Synthetic genetic constructs that induce gene expression in response to tumor-specific metabolic alterations

Abstract

Exemplary engineered synthetic genetic constructs are disclosed that can induce high levels of gene expression in response to changes in metabolism that are unique to solid tumors. These synthetic genetic constructs, hereafter referred to as promoters, can function in human cells, and can be used to specify or enhance the activity of chimeric antigen receptor (CAR) T cell therapies. CAR-T cell therapies are an important class of immunotherapies that is predicted to grow to be a 15B market by 2028 in the US. CAR-T cells and other autologous immune cell therapies have been shown to be deactivated by adenosine, an immunosuppressive small metabolite that accumulates to high levels in solid tumors. Accumulation of adenosine (e.g., ribonucleoside adenosine) can trigger a CREB-mediated signaling pathway to suppress the function of immune cells in solid tumor environments.

Description

SYNTHETIC GENETIC CONSTRUCTS THAT INDUCE GENE EXPRESSION IN RESPONSE TO TUMOR-SPECIFIC METABOEIC AETERATIONSCROSS REFERENCE TO REEATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 513,190, filed July 12, 2023, which is incorporated by reference herein in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. DP2CA280622, awarded by the National Institutes of Health. The government has certain rights in this invention.REFERENCE TO SEQUENCE LISTING

[0003] The sequence listing submitted on July 11, 2024, as an .XML file entitled “10034- 293W01_ST26.xml” created on July 9, 2024, and having a file size of 111,907 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).BACKGROUND

[0004] The ability to precisely activate protein expression in a tissue or environmentspecific manner in human cells could greatly improve therapies for disease (Gamboa et al. 2020). To this end, synthetic promoters that are activated by specific external states or exogenous inducers have recently been developed to control gene expression in human cells (Cazier et al. 2021) (Ede et al. 2016) (Miller et al. 2018). These inducible synthetic promoters typically exist in an “OFF” state - necessitating a specific input to activate and turn “ON” gene expression. For example, synthetic promoters constructed from tandem repeats of heat-shock transcription factor binding sites can be activated by with near infrared radiation (NIR)- controllable increases in temperature. These heat-inducible promoters have been utilized to turn on the expression of chimeric antigen receptors (CARs) and other T cell proteins with precise spatial control, and specifically in solid tumors (Miller et al. 2021). Synthetic promoters that have a native “ON” state and an inducible “OFF” can also be useful. In fact, induced “ON” and induced “OFF” synthetic promoters can be used in the same cell, for instance, to repress transcription with light and activate gene expression with doxycycline (Yamada et al. 2018).

[0005] There are often tradeoffs with the synthetic promoters, as they typically require user-administered inducers (e.g., heat, light, or exogenous chemicals) (Miller et al. 2018)(Yamada et al. 2018). Delivery of heat or light through skin to hard-to-reach physiological locations limits their practical and clinical application, and administration of exogenous chemical inducers can be hindered when the drug diffuses elsewhere in the body, compromising effective transcriptional control and potentially broaching safety concerns (Shen et al. 2020) (Zhang et al. 2019) (Sun et al. 2022) (Zhao et al. 2020). Synthetic promoters that can respond to local environmental cues to control gene expression in human cells may be more desirable, particularly if the local environmental cue were associated with a disease state.

[0006] Thus, there is a need for improved synthetic promoters. This need and others are at least partially satisfied by the present disclosure.SUMMARY

[0007] Exemplary engineered synthetic genetic constructs are disclosed that can induce high levels of gene expression in response to changes in metabolism that are unique to solid tumors. These synthetic genetic constructs, hereafter referred to as promoters, can function in human cells and can be used to specify or enhance the activity of chimeric antigen receptor (CAR) T cell therapies. CAR-T cell therapies are an important class of immunotherapies that is predicted to grow to be a 15B market by 2028 in the US. CAR-T cells and other autologous immune cell therapies have been shown to be deactivated by adenosine, an immunosuppressive small metabolite that accumulates to high levels in solid tumors. Accumulation of adenosine (e.g., ribonucleoside adenosine) can trigger a CREB-mediated signaling pathway to suppress the function of immune cells in solid tumor environments.

[0008] The collection of CREB -activated promoters can allow for strong and tunable adenosine-induced gene expression in human cells, and that can be used to activate cellular functions that can help CAR-T cell therapies overcome solid tumor immune suppression. By optimizing the number of CREB transcription factor binding sites and altering the core promoter region of CREB-based hybrid promoters, synthetic constructs can be created that can drive gene expression to higher levels than strong, endogenous mammalian promoters in the presence of adenosine. These synthetic adenosine-activated promoters can be induced up to 47-fold by adenosine, as the presence of CREB binding sites does not result in substantial gene expression without adenosine induction. The disclosed CREB-based promoters can further be activated by other compounds that act as signaling analogs, and the addition of low levels of adenosine and these compounds can have a synergistic impact on gene expression. CREB- based promoter strength can be moderately enhanced by incorporating other transcription factor binding sites. More broadly, the adenosine-activated promoters can be utilized to drivethe expression of any gene solely in the solid tumor environment, which can have applications in drug delivery, cell therapy, cancer detection, and basic biological research.

