS DPT0O3T46P), penpulimab (GSRS IBS1BZ4E4I), peresolimab (GSRS DI4N99Q6KK), pidilizumab (GSRS B932PAQ1BQ), retifanlimab (GSRS 2Y3T5IF01Z), rosnilimab (GSRS 93OKX5KD2L), sintilimab (GSRS 8FU7FQ8UPK), spartalizumab (GSRS QOG25L6Z8Z), tislelizumab (GSRS 0KVO411B3N), toripalimab (GSRS 8JXN261VVA), volrustomig (GSRS K96EGJ218N), vudalimab (GSRS 4I9I5X3Z6N), zeluvalimab (GSRS DX474PAV6P), and zimberelimab (GSRS ZBL7O904IL).

[0151]

[0108] In some embodiments, the therapeutic protein is or is derived from a checkpoint inhibitor, the checkpoint inhibitor may be derived from a commercially available anti-PD-Ll antibody, antibody fragment, or derivative thereof. Additional information relating to the anti-PD-Ll antibodies from which the therapeutic protein may be derived are provided at the FDA GSRS Accession numbers set forth herein, including, e.g., atezolizumab (available under the tradename TECENTRIQ; GSRS 52CMI0WC3Y), avelumab (available under the tradename BAVENCIO; GSRS KXG2PJ551I), cosibelimab (GSRS PNW7GBB44P), danburstotug (GSRS F44C4N45HG), durvalumab (GSRS 28X28X9OKV), inbakicept (GSRS QN4LI58PJ5), lodapolimab (GSRS NR4MAD6PPB), pimivalimab (GSRS MA05AF40UO), and socazolimab (GSRS 82P4S8Q3FB). Signal Peptides

[0152]

[0109] In some embodiments, the nucleic acid coding sequence further includes a sequence that encodes a signal peptide. In some embodiments, these embodiments, the nucleic acid coding sequence encodes a peptide, polypeptide, or protein connected to an in-frame nucleic acid encoding the signal peptide such that a single, contiguous peptide, polypeptide, or protein is translated. The signal peptide may be 5’ or 3’ to the nucleic acid coding sequence. Therapeutic proteins containing a signal peptide and no transmembrane domain will be secreted from a cell. Therapeutic proteins containing a signal peptide and a transmembrane domain will be transported to the cell membrane. Typically, the signal peptide is cleaved from the protein encoded by the nucleic acid coding sequence before secretion or transport to the cell membrane.

[0153] [HO] In some embodiments, the signal peptide is derived from albumin (NCBI gene ID 213), CD8a (NCBI gene ID 925), CD33 (NCBI gene ID 945), erythropoietin (EPO; NCBI gene ID 2056), IL-2 (NCBI gene ID 3558), human Ig-kappa chain V-III (IgK VIII; NCBI gene ID 28912), mouse IgK VIII (NCBI gene ID 667924), tissue plasminogen activator (tPA; NCBI gene ID 5340), or secreted alkaline phosphatase (SEAP; NCBI gene ID 250). Signal peptides may also be synthetic (i.e., non-naturally occurring).

[0154] Therapeutic Nucleic Acids

[0155] [Hl] In some embodiments, the nucleic acid coding sequence does not encode a translated product i.e., peptides, polypeptides, or proteins), but instead is therapeutic in its own right by encoding a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is a miRNA. The miRNA is expressed preferentially in ovarian cancer cells relative to non-cancerous ovarian cells and enables the transcriptional unit to express miRNAs that have anti-cancer or immune- stimulating effects. The miRNA may function as tumor suppressors to inhibit cancer cell growth, metastasis, differentiation, regenerative potential, or a combination of two or more thereof. In some embodiments, the miRNA is let-7a, miR-15a, miR-34a, or miR-200.

[0156] Regulatory Units

[0112] Tn some embodiments, the transcriptional unit further contains at least one additional regulatory unit functional in an ovarian cancer cell. The regulatory unit is a segment of nucleic acids that is capable of increasing expression of the nucleic acid coding sequence by providing a binding motif to endogenous proteins (e.g., transcription factors) to the regulatory unit. Representative regulatory units include enhancers, additional promoters, TATA motifs, and sequences that promote binding to RNA polymerase proteins. The regulatory unit may be disposed 5’ of the synthetic promoter (upstream of the synthetic promoter) or 3’ of the synthetic promoter (disposed between the synthetic promoter and the nucleic acid coding sequence).

[0157]

[0113] In some embodiments, the regulatory unit is derived from the Adenovirus major late promoter.

[0158]

[0114] The nucleic acid sequence of a representative Adenovirus major late promoter is set forth below (SEQ ID NO: 168):

[0159] 1 agacgctagc ggggggctat aaaagggggt gggggcgttc gtcctcactc t

[0160] Vectors

[0161]

[0115] The synthetic promoter or transcriptional unit may be introduced to a cell by a suitable vector. Vectors are configured to contain the elements necessary to effect transport of the synthetic promoter or transcriptional unit into the cell. Such elements may include an origin of replication, a poly-A tail sequence, a selectable marker, and one or more suitable insertion sites for the inclusion of a nucleic acid coding sequence or a transcriptional unit, such as a multiple cloning site (MCS), and additional optional regulatory elements.

[0162]

[0116] In some embodiments, the vector contains a synthetic promoter and an insertion site. The vector is therefore suitable for the inclusion of a nucleic acid coding sequence at the insertion site. In some embodiments, the vector contains a transcriptional unit (which contains a synthetic promoter and a nucleic acid coding sequence).

[0163]

[0117] In some embodiments, the vector is a viral vector, for example, a retroviral vector, a lentiviral vector, an adenoviral vector, a herpesvirus vector, an adenovirus, or an adeno-associated virus (AAV) vector.

[0164]

[0118] In some embodiments, the vector containing the transcriptional unit is a non-integrative and non-replicative recombinant lentivirus vector. The construction of lentiviral vectors has been described, for example, in U.S. Patent Nos. 5,665,577 (Sodroski et al.), 5,981,276 (Sodroski et al.), 6,013,516 (Verma et al ), 7,090,837 (Spencer et al.), 8,119,119 (Mallet et al.), and 10,954,530 (Pule et al.). Lentivirus vectors include a defective lentiviral genome, i.e., in which at least one of the lentivirus genes gag, pol, and env, has been inactivated or deleted.