[0009] In an aspect, provided is a construct including a transcriptional factor binding site and a core promoter, wherein the transcriptional factor binding site includes two or more cAMP response element (CRE) motifs.

[0010] In another aspect, provided is a vector including any of the disclosed constructs and a target gene downstream of the core promoter.

[0011] In yet another aspect, provided is an engineered cell including any of the disclosed vectors.

[0012] In yet still aspect, provided is a method of treating a solid tumor in a subject, the method including administering to the subject a therapeutically effective amount of a vector comprising a transcriptional factor binding site, a core promoter, and a target gene downstream of the core promoter, wherein the transcriptional factor binding site includes two or more cAMP response element (CRE) motifs.

[0013] Other systems, methods, features and / or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and / or advantages be included within this description and be protected by the accompanying claims.BRIEF DESCRIPTION OF DRAWINGS

[0014] FIGURES 1A-1D depict creating synthetic CREB -responsive promoters. FIG. 1A is a depiction of adenosine signaling. Adenosine (red spheres) binds adenosine receptors A2AR / A2BR, which mobilizes the associated G protein (green) to activate adenylyl cyclases (orange receptor) and convert ATP into cAMP. Alternatively, forskolin (orange spheres) can activate the adenylyl cyclases directly. cAMP binds PKA to phosphorylate CREB, which binds the palindromic DNA motif “TGACGTCA,” activating gene expression. FIG. IB shows promoter design and screening schematic. CREs (highlighted yellow) were cloned in 3x replicates, flanked with a guanine “G” (underlined) with six interspersed filler nucleotides (n). 3x CREs (gray squares) were then placed in 1-6 replicates upstream of a core promoter (blue arrow). Promoter activity was quantified with either Gaussian Luciferase (GLuc) or green fluorescent protein (eGFP). FIGS. 1C-1D show that HEK293T cells were reverse transfected with the indicated constructs (x-axis) in 96 well plates. 48hrs after transfection, the cell media was changed to media with vehicle (DMSO, light blue bars) or 20pM forskolin (Fsk, dark bluebars). Eight hours later, media was sampled and tested for GLuc activity (by RLU). Bars represent average of n=3 experimental replicates and error bars represent standard error (SEM).

[0015] FIGURE 2 depicts that promoters harboring less than three response elements are nonfunctional. Jurkat cells were transduced with promoter constructs bearing two CRE sequence elements (2x-CRE_YB) or the YB core promoter with zero response elements (YB). As a positive control, they were also transduced with the murine PGK promoter. Transduced cells were sorted by tagBFP, and then seeded with the indicated media at 1 x 106cells / mL in a 96 well plate. 24 hours later, cell supernatant was sampled and analyzed for the quantity of GLuc by femtograms per cell. Bars represent the average of n=3 experimental replicates and error bars are SEM. ND = not detectable, or below standard curve.

[0016] FIGURES 3A-3C depict synthetic promoter responsiveness to adenosine. FIGS. 3A-3B show promoter inductions by flow cytometry. HEK293T cells were reverse transfected with the indicated constructs (x-axis) in 96 well plates. 48hrs after transfection, cell media was changed to untreated media (light blue bars), or media supplemented with 0.750mM adenosine (ADO, dark blue bars). Eight hours later, cells were trypsinized and resuspended in FACS buffer for flow cytometry. Y-axis represents the eGFP median fluorescent intensity of forward scatter (FSC) singlets. Bars represent average of n=3 experimental replicates and error bars represent standard error (SEM). FIG. 3C shows promoter dose responsiveness to adenosine. HEK293T cells were reverse transfected in 96 well plates with the constructs indicated in the legend and then cultured for 48 hours. Media was then changed to add different adenosine concentrations, sampled after 8 hours, and tested for GLuc activity (RLU).

[0017] FIGURES 4A-4D depict that FBS-mediated adenosine degradation affects cellular responsiveness to adenosine. FIG. 4A shows media background adenosine degradation. HEK293T cell media (DMEM) was supplemented with ImM adenosine and (i) no FBS (purple), (ii) 10% FBS (white diamonds), (iii) 10% FBS with 30-minute heat deactivation (HD) (light blue circles), (iv) 10% FBS with 6hr HD (medium blue triangles), or (v) 10% FBS with 24hr HD (dark blue squares). Media was sampled at the indicated time points, diluted in PBS, and read by plate reader for absorbance at 265nm. Each point represents n=3 experimental replicates and error bars represent standard error (SEM). FIG. 4B shows promoter dose responsiveness to adenosine in fully heat-deactivated media. HEK293T cells were reverse transfected with the indicated constructs (9x-CRE_minCMV = green, 12x-CRE_YB = dark blue, minCMV = light green, and YB = light blue) in 96 well plates. 48 hours later, media was changed to media with the adenosine concentrations indicated on the x-axis. FIG. 4C shows 12xYB promoter responsiveness to fully heat deactivated (HD) or regular media. The 12x-CRE_YB promoter was transfected into HEK293T cells in 96 well plates. 48 hours later, adenosine was titered in either HD (blue squares) or regular media (light blue circles). 8 hours later, the cells were collected and analyzed by flow cytometry to eGFP median fluorescent intensity (MFI). Each point represents the mean of n=3 biological replicates, and error bars represent SEM. FIG. 4D shows 9x-CRE_minCMV promoter responsiveness compared in HD or regular media. The experiment is performed as in FIG. 4C.

[0018] FIGURES 5A-5D depict promoter responsiveness to other cAMP stimulating compounds. FIGS. 5A-5B show promoter responsiveness to prostaglandin E2 (PGE2) or norepinephrine (Norep). HEK293T cells were reverse transfected with either the 9x- CRE_minCMV (dark blue circles) or 12xYB (light blue circles) promoters. 48 hours later, media was changed to the indicated PGE2 or Norep concentration (x-axis) and analyzed by GLuc 8 hours later. Fold induction is relative to the untreated control. Each point is the average of n=3 experimental replicates and error bars are standard error (SEM). FIG. 5C shows fold induction comparisons between adenosine (ADO), PGE2 and Norep. HEK293T cells were reverse transfected with the 9x-CRE_minCMV promoter, media changed 48 hours after with the indicated media, and read for GLuc activity. Fold induction is quantified by untreated controls. Each bar is the average of n=3 experimental replicates and error bars = SEM. FIG. 5D shows promoter responsiveness to different combinations of each cAMP-stimulating compound. HEK293T cells were transfected with the 9x-CRE_minCMV promoter and then cultured for 48 hours. Then, the media was changed to the supplemented media indicated on the x-axis and, after 8 hours, read for GLuc activity (by RLU, y-axis).

[0019] FIGURES 6A-6B depict that the 9x-CRE_minCMV promoter outperforms endogenous promoters and has favorable on / off kinetics within engineered cell lines. FIG. 6A shows that the 9x-CRE_minCMV promoter outperforms a constitutively active endogenous promoter when in an induced state. 9x-CRE_minCMV and PGK viral constructs were transduced into HEK293T and sorted based on tagBFP. 5 x 104sorted cells were seeded in 96 well plates and treated with (dark blue) or without (light blue) 20pM forskolin (Fsk). After 24- hour treatment, the cells were analyzed by flow cytometry for eGFP median fluorescent intensity (MFI). Bars are the average of n=3 experimental replicates and error bars = standard error (SEM). FIG. 6B shows HEK293T cell lines engineered with the 9x-CRE_minCMV promoter were treated with either (i) 20pM Fsk, dark blue circles and line (ii) 250pM adenosine (ADO), medium blue squares and line every 12 hours or (iii) 25|lM ADO, 4.17p.M PGE2, and 6. ! 25pM Norep every 12 hours (Combo), light blue triangles. After the indicated treatmenttimes, the cells were analyzed by flow cytometry for eGFP MFI, normalized to untreated cells to achieve fold induction. The black arrow represents when media was changed to adenosine + recombinant ADA1 media to observe promoter shut-off. Each point represents the average of 3 experimental replicates and error bars = SEM.

[0020] FIGURE 7 depicts that stable cell lines harboring the PGK and minCMV promoters are not sensitive to adenosine or combinatorial induction. HEK293T stable cell lines expressing either the minCMV or PGK promoters were seeded in 96 well plates. After attachment, the cells were exposed to the indicated compound for 24 hours until harvesting for flow cytometry. Fold induction is compared to untreated control. Bars represent the average of n=3 experimental replicates and error bars = SEM.

[0021] FIGURES 8A-8C depict improving the strength of the 12x-CRE_YB promoter through incorporation of RELA sites. FIG. 8A is an image depiction of the expected mechanism of RELA promoter strength augmentation. NFkB (RELA, purple) is recruited to proximal DNA binding sequences (dark blue), allowing recruitment of CREB binding protein (CREB-BP, light gray), which through its lysine acetyltransferase (KAT) domain, acetylate CREB dimers at palindromic CRE sites (light blue, dark gray) to improve their transcriptional activity. FIG. 8B is a schematic of promoter designs. Three RELA sites (dark blue) were interspersed between 6xCREs (dark gray) and called the 3x-RELA-12x-CRE_YB. RELA sites were removed from 3’ to 5’ by one to form 2x and lx sequences, respectively. FIG. 8C shows a comparison of RELA-supplemented 12x-CRE_YB promoters. HEK293T cells were transfected with the indicated promoter construct and titrated at different adenosine concentrations for eight hours. After which, the media was sampled for GLuc activity. Each point represents the average of n=3 experimental replicates and error bars are SEM.

[0022] FIGURE 9 shows that the CMV promoter is weakly activated by adenosine. HEK293T cells were transfected with the CMV promoter driving expression of GLuc-P2A- eGFP. Then, the cells were titrated with the indicated concentration of adenosine. After eight hours, the supernatant was harvested and analyzed for GLuc activity. Fold induction is compared to untreated control. Each point represents the average of n=3 experiments and error bars = SEM.

[0023] FIGURES 10A-10C depict that CREB -activated promoters are functional in other cells. Each cell line was transduced with either the 9x-CRE_minCMV (dark blue) or 12x- CRE_YB promoter (light blue) and sorted on the basis of tagBFP. Each cell type was seeded at 5 x 104cells / well in a 96 well plate. For Jurkat cells, they were directly seeded in the titrated forskolin media, but for 4T1 and MAD109, the cell lines were allowed to attach before themedia was changed to forskolin-containing media. After 24 hours, the media was sampled and analyzed for GLuc activity by RLU. Each point represents the average of n=3 experimental replicates and error bars = SEM.