[0165]

[0119] A lentiviral vector can exhibit functions additional to, or different from, a naturally occurring lentivirus. For example, a lentiviral vector can be modified to change or reduce a lentivirus characteristic. A lentiviral vector also can be modified to exhibit characteristics of one or more other retroviruses, retroviral vectors, host cells or heterologous cells. Modifications can include, for example, pseudotyping, modifying binding and / or fusion functions of the envelope polypeptide, incorporating heterologous, chimeric, or multifunctional polypeptides into the vector, incorporating non-lentivirus genomes, or incorporating heterologous genes into the lentiviral vector genome.

[0166]

[0120] In some embodiments, the vector containing the transcriptional unit is a Herpes Simplex virus (HSV) vector. Construction of HSV vectors has been described, for example, in U.S. Patent Nos. 5,849,572 (Glorioso et al ), 6,261,552 (DeLuca), 6,764,675 (Whitley et al.), 7,531,167 (Glorioso et al.), 7,749,745 (Johnson et al.), 10, 174,341 (Glorioso et al.), and 10,172,893 (Uchida et al.) and U.S. Patent Application Publication Nos. 2019 / 0106709 (Jiaet al.), 2020 / 0071679 (Huang et al.), and 2022 / 0213508 (Glorioso et al.).

[0167]

[0121] In other embodiments, the vector is a non-viral vector, representative examples of which include plasmids, mRNA, linear single stranded (ss) DNA or linear double stranded (ds) DNA, minicircles, and transposon-based vectors, such as Sleeping Beauty (SB)-based vectors and piggyBac (PB)-based vectors. In yet other embodiments, the vector may include both viral and non- viral elements.

[0168]

[0122] In some embodiments the vector is a plasmid. In addition to a promoter operatively linked to the nucleic acids, the plasmid may also contain other elements e.g., that facilitate transport and expression of the nucleic acid in an immune cell. The plasmid may be linearized with restriction enzymes, in vitro transcribed to produce mRNA, and then modified with a 5’ cap and 3’ poly-A tail.

[0169]

[0123] In some embodiments, the vector may be disposed (e.g., encapsulated) in a carrier. The carrier may be lipid-based, e.g., lipid nanoparticles (LNPs), liposomes, lipid vesicles, or lipoplexes. In some embodiments, the carrier is an LNP. In certain embodiments, an LNP includes two or more concentric bilayers separated by aqueous compartments. Lipid bilayers may be functionalized and / or crosslinked to one another. Lipid bilayers may include one or more ligands, proteins, or channels.

[0124] Lipid carriers, e.g., LNPs may include one or more cationic / ionizable lipids, one or more polymer conjugated lipids, one or more structural lipids, and / or one or more phospholipids. A “cationic lipid” refers to positively charged lipid or a lipid capable of holding a positive charge. Cationic lipids include one or more amine group(s) which bear the positive charge, depending on pH. A “polymer conjugated lipid” refers to a lipid with a conjugated polymer portion. Polymer conjugated lipids include a pegylated lipids, which are lipids conjugated to polyethylene glycol. A “structure lipid” refers to a non-cationic lipid that does not have a net charge at physiological pH. Exemplary structural lipids include cholesterol, fecosterol, sitosterol, ergosterol, campesterol, and the like. A “phospholipid” refers to lipids that have a triester of glycerol with two fatty acids and one phosph ate ion. Phospholipids in LNPs assemble the lipids into one or more lipid bilayers. LNPs, their method of preparation, formulation, and delivery are disclosed in, e.g., U.S. Patent Application Publication Nos. 2004 / 0142025 (Maclachlan et al ), 2007 / 0042031 (Maclachlan et al ), and 2020 / 0237679 (Kaufman) and U.S. Patent Nos. 9,364,435 (Yaworski et al ), 9,518,272 (Yaworski et al.), 10,022,435 (Ciaramella et al.), and 11,191,849 (Abrams et al.).

[0170]

[0125] Lipoplexes, liposomes, and lipid nanoparticles may include a combination of lipid molecules, e.g., a cationic lipid, a neutral lipid, an anionic lipid, polypeptide-lipid conjugates, and other stabilization components. Representative stabilization components include antioxidants, surfactants, and salts. Compositions and preparation methods of lipoplexes, liposomes, and lipid nanoparticles are known in the art. See, e. ., U.S. Patent Nos. 8,058,069 (Yaworski et al.), 8,969,353 (Mahon et al.), 9,682,139 (Manoharan et al ), 10,238,754 (Guild et al ), and U.S. Patent Application Publication Nos. 2005 / 0064026 (Garidel et al.) and 2018 / 0291086 (Anchordoquy et al.), and Lasic, Trends Biotechnol. 760:307-21 (1998), Lasic et al., FEBS Lett. 372(2-3) :255-8 (1992), and Drummond et al., Pharmacol. Rev. 5 / 0:691 -743 (1999).

[0171] Cancer

[0172]

[0126] There are also provided methods of transcribing a nucleic acid coding sequence operatively linked to the synthetic promoter in an ovarian cancer cell. The method entails contacting the ovarian cancer cell with a vector encoding the transcriptional unit.

[0127] In some embodiments, the ovarian cancer cell is contacted with the vector in vitro. In some embodiments, a subject in need thereof is administered a pharmaceutical composition containing the vector and a pharmaceutically acceptable carrier (z.c., in vivo) to treat ovarian cancer.

[0173] Screening Methods

[0174]

[0128] There are also provided methods of testing the activity of one or more synthetic promoters in ovarian cancer cells. In some embodiments, a method includes measuring the activity of one or more synthetic promoters in an ovarian cancer cell. In some embodiments, a method includes measuring the activity of more than one synthetic promoter in ovarian cancer cells and comparing the activity of each promoter. For example, a method may include ranking the activity of each promoter. In some embodiments, a method includes identifying a synthetic promoter having the highest activity in an ovarian cancer cell from a group of more than one synthetic promoter, such as a group of five or more, ten or more, or fifteen or more synthetic promoters.