[0024] FIGURE 11 depicts that the 9x-CRE_minCMV promoter outperforms endogenous promoters and can produce immunomodulatory cytokines within engineered cell lines. HEK293T cell lines engineered to express human interleukin 2 (IL-2) from the indicated promoter on the x-axis were treated with the indicated compound. Cell lines were treated with 20pM Forskolin (Fsk), 250pM adenosine (ADO) every 12 hours, or left untreated. Supernatant was harvested for Fsk treated samples after 24 hours, while ADO and untreated samples were harvested after 48 hours. IL-2 concentration (ng / mL) was determined by ELISA and ND = not detected within our lowest standard concentration. Bars represent the average of N=3 experimental replicates and error bars = SEM.

[0025] FIGURE 12 depicts localization analysis of 9x-CRE_minCMV induced promoter compared to PGK control. Enlargement of IVIS images from CAR T cell localization study showing better localization of 9x-CRE_minCMV (middle) compared to PGK control. The numbers below the image represent the tumor or full body radii of interest (ROIs) in photons per second. The far-right numbers represent an average of N=3 mice that received CAR T cells engineered without Flue along with a representative image. Percent localization (% loc) was calculated by subtracting CAR only average background signals from each respective tumor or body ROI signal.DETAILED DESCRIPTION

[0026] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment. To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.DEFINITIONS

[0027] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like.

[0028] Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0029] ‘Administration” or “administering” to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra- articular, intra- synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. "Concurrent administration", "administration in combination", "simultaneous administration" or "administered simultaneously" as used herein, means that thecompounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. “Systemic administration” refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject’s body, for example through entrance into the circulatory or lymph systems. By contrast, “local administration” refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject’s body. Administration includes self-administration and the administration by another.

[0030] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0031] The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

[0032] “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0033] A DNA sequence that "encodes" a particular RNA is a DNA nucleic acid sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or aDNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a "non-coding" RNA (ncRNA), a guide RNA, etc.).

[0034] "Expression vector" or “vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.)

[0035] The “fragments,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc. In any case, the fragment must possess a bioactive property, such as regulating the transcription of the target gene.

[0036] The term "gene" or "gene sequence" refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a "gene" as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term "gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site).

[0037] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are thensaid to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) nucleotide sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the nucleotides in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0038] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0039] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0040] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0041] The term “increased” or “increase” as used herein generally means an increase by a statically significant amount; for the avoidance of any doubt, “increased” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level so long as the increase is statistically significant.

[0042] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decreasecan be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0043] The term "nucleic acid" as used herein means a polymer composed of nucleotides, e.g., deoxy ribonucleotides (DNA) or ribonucleotides (RNA). The terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed of ribonucleotides. The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxyribonucleotides .

[0044] “Optional” or “optionally” means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0045] As used herein, "operatively linked" can indicate that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence. This same definition is sometimes applied to the arrangement of coding sequences and / or transcription control elements (e.g. promoters, enhancers, and termination elements), and / or selectable markers in an expression vector. The term "operatively linked" can also refer to the arrangement of polypeptide segments within a single polypeptide chain, where the individual polypeptide segments can be, without limitation, a protein, fragments thereof, linking peptides, and / or signal peptides. The term operatively linked can refer to direct fusion of different individual polypeptides within the single polypeptides or fragments thereof where there are no intervening amino acids between the different segments as well as when the individual polypeptides are connected to one another via one or more intervening amino acids.

[0046] The term "polynucleotide" refers to a single or double stranded polymer composed of nucleotide monomers.

[0047] The term "polypeptide" refers to a compound made up of a single chain of D- or L- amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

[0048] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0049] A “chimeric antigen receptor” is an artificial cell receptor used for immunotherapy. CAR are protein receptors that have been engineered to give immune cells (e.g., T cells) anenhanced ability to target a specific protein. CAR receptors are chimeric because the antigen binding and cell activating functions have been combined into a single receptor.

[0050] The terms "cell," "cell line" and "cell culture" include progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological property, as screened for in the originally transformed cell, are included. The "host cells" used in the present invention generally are prokaryotic or eukaryotic hosts.

[0051] "Pharmaceutically acceptable" component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to a human, the term generally implies the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0052] "Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and / or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil / water or water / oil emulsion) and / or various types of wetting agents. As used herein, the term "carrier" encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.

[0053] “Pharmacologically active” (or simply “active”), as in a “pharmacologically active” derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.

[0054] As used herein, by a “subject” is meant an individual. Thus, the “subject” can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, chickens, ducks, geese, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. “Subject” can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient.

[0055] The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating, or reducing the intensity of one or more attendant symptoms of a disorder or condition and / or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively, or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g., upon initial signs and symptoms of cancer), or after an established development of cancer. Prophylactic administration can occur for several days to years prior to the manifestation of symptoms of a cancer.

[0056] ‘Effective amount” of an agent refers to a sufficient amount of an agent to provide a desired effect. The amount of agent that is “effective” will vary from subject to subject, depending on many factors such as the age and general condition of the subject, the particular agent, or agents, and the like. Thus, it is not always possible to specify a quantified “effective amount.” However, an appropriate “effective amount” in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of an agent can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of an agent necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0057] “Therapeutic agent” refers to any composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a cancer). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “therapeutic agent” is used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0058] “Therapeutically effective amount” or “therapeutically effective dose” of a composition (e.g., a composition comprising an agent) refers to an amount that is effective to achieve a desired therapeutic result. In some embodiments, a desired therapeutic result is the control of cancer. In some embodiments, a desired therapeutic result is the control of metastasis. In some embodiments, a desired therapeutic result is the reduction of tumor size. In some embodiments, a desired therapeutic result is the prevention and / or treatment of relapse. Therapeutically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect, such as pain relief. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the agent and / or agent formulation to be administered (e.g., the potency of the therapeutic agent, the concentration of agent in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years.CONSTRUCTS, VECTORS, AND CELLS

[0059] The ribonucleoside adenosine can accumulate to levels approximately 100 to 1000- fold in solid tumors than it does in healthy tissue (i.e., nanomolarmicromolar levels), and tumor- accumulated adenosine has been shown to directly stimulate receptor-based signaling in several immune cells, particularly in T cells, inhibiting their inflammatory or cytotoxic capabilities (Jennings et al. 2021). When adenosine binds to the cell-surface adenosine receptors A2AR or A2BR, their coupled Gsproteins activate adenylyl cyclases that produce 3’5’- cyclic adenosine monophosphate (cAMP), the ligand for protein kinase A (PKA) (Sheth et al. 2014). Activated PKA phosphorylates the cAMP response element binding protein (CREB) at Serine 133 (Oberprieler et al. 2010). pCREB is a transcription factor, binding the palindromic cAMP response element motif (CRE motif), 5’-TGACGTCA-3’, and recruiting other coactivators to upstream enhancers / promoters to induce transcription (Mayr et al. 2001) (Gonzalez et al. 1989). In summation, through the cAMP / PKA signaling pathway, extracellular adenosine can control cellular gene expression (FIGS. 1A-1B). Thus, by incorporating CREs within a synthetic promoter, it may be possible to engineer cells that exhibit tumor-activated gene expression through adenosine induction.

[0060] In an aspect, provided is a construct including a transcriptional factor binding site and a core promoter, wherein the transcriptional factor binding site includes two or more cAMP response element (CRE) motifs.

[0061] “Transcriptional factors” are proteins that bind to a specific sequence of doublestranded DNA molecules, and modulate or regulate positively or negatively the transcription of DNA. Transcriptional factors are activated through different mechanisms such as translocations, post-translational modifications like phosphorylations, and association with cofactors, such as co-activators and co-repressors, which respectively increase and decrease the transcriptional regulation. Thus, a “transcriptional factor binding site” refers to a site in a double-stranded DNA molecule (e.g., the disclosed constructs) to which a transcriptional factor can bind.

[0062] In some aspects, the transcriptional factor binding site can include two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more) CRE motifts.

[0063] In some aspects, each CRE motif can include a sequence at least about 80% identical (e.g., at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical) to SEQ ID NO: 4 or a fragment thereof. In some aspects, each CRE motif can include SEQ ID NO: 4 or a fragment thereof.

[0064] In some aspects, each CRE motif can be the same. In other aspects, some or all of the CRE motifs may be different.

[0065] In some aspects, the transcriptional factor binding site can further include a spacer between adjacent CRE motifs. In some aspects, each spacer can independently include at least about 10 nucleotides (e.g., at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides). In some aspects, each spacer can independently include up to about 20 nucleotides (e.g., up to about 19 nucleotides, up to about 18 nucleotides, up to about 17 nucleotides, up to about 16 nucleotides, up to about 15 nucleotides, up to about14 nucleotides, up to about 13 nucleotides, up to about 12 nucleotides, up to about 11 nucleotides, up to about 10 nucleotides).

[0066] It is considered that each spacer can independently include an amount of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, each spacer can independently include from about 10 nucleotides to about 20 nucleotides (e.g., from about 11 nucleotides to about 19 nucleotides, from about 12 nucleotides to about 18 nucleotides, from about 13 nucleotides to about 17 nucleotides, from about 14 nucleotides to about 16 nucleotides, from about 10 nucleotides to about 15 nucleotides, from about 11 nucleotides to about 14 nucleotides, from about 12 nucleotides to about 13 nucleotides, from about 15 nucleotides to about 20 nucleotides, from about 16 nucleotides to about 19 nucleotides, from about 17 nucleotides to about 18 nucleotides).

[0067] In some aspects, the transcriptional factor binding site can include at least about 4 nuleotides (e.g., at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides). In some aspects, the transcriptional factor binding site can include up to about 100 nucleotides (e.g., up to about 95 nucleotides, up to about 90 nucleotides, up to about 85 nucleotides, up to about 80 nucleotides, up to about 75 nucleotides, up to about 70 nucleotides, up to about 65 nucleotides, up to about 60 nucleotides, up to about 55 nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, up to about 35 nucleotides, up to about 30 nucleotides, up to about 25 nucleotides, up to about 20 nucleotides, up to about 15 nucleotides, up to about 10 nucleotides, up to about 9 nucleotides, up to about 8 nucleotides, up to about 7 nucleotides, up to about 6 nucleotides, up to about 5 nucleotides, up to about 4 nucleotides).