[0175]

[0129] In some embodiments, a method of measuring the activity of a synthetic promoter in an ovarian cancer cell, includes contacting the ovarian cancer cell with a vector including a gene under the control of the synthetic promoter; and measuring expression of the gene in the ovarian cancer cell, thereby measuring the activity of the synthetic promoter. The synthetic promoters used in such methods may be any disclosed herein. In some embodiments, the synthetic promoter includes a nucleic acid including 3 to 15 transcription factor binding sites (TFBS); wherein each TFBS has any one of the following nucleic acid sequences: CCACGTGNi (SEQ ID NO: 1), GCACGTG (SEQ ID NO: 2), AN2N3TGGCGGGAAAC (SEQ ID NO: 3), N4N5TTTTGGCGCCAAAAN6N7 (SEQ ID NO: 4), N8N9TTTTGGCGCCAAAATN10 (SEQ ID NO: 5), CCACGTGNi 1N12N13 (SEQ ID NO: 6), N14GCACGTGGN15 (SEQ ID NO: 7), N16N17TTCCCGCCN18AN19N20 (SEQ ID NO: 8), N21N22GGCGGGAA (SEQ ID NO: 9), GAAAATGGCGCCATTTTT (SEQ ID NO: 10), TN23TTCCCGCCAAAN24 (SEQ ID NO: 11), AGGCGGGAAN25N26 (SEQ ID NO: 12), N27TTN28GCGCN29AN30A (SEQ ID NO: 13), N31TTCCCGCCN32AA (SEQ ID NO: 14), N33N34TCGCGN35GA (SEQ ID NO: 15), TN36TCN37N38GAN39A (SEQ ID NO: 16), or AATATCGCGAGA (SEQ ID NO: 219); wherein each of Ni - N39 independently represents any nucleotide, and which for any of SEQ ID NOs: 1-16 and 219 may be the same or different; and each TFBS may be the same or different. In some embodiments, the synthetic promoter includes any one of SEQ ID NO: 150-218 and 220-273. In some embodiments, the synthetic promoter includes any one of SEQ ID NO: 150-218 and 220-223. In some embodiments, the synthetic promoter includes any one of SEQ ID NO: 150-168 and 220-223.

[0176]

[0130] In some embodiments, a method of ranking the activity of more than one synthetic promoter in ovarian cancer cells, includes for each synthetic promoter of interest, contacting am ovarian cancer cell with a vector including a gene under the control of the synthetic promoter; measuring expression of the gene in each ovarian cancer cell; and comparing expression of the gene in each ovarian cancer cell, thereby comparing the activity of each synthetic promoter in ovarian cancer cells. In some embodiments, the synthetic promoter includes a nucleic acid including 3 to 15 transcription factor binding sites (TFBS); wherein each TFBS has any one of the following nucleic acid sequences: CCACGTGNi (SEQ ID NO: 1), GCACGTG (SEQ ID NO: 2), AN2N3TGGCGGGAAAC (SEQ ID NO: 3), N4N5TTTTGGCGCCAAAAN6N7 (SEQ ID NO: 4), N8N9TTTTGGCGCCAAAATN10 (SEQ ID NO: 5), CCACGTGNi 1N12N13 (SEQ ID NO: 6), N14GCACGTGGN15 (SEQ ID NO: 7), N16N17TTCCCGCCN18AN19N20 (SEQ ID NO: 8), N21N22GGCGGGAA (SEQ ID NO: 9), GAAAATGGCGCCATTTTT (SEQ ID NO: 10), TN23TTCCCGCCAAAN24 (SEQ ID NO: 11), AGGCGGGAAN25N26 (SEQ ID NO: 12), N27TTN28GCGCN29AN30A (SEQ ID NO: 13), N31TTCCCGCCN32AA (SEQ ID NO: 14), N33N34TCGCGN35GA (SEQ ID NO: 15), TN36TCN37N38GAN39A (SEQ ID NO: 16), or AATATCGCGAGA (SEQ ID NO: 219); wherein each of Ni - N39 independently represents any nucleotide, and which for any of SEQ ID NOs: 1-16 and 219 may be the same or different; and each TFBS may be the same or different. In some embodiments, the synthetic promoters include one or more of SEQ ID NO: 150-218 and 220-273. In some embodiments, the synthetic promoters include one or more SEQ ID NO: 150-218 and 220-223. In some embodiments, the synthetic promoters include one or more SEQ ID NO: 150-168 and 220-223.

[0177] Pharmaceutical Compositions

[0178]

[0131] Pharmaceutical compositions include effective amounts of the vector containing the synthetic promoter or transcriptional unit and a pharmaceutically acceptable carrier. The amount of vector in the pharmaceutical composition administered to a subject will vary between wide limits, depending upon one or more factors that may include the location, type, and severity of the cancer, the age, body weight, and condition of the individual to be treated. A physician will ultimately determine appropriate doses to be used.

[0179]

[0132] Compositions may be provided as sterile lyophilized or liquid preparations. Lyophilized preparations may be reconstituted and diluted into a liquid preparation before use, e.g., with carriers containing isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous solutions, which may be buffered to a selected pH. Liquid carriers include aqueous or non-aqueous carriers alike. Representative examples of liquid carriers include sterile water for injection, saline, Lactated Ringer Injection solution, phosphate buffered saline, a soluble protein, soluble sugars (c.g., dextrose), dimethyl sulfoxide (DMSO), polyol ( .g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), ethanol, and suitable mixtures thereof. In some embodiments, the liquid carrier includes a protein dissolved or dispersed therein, representative examples include serum albumin (e.g, human serum albumin, recombinant human albumin), gelatin, and casein. The compositions are typically isotonic, i.e., they have the same osmotic pressure as blood. Citric acid, sodium chloride, sugars, polyalcohols, and isotonic electrolyte solutions (e.g, that available under the tradename PLASMA-LYTE) may be used to achieve the desired isotonicity. Depending on the carrier, and the vector other excipients may be added, e.g, wetting, dispersing, or emulsifying agents, gelling and viscosity enhancing agents, preservatives and the like as known in the art. In some embodiments, the compositions include the vector, sucrose, polysorbate-20, potassium chloride, potassium phosphate, sodium chloride, and sodium phosphate. In some embodiments, the composition is further diluted in a 0.9% normal saline solution.

[0180]

[0133] Additional pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions, their formulation, and delivery of expression vectors such as viral vectors are known in the art. See, for example, U.S. Patent Application Publication Nos. 2013 / 0090374 (Sena-Esteves et al.), and 2012 / 0309050 (Kumon et al.), and U.S. Patent Nos. 7,858,367 (Amalfitano et al.), 11,020,443 (Sena-Esteves et al.), and 11,041,171 (Gao et al.).

[0181] Administration

[0182]

[0134] The amount of pharmaceutical composition containing the vector administered to an ovarian cancer cell or subject may vary between wide limits.

[0135] Tn embodiments where the ovarian cancer cell is contacted with the vector in vitro, the effective amount of the vector is between approximately 0.3 to approximately 5 genome copies (gc) per cell.

[0183]

[0136] In some embodiments, where the ovarian cancer cell is contacted with the vector in vivo, as in a subject to be treated, the amount of pharmaceutical composition or vector will vary between wide limits, depending on factors that may include the location, type, and severity of the ovarian cancer, the age, body weight, and condition of the individual to be treated. A physician will ultimately determine appropriate amount of vector to be used. Typically, the vector will be given in a single dose. In some embodiments, an effective amount of the vector in the pharmaceutical composition may range from approximately! x 106to approximately 5 x 1014gc per kg body weight (gc / kg). In some embodiments, the effective amount of the vector in the pharmaceutical composition may range from approximately 1 x 1010to approximately 2 x 1013gc / kg. In some embodiments, the effective amount of the vector in the pharmaceutical composition is approximately 2 x 1013gc / kg. In some embodiments, the effective amount of the vector in the pharmaceutical composition is approximately 1 x 107gc per subject. In some embodiments, the effective amount of the vector in the pharmaceutical composition is approximately 2 x 108gc per subject. In some embodiments, the effective amount of the vector in the pharmaceutical composition is approximately 1 x 109gc per subject. In some embodiments, the effective amount of the vector in the pharmaceutical composition is approximately 1.5 x 1011gc per subject.

[0184]

[0137] Pharmaceutical compositions containing an effective amount of the vector may be brought into contact with an ovarian cancer cell in vivo by any medically acceptable route. In some embodiments, the pharmaceutical compositions are administered to a subject for the treatment of ovarian cancer. The pharmaceutical composition is may often be delivered intravenously or intraperitoneally, although they may also be introduced into other convenient sites (e.g., intratumorally to an affected organ or tissue) or modes, as determined by an attending physician.

[0185]

[0138] In some embodiments, the pharmaceutical composition is administered as an infusion (e.g., intravenously, or intraperitoneally) to the subject over a predetermined period of time. Representative infusion times are 30 minutes, 60 minutes, 90 minutes, and 120 minutes. In some embodiments, the infusion time is between 60 and 120 minutes. In some embodiments, the pharmaceutical composition is administered intratumorally. Representative intratumoral injection volumes are 0.1 milliliter (mL), 0.2 mb, 0.3 mL, 0.4 mL, 0.5 mL, 1 mL, 2 mL, 5 mL, and 10 mL. Combination Therapy

[0186]

[0139] In some embodiments, the present methods may include co-administration of the pharmaceutical composition along with one or more anti-cancer agents. The term “co-administered” includes substantially contemporaneous administration, by the same or separate dosage forms, or sequentially, e.g., as part of the same treatment regimen or by way of successive treatment regimens. The sequence and time interval may be determined such that the co-administered therapies can act together (e.g., synergistically to provide an increased benefit than if they were administered otherwise). For example, the therapeutics may be administered at the same time or sequentially in any order at different points in time; however, if not administered at the same time, they may be administered sufficiently close in time so as to provide the desired therapeutic effect, which may be in a synergistic fashion.

[0187]

[0140] Anti-cancer agents that may be used in combination with the inventive pharmaceutical compositions are known in the art. See, e. ., U.S. Patent No. 9,101,622 (Zeldis; Section 5.2 thereof). An “anti-cancer” agent is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0188]

[0141] In some embodiments, the provided methods are used before, in conjunction with, or following therapies known in the art, such as chemotherapeutic, radiotherapeutic, immunotherapeutic intervention, targeted therapy, pro-apoptotic therapy, or cell cycle regulation therapy.

[0189]

[0142] These and other aspects of the present application will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain embodiments of the application but are not intended to limit its scope, as defined by the claims.

[0190] EXAMPLES

[0191] Example 1 : Materials and Methods

[0143] Promoter cloning. Synthetic promoters were synthesized by Integrated DNA Technologies (IDT; Coralville, IA) as pooled DNA oligonucleotides. Each individual synthetic promoter was amplified by polymerase chain reaction (PCR). The PCR product was further cloned into vectors with conventional restriction enzyme cloning, upstream of an adenovirus minimal promoter to control the expression of a nucleic acid coding sequence encoding a green fluorescent protein (GFP) gene.

[0192]

[0144] Lentiviral production and infection. Lentiviruses containing a synthetic promoter were produced in HEK-293T cells using co-transfection in a 24-well plate format. In brief, 2.4 pL of FuGENE HD (Promega, Madison, WI) was mixed with 20 pL of Opti-MEM medium (Thermo Fisher Scientific, Waltham, MA) and added to a mixture of the 3 plasmids 0.1 pg of pCMV-VSV- G vector, 0.1 pg of lentiviral packaging psPAX2 vector, and 0.1 pg of lentiviral expression vector containing a synthetic promoter and incubated for 20 minutes. After which, HEK-293 cells were harvested and diluted to a density of 3.6 x io6cells / ml in cell culture medium. 100 pL of diluted cells (containing 3.6 x io5cells) were added to each FuGENE HD / DNA mixture. The culture medium of transfected cells was replaced with 400 pL fresh culture medium 18 h post-transfection. Supernatants containing newly produced viruses were collected 48 h post-transfection and filtered through a 0.45 pm syringe filter. The viral supernatant was used to infect target cells with 8 pg / mL of Polybrene (Sigma) overnight.

[0193]

[0145] Quantifying the tumor-specificity of identified promoters. Each synthetic promoter was operatively linked to a nucleic acid encoding the GFP. Flow cytometry was used to measure the GFP intensity to characterize the synthetic promoter activity in tumor cells and normal cells. To quantify the tumor-specificity of identified promoters, the mean fluorescence intensity (MFI) measured in tumor cells was divided by the MFI measured in normal cells. Data analysis was performed by FlowJo software (TreeStar Inc, Ashland, OR).

[0194] Example 2: Preferential Expression of Synthetic Promoters in Ovarian Cancer Cells

[0195]

[0146] Synthetic promoters having the nucleic acid sequences of SEQ ID NOs: 150-167, were operatively linked to a nucleic acid coding sequence encoding a GFP protein. The transcriptional units were introduced into the ovarian cancer cell line OVCAR8 and the non-cancerous human immortalized ovarian surface epithelium (IOSE) cells and assayed for activity. Normalized activity for each synthetic promoter is illustrated in FIG. 1.

[0196]

[0147] To analyze the preferential expression of the synthetic promoters for ovarian cancer cells as compared to non-cancerous ovarian cells, the expression levels were calculated in terms of a foldincrease based on the activity in the 0VCAR8 ovarian cancer cells against the activity in the non- cancerous ovarian IOSE cells (FIG. 2). The fold-increases of expression of an operatively linked nucleic acid for each synthetic promoter in the ovarian cancer 0VCAR8 cells relative to non- cancerous IOSE cells are summarized in Table 7.