[0068] It is considered that the transcriptional factor binding site can include an amount of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the transcriptional binding factor can include from about 4 nucleotides to about 100 nucleotides (e.g., from about 5nucleotides to about 95 nucleotides, from about 6 nucleotides to about 90 nucleotides, from about 7 nucleotides to about 85 nucleotides, from about 8 nucleotides to about 80 nucleotides, from about 9 nucleotides to about 75 nucleotides, from about 10 nucleotides to about 70 nucleotides, from about 15 nucleotides to about 65 nucleotides, from about 20 nucleotides to about 60 nucleotides, from about 25 nucleotides to about 55 nucleotides, from about 30 nucleotides to about 50 nucleotides, from about 35 nucleotides to about 45 nucleotides, from about 4 nucleotides to about 40 nucleotides, from about 5 nucleotides to about 35 nucleotides, from about 6 nucleotides to about 30 nucleotides, from about 7 nucleotides to about 25 nucleotides, from about 8 nucleotides to about 20 nucleotides, from about 9 nucleotides to about 15 nucleotides, from about 40 nucleotides to about 100 nucleotides, from about 45 nucleotides to about 95 nucleotides, from about 50 nucleotides to about 90 nucleotides, from about 55 nucleotides to about 85 nucleotides, from about 60 nucleotides to about 80 nucleotides, from about 65 nucleotides to about 75 nucleotides).

[0069] As used herein, the terms “promoter,” “promoter element,” or “promoter sequence” refer to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA. A promoter is typically, though not necessarily, located 5' (i.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. Promoters may be constitutive or replatable. The “core promoter” refers to the minimal portion of the promoter required to initiate transcription. In some aspects, the core promoter can be a truncated version of any suitable mammalian promoter, for example, a minimal cytomegalovirus (minCMV) core promoter, a Y-box (YB) core promoter, or a minimal thymidine kinase (miniTK) core promoter.

[0070] In some aspects, the construct can further include two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more) core promoters. In some aspects, each core promoter can be the same. In other aspects, some or all of the core promoters may be different.

[0071] In some aspects, the construct can include a sequence at least 80% identical (e.g., at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at leastabout 97% identical, at least about 98% identical, at least about 99% identical) to any one of SEQ ID NOs: 6 and 8-19 or a fragment thereof. In some aspects, the construct can include any one of SEQ ID NOs: 6 and 8-19 or a fragment thereof.

[0072] In some aspects, the construct can further include one or more RelA binding sites. RelA (the p65 subunit of NKKB) is a transcription factor which is another a target of PKA other than CREB, and which has been shown to recruit a CREB interaction partner, CREB binding protein (CBP).

[0073] In some aspects, the RelA binding site can include a sequence at least 80% identical (e.g., at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical) to SEQ ID NO: 3 or a fragment thereof. In some aspects, the RelA binding site can include SEQ ID NO: 3 or a fragment thereof.

[0074] In another aspect, provided is a vector including any of the disclosed constructs and a target gene downstream of the core promoter.

[0075] In some aspects, the target gene can be at least about 50 nucleotides (e.g., at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides) away from the core promoter. In some aspects, the target gene can be up to 250 nucleotides (e.g., up to about 240 nucleotides, up to about 230 nucleotides, up to about 220 nucleotides, up to about 210 nucleotides, up to about 200 nucleotides, up to about 190 nucleotides, up to about 180 nucleotides, up to about 170 nucleotides, up to about 160 nucleotides, up to about 150 nucleotides, up to about 140 nucleotides, up to about 130 nucleotides, up to about 120 nucleotides, up to about 110 nucleotides, up to about 100 nucleotides, up to about 90 nucleotides, up to about 80 nucleotides, up to about 70 nucleotides, up to about 60 nucleotides, up to about 50 nucleotides) away from the core promoter.

[0076] It is considered that the target gene can be an amount of nucleotides ranging from any of the minimum values described above to any of the maximum values described above away from the core promoter. For example, in some aspects, the target gene can be from about 50 nucleotides to about 250 nucleotides (e.g., from about 60 nucleotides to about 240 nucleotides, from about 70 nucleotides to about 230 nucleotides, from about 80 nucleotides to about 220 nucleotides, from about 90 nucleotides to about 210 nucleotides, from about 100 nucleotides to about 200 nucleotides, from about 110 nucleotides to about 190 nucleotides, from about 120 nucleotides to about 180 nucleotides, from about 130 nucleotides to about 170 nucleotides, from about 140 nucleotides to about 160 nucleotides, from about 50 nucleotides to about 150 nucleotides, from about 60 nucleotides to about 140 nucleotides, from about 70 nucleotides to about 130 nucleotides, from about 80 nucleotides to about 120 nucleotides, from about 90 nucleotides to about 110 nucleotides, from about 150 nucleotides to about 250 nucleotides, from about 160 nucleotides to about 240 nucleotides, from about 170 nucleotides to about 230 nucleotides, from about 180 nucleotides to about 220 nucleotides, from about 190 nucleotides to about 210 nucleotides) away from the core promoter.