[0197] Table 7: Fold-increase of Synthetic Promoters

[0198] Example 3: Sequence-specific Effects on TFBS Preferential Expression

[0148] To analyze the sequence-specific effects of the TFBSs on preferential expression in ovarian cancer cells, the nucleic acid sequences of select TFBSs (i.e., SEQ ID Nos: 17, 2, 19, 23, 41, 50, 59, 63, 79, 64, 10, 85, 89, 94, 111, 51, 115, and 137) were altered and combined with linkers to produce expanded sets of synthetic promoters described in the paragraphs below, and that contain varying numbers of repeating TFBSs as listed in Tables 8-23, each TFBS separated by a linker. Transcriptional units were then synthesized as per Example 1. The fold-increase in expression was calculated based on a next-generation sequencing-based activity measurement in OVCAR8 cells against the activity in IOSE cells. Tumor-preferentiality indexes were calculated as the percent change of the fold-increase of activity in OVCAR8 cells related to the unaltered synthetic promoter. The data obtained are shown in Tables 8-23, below.

[0199]

[0149] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 1 are set forth in Table 8. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 150 and 169. Each synthetic promoter contains 11 TFBSs, each with the nucleic acid sequences set forth in the table, separated by linkers three nucleotides in length. By way of example, for Table 8, and similarly for Table 9 - Table 23 below, the first row in Table 8 refers to a synthetic promoter having the nucleic acid sequence SEQ ID NO: 150, which has 11 TFBSs each having the sequence identified by SEQ ID NO: 17, wherein the first and second TFBS are connected via the linker AGA, the second and third TFBS are connected via the linker TCG, the third and fourth TFBS are connected via the linker GAC, the fourth and the fifth TFBS are connected via the linker ATA, the fifth and the sixth TFBS are connected via the linker ACT, the sixth and the seventh TFBS are connected via the linker TAC, the seventh and the eighth TFBS are connected via the linker TAG, the eighth and the ninth TFBS are connected via the linker CAT, the ninth and the tenth TFBS are connected via the linker ACT, and the tenth and the eleventh TFBS are connected via the linker GAT. For example, the nucleic acid sequence of SEQ ID NO: 169 includes 11 TFBSs of nucleic acid sequence CACGTGT (SEQ ID NO: 18), and is represented by TFBSis-AGA-TFBSis-TCG- TFBS18-GAC-TFBS18-ATA-TFBS18-ACT-TFBS18-TAC-TFBS18-TAG-TFBS18-CAT-TFBS18- ACT-TFBSis-GAT-TFBSis or has the following nucleic acid sequence:

[0200] CACGTGTAGACACGTGTTCGCACGTGTGACCACGTGTATACACGTGTACTCACGTGTTAC CACGTGTTAGCACGTGTCATCACGTGTACTCACGTGTGATCACGTGT (SEQ ID NO: 169)

[0201]

[0150] The altered tumor-specificity index for each synthetic promoter was calculated as tumorspecificity of the given synthetic promoter divided by the tumor-specificity of synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 150, multiplied by 100 and expressed as a percentage. These data are set forth in Table 8.

[0202] Table 8: Tumor-Specificity of Synthetic Promoters

[0203]

[0151] The synthetic promoter that contains TFBSs that fall within the scope of SEQ ID NO: 2 is set forth in Table 9. Its nucleic acid sequence is set forth in the sequence listing as SEQ ID NO: 151, which contains 11 TFBSs each with the nucleic acid sequence of SEQ ID NO: 2 separated by linkers three nucleotides in length. The altered tumor-specificity index for SEQ ID NO: 151 was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 151, multiplied by 100 and expressed as a percentage.

[0204] Table 9: Tumor-Specificity of Synthetic Promoters

[0205]

[0152] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 3 are set forth in Table 10. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 152 and 170-172. Each synthetic promoter contains 6 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 152, multiplied by 100 and expressed as a percentage.

[0206] Table 10: Tumor-Specificity of Synthetic Promoters

[0153] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 4 are set forth in Table 11. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 153 and 173-179. Each synthetic promoter contains 4 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 153, multiplied by 100 and expressed as a percentage.

[0207] Table 11 : Tumor-Specificity of Synthetic Promoters

[0208]

[0154] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 5 are set forth in Table 12. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 154 and 180-182. Each synthetic promoter contains 4 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides. The altered tumorspecificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 154, multiplied by 100 and expressed as a percentage.

[0209] Table 12: Tumor-Specificity of Synthetic Promoters

[0210]

[0155] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 6 are set forth in Table 13. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 155 and 183-187. Each synthetic promoter contains 8 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 155, multiplied by 100 and expressed as a percentage.

[0211] Table 13: Tumor-Specificity of Synthetic Promoters

[0212]

[0156] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 7 are set forth in Table 14. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 156 and 188-189. Each synthetic promoter contains 8 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 156, multiplied by 100 and expressed as a percentage.

[0213] Table 14: Tumor-Specificity of Synthetic Promoters

[0214]

[0157] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 8 are set forth in Table 15. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 157 and 190-194. Each synthetic promoter contains 6 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 157, multiplied by 100 and expressed as a percentage.

[0215] Table 15: Tumor-Specificity of Synthetic Promoters

[0216]

[0158] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 9 are set forth in Table 16. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 158 and 195-198. Each synthetic promoter contains 8 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 158, multiplied by 100 and expressed as a percentage.

[0217] Table 16: Tumor-Specificity of Synthetic Promoters

[0218] 1159] The synthetic promoter that contains TFBSs that fall within the scope of SEQ ID NO: 10 is set forth in Table 17. Its nucleic acid sequence is set forth in the sequence listing as SEQ ID NO: 160, which contains 4 TFBSs each with the nucleic acid sequence of SEQ ID NO: 10 separated by linkers three nucleotides in length. The altered tumor-specificity index for SEQ ID NO: 160 was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 160, multiplied by 100 and expressed as a percentage.

[0219] Table 17: Tumor-Specificity of Synthetic Promoters

[0220]

[0160] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 11 are set forth in Table 18. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 161 and 199. Each synthetic promoter contains 6 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumorspecificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 161, multiplied by 100 and expressed as a percentage.

[0221] Table 18: Tumor-Specificity of Synthetic Promoters

[0222]

[0161] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 12 are set forth in Table 19. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 162 and 200-202. Each synthetic promoter contains 7 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 162, multiplied by 100 and expressed as a percentage.

[0223] Table 19: Tumor-Specificity of Synthetic Promoters

[0224]

[0162] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 13 are set forth in Table 20. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 163 and 203-206. Each synthetic promoter contains 7 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 163, multiplied by 100 and expressed as a percentage.

[0225] Table 20: Tumor-Specificity of Synthetic Promoters

[0226]

[0163] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 14 are set forth in Table 21. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 164 and 207. Each synthetic promoter contains 7 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumorspecificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 164, multiplied by 100 and expressed as a percentage.

[0227] Table 21 : Tumor-Specificity of Synthetic Promoters

[0228]

[0164] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 15 are set forth in Table 22. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 166 and 208-213. Each synthetic promoter and contains 8 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 166, multiplied by 100 and expressed as a percentage. Table 22: Tumor-Specificity of Synthetic Promoters

[0229]

[0165] The select synthetic promoters that contain TFBSs that fall within the scope of SEQ ID NO: 16 are set forth in Table 23. Their nucleic acid sequences are set forth in the sequence listing as SEQ ID NOs: 167 and 214-218. Each synthetic promoter contains 8 TFBSs each with the nucleic acid sequences set forth in the table separated by linkers three nucleotides in length. The altered tumor-specificity index for each synthetic promoter was calculated as tumor-specificity of the given synthetic promoter divided by the tumor-specificity of the synthetic promoter that has the nucleic acid sequence of SEQ ID NO: 167, multiplied by 100 and expressed as a percentage.

[0230] Table 23: Tumor-Specificity of Synthetic Promoters

[0231] Example 4: In vivo Promoter Activity

[0232]

[0166] Promoters listed in Table 24 were identified by performing a synthetic promoter library screening consisting of approximately 57000 synthetic promoters on a murine ovarian cancer cell line (ID8), and then validated the top hits on three additional murine ovarian cancer cell lines (BPPNM, KPCA, and PPNM). Table 24: Tn Vivo Synthetic Promoter and TFBS Sequences

[0233]

[0167] For cell assays, lentivirus encoding a GFP reporter gene under the control of each synthetic promoter of interest was prepared in HEK-293T cells. HEK-293T cells were transfected to produce lentivirus according to standard protocols. Lentivirus was collected 48 hours after transduction.

[0234]

[0168] ID8 cells were obtained from Millipore Sigma. BPPNM, KPCA, and PPNM cells were obtained from the Robert Weinberg lab. ID8 cells were cultured in DMEM supplemented with 10% fetal bovine serum (Thermo Fisher Scientific; IFS, F4135). BPPNM, KPCA, and PPNM cells were cultured in fallopian tube cells media (FT-media) - DMEM supplemented with 1% Insulin- Transferrin-Selenium (Thermo Fisher Scientific; ITS-G, 41400045), lOOpl EGF (lOug / ml), 4% heat-inactivated fetal bovine serum (Thermo Fisher Scientific; IFS, F4135) and 1% penicillin and streptomycin.

[0235]

[0169] To test the activity of each promoter in cells, harvested lentivirus was diluted 1:4 in DMEM, and added to cultured BPPNM, KPCA, or PPNM tumor cells. BPPNM, KPCA, and PPNM cells were chosen because they are all clinically relevant murine ovarian cancer cell lines. Five days after infection, expression of the GFP reporter gene was measured using flow cytometry. Immortalized Human Ovarian Epithelial Cells (obtained from abm; T1074) were used as normal cell control here. In each cell line, expression was highest when the ZBTB33 V2 promoter (SEQ ID NO: 223) was used (FIG. 3). However, some expression was observed in all cell lines for all promoters (FIG. 3). The Tumor / Normal ratio denotes in the Y-axis of figure 3 was calculated by dividing the mean fluorescence intensities of GFP from the tumor cells to that of the normal cell control.

[0236]

[0170] To test the in vivo activity of each promoter, lentivirus encoding a luciferase reporter gene under the control of each synthetic promoter was produced as described above. A first cohort of mice were injected with BPPNM cells intraperitoneally (i.p.) to create a disseminated ovarian cancer model. Each mouse was injected i.p. with undiluted lentivirus produced as previously described. As a control, a second cohort of mice without murine ovarian cancer tumors were injected with the same dose of lentivirus. Six days after infection, all mice were injected with 3 mg of D-luciferin and bioluminescence was measured using a live animal imaging system (IVIS spectrum optical imaging system, Alameda, CA). For each promoter, minimal bioluminescence was observed in mice without an ovarian cancer cell tumor, but strong bioluminescence was observed in mice harboring an ovarian cancer cell tumor. MYC V2 (SEQ ID NO: 220) and MYC_v4 (SEQ ID NO: 221) showed the strongest radiance per area (FIG. 4).

[0237]

[0171] The results are shown in FIG. 3, which shows the promoters identified by the screening platform active in murine ovarian cancer cell lines (BPPNM, KPCA, and PPNM). Promoter names are listed on the X-axis.

[0238]

[0172] FIG. 4 shows the radiance observed in mice harboring ovarian cancer (BPPNM) or without cancer (control) infected with a lentivirus containing luciferase reporter under the control of a promoter of interest (MYC_v2, MYC_v4, ZBTB33_vl, or ZBTB33_v2). On-target infection and off-target effects in the whole mouse body were identified with luminescence imaging.

[0239]

[0173] Tables 25 and 26 below include the sequences of synthetic promoters encompassed by this disclosure.

[0240] Table 25: Synthetic promoters including defined trinucleotide linkers Table 26: Synthetic promoters including undefined trinucleotide linkers

[0241]

[0174] All patent publications and non-patent publications are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All these publications and Accession numbers, including gene ID numbers, are herein incorporated by reference to the same extent as if each individual publication were specifically and individually indicated as being incorporated by reference.