[0077] In some aspects, the target gene can encode a cytokine (e.g., IL-2, IL- 12, IL- 10), a chemokine, an antibody, a nanobody, a bispecific engager (BiTEs), a cytotoxic compound, a transcription factor for promoting T cell health, or an enzyme having antitumor or pro-T cell health activity.

[0078] In some aspects, the construct can further include two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more) core promoters, and the vector can further include a target gene downstream of each core promoter. In some aspects, each core promoter can be the same. In other aspects, some or all of the core promoters may be different. In some aspects, each target gene can be the same. In other aspects, some or all of the target genes may be different.

[0079] In some aspects, the target gene may be expressed only in the presence of a high concentration of adenosine. In some aspects, the high concentration of adenosine can be at least about 10 pM (e.g., at least about 15 pM, at least about 20 pM, at least about 25 pM, at least about 30 pM, at least about 35 pM, at least about 40 pM, at least about 45 pM, at least about 50 pM, at least about 55 pM, at least about 60 pM, at least about 65 pM, at least about 70 pM, at least about 75 pM, at least about 80 pM, at least about 85 pM, at least about 90 pM, at least about 95 pM, at least about 100 pM). In some aspects, the high concentration of adenosine can be up to about 100 pM (e.g., up to about 95 pM, up to about 90 pM, up to about 85 pM, up to about 80 pM, up to about 75 pM, up to about 70 pM, up to about 65 pM, up to about 60 pM,up to about 55 pM, up to about 50 pM, up to about 45 pM, up to about 40 pM, up to about 35 pM, up to about 30 pM, up to about 25 pM, up to about 20 pM, up to about 15 pM, up to about 10 pM).

[0080] It is considered that the high concentration of adenosine can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the high concentration of adenosine can be from about 10 pM to about 100 pM (e.g., from about 15 pM to about 95 pM, from about 20 pM to about 90 pM, from about 25 pM to about 85 pM, from about 30 pM to about 80 pM, from about 35 pM to about 75 pM, from about 40 pM to about 70 pM, from about 45 pM to about 65 pM, from about 50 pM to about 60 pM, from about 10 pM to about 55 pM, from about 15 pM to about 50 pM, from about 20 pM to about 45 pM, from about 25 pM to about 40 pM, from about 30 pM to about 35 pM, from about 55 pM to about 100 pM, from about 60 pM to about 95 pM, from about 65 pM to about 90 pM, from about 70 pM to about 85 pM, from about 75 pM to about 80 pM).

[0081] In yet another aspect, provided is an engineered cell including any of the disclosed vectors. In some aspects, the engineered cell can be a T cell. In some aspects, the engineered cell can be a CAR-T cell. Engineered cells are described in more detail below.

[0082] PKA / pCREB signaling and transcriptional activation can also be mediated by other metabolites, specifically by prostaglandin E2 (PGE2), epinephrine, and norepinephrine (Norep) (Jennings et al. 2021). When prostaglandin E2 (PGE2) binds EP2 or EP4 receptors or epinephrine or Norep bind beta-adrenergic receptors, the receptors’ coupled Gsproteins activate adenylyl cyclases to generate cAMP (Kalinski 2012) (Dror et al. 2011). PGE2, epinephrine, and Norep have also been demonstrated to suppress immune cell antitumor immune function.

[0083] Synthetic promoters as disclosed herein can exhibit CREB / adenosine-induced logfold increases in gene expression. These genetically compact and effective CREB -activated promoters can be capable of at least 5-fold (e.g., at least 10-fold, at least 15-fold, at least 20- fold, at least 25-fold, at least 30-fold, at least 35-fold, at least 40-fold, at least 45-fold, at least 50-fold, at least 55-fold, at least 60-fold, at least 65-fold, at least 70-fold, at least 75-fold) gene expression induction, resulting in higher reporter expression than either constitutive promoters or other endogenous adenosine-responsive promoters. In some specific aspects, the CREB- activated promoters can be capable of at least 47-fold gene expression induction. In some aspects, the optimal number of tandem-repeated CRE sequences in a synthetic promoter canvary slightly by the core promoter region they induce (minCMV and YB) using a smallmolecule inducer of adenylate cyclase.

[0084] Notably, these engineered promoters can be highly induced by adenosine, a tumoraccumulating metabolite, as well as a combination of adenosine and other immunosuppressive molecules. In some embodiments, these promoters can afford up to 32-fold induction of reporter gene expression in response to high levels of adenosine addition, with reporter protein levels surpassing those seen with strong, endogenous human and murine promoters. In other embodiments, these promoters can be fully induced by physiologically relevant adenosine levels.

[0085] In yet other embodiments, the promoters can be activated both by other AC- stimulating compounds in a dose-dependent manner with favorable on / off kinetics. In yet other embodiments, the addition of additional transcription factor binding sites, apart from CRE sequences, can further heighten adenosine-induced gene expression without severely impacting promoter off-state. In still further embodiments, the removal of adenosine can reduce gene expression within 12 hours. In yet other embodiments, the promoters can be functional in several mammalian cell types. Adenosine-induced gene expression achieved by the disclosed promoters in other cell types, such as primary T cells, can be significantly more responsive, particularly if they express the A2AR rather than the A2BR, considering their differing affinities (EC50 = O.73|1M vs. EC50 = 23.5|1M)26.