[0242]

[0175] Various aspects of the foregoing description may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described. The provided synthetic promoters, transcriptional units, vectors, pharmaceutical compositions, and methods are, therefore, not limited in application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

What is claimed is:

1. A synthetic promoter comprising a nucleic acid comprising 3 to 15 transcription factor binding sites (TFBS); wherein each TFBS has any one of the following nucleic acid sequences: CCACGTGNi (SEQ ID NO: 1), GCACGTG (SEQ ID NO: 2), AN2N3TGGCGGGAAAC (SEQ ID NO: 3), N4N5TTTTGGCGCCAAAAN6N7 (SEQ ID NO: 4), NSNQTTTTGGCGCCAAAATNIO (SEQ ID NO: 5), CCACGTGNi 1N12N13 (SEQ ID NO: 6), N14GCACGTGGN15 (SEQ ID NO: 7), N16N17TTCCCGCCN18AN19N20 (SEQ ID NO: 8), N21N22GGCGGGAA (SEQ ID NO: 9), GAAAATGGCGCCATTTTT (SEQ ID NO: 10), TN23TTCCCGCCAAAN24 (SEQ ID NO: 11), AGGCGGGAAN25N26 (SEQ ID NO: 12), N27TTN28GCGCN29AN30A (SEQ ID NO: 13), N31TTCCCGCCN32AA (SEQ ID NO: 14), N33N34TCGCGN35GA (SEQ ID NO: 15), TN36TCN37N38GAN39A (SEQ ID NO: 16), or AATATCGCGAGA (SEQ ID NO: 219); wherein each of Ni - N39 independently represents any nucleotide, and which for any of SEQ ID NOs: 1-16 and 219 may be the same or different; and each TFBS may be the same or different.

2. The synthetic promoter of claim 1, wherein each TFBS is connected to an adjacent TFBS via a linker comprising at least one nucleotide, wherein the linkers may be the same or different.

3. The synthetic promoter of claim 2, wherein the linkers are from two to eight nucleotides in length.

4. The synthetic promoter of claim 2, wherein the linkers are two to four nucleotides in length.

5. The synthetic promoter of claim 2, wherein the linkers are three nucleotides in length.

6. The synthetic promoter of claim 2, wherein the linkers are seven nucleotides in length.

7. The synthetic promoter of any one of claims 1-6, which is about 27 to about 314 nucleotides in length.

8. The synthetic promoter of any one of claims 1 -6, which is about 33 to about 342 nucleotides in length.

9. The synthetic promoter of any one of claims 1-6, which is about 37 to about 370 nucleotides in length.

10. The synthetic promoter of any one of claims 1-6, which contains 3 TFBSs.

11. The synthetic promoter of claim 10, which is about 27 to about 70 nucleotides in length.

12. The synthetic promoter of any one of claims 1-11, wherein at least two TFBSs are the same.

13. The synthetic promoter of any one of claims 1-12, wherein at least three TFBSs are the same.

14. The synthetic promoter of any one of claims 1-13, wherein the TFBSs are the same.

15. The synthetic promoter of any one of claims 1-11, wherein each TFBS is different.

16. The synthetic promoter of any one of claims 1-15, which exhibits at least about a 5-fold increase in activity in an ovarian cancer cell relative to the activity of the synthetic promoter in a non-cancerous ovarian cell.

17. The synthetic promoter of any one of claims 1-15, which exhibits at least about a 10-fold increase in activity in an ovarian cancer cell relative to the activity of the synthetic promoter in a non-cancerous ovarian cell.

18. The synthetic promoter of any one of claims 1-15, which exhibits at least about a 20-fold increase in activity in an ovarian cancer cell relative to the activity of the synthetic promoter in a non-cancerous ovarian cell.

19. The synthetic promoter of any one of claims 1 -15, which exhibits at least about a 50-fold increase in activity in an ovarian cancer cell relative to the activity of the synthetic promoter in a non-cancerous ovarian cell.

20. The synthetic promoter of any one of claims 1-19, wherein for SEQ ID NO: 1, Ni is C or T, wherein for SEQ ID NO: 3, N2 is A or T, and N3 is A or T, wherein for SEQ ID NO: 4, N4 is A, C, or G, Ns is A, G, or T, Ne is A, C, or T, and N7 is any nucleotide, wherein for SEQ ID NO: 5, Ns is A or G, N9 is A, G, or T, and N10 is C or G, wherein for SEQ ID NO: 6, N11 is C or G, N12 is C or T, and N13 is C, G, or T, wherein for SEQ ID NO: 7, N14 is A or C and Nis is C or T, wherein for SEQ ID NO: 8, Ni6 is any nucleotide, N17 is A, G, or T, Nis is A or T, N19 is A or T, and N20 is A or T, wherein for SEQ ID NO: 9, N21 is A, G, or T and N22 is A or T, wherein for SEQ ID NO: 11, N23 is A or G, N24 is A or T, and N41 is A or T, wherein for SEQ ID NO: 12, N25 is A or G and N26 is A, C, or G, wherein for SEQ ID NO: 13, N27 is A, G, or T, N28 is C or G, N29 is C or G, and N30 is A, C, or T, wherein for SEQ ID NO: 14, N31 is G or T and N32 is A or T, wherein for SEQ ID NO: 15, N33 is any nucleotide, N34 is A, C, or G, and N35 is A or G, and wherein for SEQ ID NO: 16, N36 is C or G, N37 is A or G, N38 is C or T, and N39 is C, G, or T.

21. The synthetic promoter of any one of claims 1-20, wherein the TFBSs have one or more of the nucleic acid sequences of SEQ ID NO: 17-149.

22. The synthetic promoter of any one of claims 1-21, wherein each TFBS has any one of the nucleic acid sequences of SEQ ID NOs: 2, 10, 17, 19, 23, 41, 50, 51, 59, 63, 64, 79, 85, 89, 94, 111, 115, and 137.

23. The synthetic promoter of claim 1, in which the synthetic promoter is represented by the formula:TFB S i-Li-TFB S2-L2-TFB S3-L3-TFB S4-L4-TFB S5-L5-TFB S6-L6-TFB S7-L7-TFB Ss-Ls- TFBS9-L9-TFBS10-L10-TFBS11-L11-TFBS12-L12-TFBS13-L13-TFBS14-L14-TFBS15; wherein each of TFBS1-TFBS15 independently has any one of the nucleic acid sequences of SEQ ID NOs: 1-149, and wherein any one or more of TFBS4-TFBS15 may be absent; wherein each of L1-L14 independently represents a linker from two to eight nucleotides in length, and wherein each linker independently represents N40N41N42N43N44N45N46N47, wherein each of N40-N47 may be any one of A, C, T, or G, and may be the same or different, and wherein any one or more of N42-N47 may be absent.

24. The synthetic promoter of claim 1, which has the sequence of any one of the nucleic acid sequences of SEQ ID NOs: 150-167, 169-218, or 220-223.

25. The synthetic promoter of claim 1, which has the sequence of any one of the nucleic acid sequences of SEQ ID NOs: 150-167 or 220-223.

26. A transcriptional unit comprising the synthetic promoter of any one of claims 1-25 operatively linked to a nucleic acid coding sequence that encodes a therapeutic nucleic acid, peptide, polypeptide, or protein.

27. The transcriptional unit of claim 26, further comprising at least one spacer; wherein the at least one spacer comprises 30 nucleotides or less, and which is disposed 3’ and / or 5’ of the synthetic promoter.

28. The transcriptional unit of claim 26 or 27, wherein the nucleic acid coding sequence encodes a therapeutic protein or therapeutic nucleic acid.

29. The transcriptional unit of claim 28, wherein the therapeutic protein is a transcription factor.

30. The transcriptional unit of claim 29, wherein the transcription factor is a GAL4-VP16, GAL4-VP64, or GAL4VPR fusion protein.

31. The transcriptional unit of claim 28, wherein the therapeutic protein comprises a surface T cell engager (STE), a cytokine, or a checkpoint inhibitor.

32. The transcriptional unit of claim 31, wherein the therapeutic protein is a STE comprising an anti-CD3 single-chain variable antibody fragment (scFv) and a membrane anchor domain.

33. The transcriptional unit of claim 31, wherein the therapeutic protein comprises CCL21.

34. The transcriptional unit of claim 31, wherein the therapeutic protein comprises IL-12.

35. The transcriptional unit of claim 31, wherein the therapeutic protein is Gasdermin-D (GSDMD).

36. The transcriptional unit of claim 31, wherein the therapeutic protein comprises a checkpoint inhibitor comprising an EGFR inhibitor, a NKG2A inhibitor, PD-1 inhibitor, or a PD-L1 inhibitor.

37. The transcriptional unit of any one of claims 31-36, wherein nucleic acid coding sequence further comprises nucleic acid encoding a signal peptide.

38. The transcriptional unit of claim 28, wherein the nucleic acid coding sequence encodes a miRNA.

39. The transcriptional unit of claim 38, wherein the miRNA is let-7, miR-15 / 16, miR-34, or miR-200.

40. The transcriptional unit of any one of claims 26-39, further comprising a regulatory unit functional in an ovarian cancer cell.

41. The transcriptional unit of claim 40, wherein the regulatory unit is derived from the Adenovirus major late promoter.

42. A vector comprising the synthetic promoter of any one of claims 1-25.

43. A vector comprising the transcriptional unit of any one of claims 26-41.

44. A pharmaceutical composition comprising the vector of any one of claims 42-43 and a pharmaceutically acceptable carrier.

45. A method of transcribing a nucleic acid coding sequence in an ovarian cancer cell, comprising: contacting the ovarian cancer cell with the vector of any one of claims 42-43.

46. The method of claim 45, wherein the contacting is conducted in vitro.

47. The method of claim 45, wherein the contacting is conducted in vivo in a subject.

48. A method of measuring the activity of a synthetic promoter in an ovarian cancer cell, comprising: contacting the ovarian cancer cell with a vector comprising a gene under the control of the synthetic promoter; and measuring expression of the gene in the ovarian cancer cell, thereby measuring the activity of the synthetic promoter; wherein the synthetic promoter comprises a nucleic acid comprising 3 to 15 transcription factor binding sites (TFBS); wherein each TFBS has any one of the following nucleic acid sequences: CCACGTGNi (SEQ ID NO: 1), GCACGTG (SEQ ID NO: 2), AN2N3TGGCGGGAAAC(SEQ ID NO: 3), N4N5TTTTGGCGCCAAAAN6N7 (SEQ ID NO: 4), N8N9TTTTGGCGCCAAAATN10 (SEQ ID NO: 5), CCACGTGN11N12N13 (SEQ ID NO: 6), N14GCACGTGGN15 (SEQ ID NO: 7), N16N17TTCCCGCCN18AN19N20 (SEQ ID NO: 8), N21N22GGCGGGAA (SEQ ID NO: 9), GAAAATGGCGCCATTTTT (SEQ ID NO: 10), TN23TTCCCGCCAAAN24 (SEQ ID NO: 11), AGGCGGGAAN25N26 (SEQ ID NO: 12), N27TTN28GCGCN29AN30A (SEQ ID NO: 13), N31TTCCCGCCN32AA (SEQ ID NO: 14), N33N34TCGCGN35GA (SEQ ID NO: 15), TN36TCN37N38GAN39A (SEQ ID NO: 16), or AATATCGCGAGA (SEQ ID NO: 219); wherein each of Ni - N39 independently represents any nucleotide, and which for any of SEQ ID NOs: 1-16 and 219 may be the same or different; and each TFBS may be the same or different.

49. A method of ranking the activity of more than one synthetic promoter in ovarian cancer cells, comprising: for each synthetic promoter of interest, contacting am ovarian cancer cell with a vector comprising a gene under the control of the synthetic promoter; measuring expression of the gene in each ovarian cancer cell; and comparing expression of the gene in each ovarian cancer cell, thereby comparing the activity of each synthetic promoter in ovarian cancer cells; wherein the synthetic promoter comprises a nucleic acid comprising 3 to 15 transcription factor binding sites (TFBS); wherein each TFBS has any one of the following nucleic acid sequences: CCACGTGNi (SEQ ID NO: 1), GCACGTG (SEQ ID NO: 2), AN2N3TGGCGGGAAAC (SEQ ID NO: 3), N4N5TTTTGGCGCCAAAAN6N7 (SEQ ID NO: 4), N8N9TTTTGGCGCCAAAATN10 (SEQ ID NO: 5), CCACGTGNi 1N12N13 (SEQ ID NO: 6), N14GCACGTGGN15 (SEQ ID NO: 7), N16N17TTCCCGCCN18AN19N20 (SEQ ID NO: 8), N21N22GGCGGGAA (SEQ ID NO: 9), GAAAATGGCGCCATTTTT (SEQ ID NO: 10), TN23TTCCCGCCAAAN24 (SEQ ID NO: 11), AGGCGGGAAN25N26 (SEQ ID NO: 12), N27TTN28GCGCN29AN30A (SEQ ID NO: 13), N31TTCCCGCCN32AA (SEQ ID NO: 14), N33N34TCGCGN35GA (SEQ ID NO: 15), TN36TCN37N38GAN39A (SEQ ID NO: 16), or AATATCGCGAGA (SEQ ID NO: 219);wherein each of Ni - N39 independently represents any nucleotide, and which for any of SEQ ID NOs: 1-16 and 219 may be the same or different; and each TFBS may be the same or different.

Images