[0086] Importantly, it can be shown that background adenosine degradation from latent adenosine deaminase activity prevalent in FBS (tested across several lots) may be considered for in vtiro studies involving adenosine. FBS-mediate degradation of adenosine may have been a confounding factor in prior efforts studying immunosuppression mediated by adenosine as well as adenosine dose-responsiveness (Stagg et al. 2010) (Gaudreau et al. 2016) (Tardif et al. 2019) (Ohta 2016). Typical heat-deactivation protocols performed by FBS suppliers (30 minutes at 56°C) do not eliminate this adenosine degradation activity, potentially due to the higher than 56°C melting temperature of mammalian adenosine deaminase enzymes (Ma et al. 2022).

[0087] Adenosine and CREB- signaling are highly implicated in the immune suppression of T cells in tumor environments and have been shown to limit CAR T cell function via A2AR (Giuffrida et al. 2021) (Qu et al. 2021). It can be shown that the disclosed synthetic promoters can be strongly induced at physiologically relevant levels of adenosine, i.e., at levels of adenosine found in solid tumors. In other embodiments, the synthetic promoters can have a lowoff-state expression level, i.e., without exposure to adenosine. As such, an adenosine-activated synthetic promoter can be utilized to drive cell-based, tumor-localized expression of agents that have antitumor activity but are toxic when present systemically (Buchbinder et al. 2019) (Ngyuen et al. 2019). In this vein, the 9x-CRE_minCMV promoter is genetically compact, making it attractive to use in cell therapy applications. The full suites of YB- and minCMV- based promoters also fully sample a broad range of expression, which can also enable tunable, CREB -activated protein production.

[0088] In some embodiments, the incorporation of RELA response elements can moderately improve the strength and fold-induction of the 12x-CRE_YB promoter. Incorporation of other heterologous response elements (e.g., dioxin responsive element or hypoxic responsive element) that allow for response to other mechanisms of immune suppression tumors can also expand the function and utility of these synthetic promoters (Triplett et al. 2018) (He et al. 2021) (Yang et al. 2022). In sum, the present embodiments include effective adenosine-activated promoters with high induction strengths that can be activated in response to tumoral environmental conditions. The adenosine-activated promoters can be put forward in consideration for the testing of adenosine cellular responses in vitro with FBS.

[0089] It is contemplated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure.METHODS

[0090] In an aspect, provided is a method of treating a solid tumor in a subject, the method including administering to the subject a therapeutically effective amount of a vector comprising a transcriptional factor binding site, a core promoter, and a target gene downstream of the core promoter, wherein the transcriptional factor binding site includes two or more cAMP response element (CRE) motifs.

[0091] In some aspects, the transcriptional factor binding site can include two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more) CRE motifts.

[0092] In some aspects, each CRE motif can include a sequence at least about 80% identical (e.g., at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical) to SEQ ID NO: 4 or a fragment thereof. In some aspects, each CRE motif can include SEQ ID NO: 4 or a fragment thereof.

[0093] In some aspects, each CRE motif can be the same. In other aspects, some or all of the CRE motifs may be different.

[0094] In some aspects, the transcriptional factor binding site can further include a spacer between adjacent CRE motifs. In some aspects, each spacer can independently include at least about 10 nucleotides (e.g., at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 16 nucleotides, at least about 17 nucleotides, at least about 18 nucleotides, at least about 19 nucleotides, at least about 20 nucleotides). In some aspects, each spacer can independently include up to about 20 nucleotides (e.g., up to about 19 nucleotides, up to about 18 nucleotides, up to about 17 nucleotides, up to about 16 nucleotides, up to about 15 nucleotides, up to about 14 nucleotides, up to about 13 nucleotides, up to about 12 nucleotides, up to about 11 nucleotides, up to about 10 nucleotides).

[0095] It is considered that each spacer can independently include an amount of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, each spacer can independently include from about 10 nucleotides to about 20 nucleotides (e.g., from about 11 nucleotides to about 19 nucleotides, from about 12 nucleotides to about 18 nucleotides, from about 13 nucleotides to about 17 nucleotides, from about 14 nucleotides to about 16 nucleotides, from about 10 nucleotides to about 15 nucleotides, from about 11 nucleotides to about 14 nucleotides, from about 12 nucleotides to about 13 nucleotides, from about 15 nucleotides to about 20 nucleotides, from about 16 nucleotides to about 19 nucleotides, from about 17 nucleotides to about 18 nucleotides).

[0096] In some aspects, the transcriptional factor binding site can include at least about 10 nucleotides (e.g., at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40nucleotides, at least about 45 nucleotides, at least about 50 nucleotides, at least about 55 nucleotides, at least about 60 nucleotides, at least about 65 nucleotides, at least about 70 nucleotides, at least about 75 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 100 nucleotides). In some aspects, the transcriptional factor binding site can include up to about 100 nucleotides (e.g., up to about 95 nucleotides, up to about 90 nucleotides, up to about 85 nucleotides, up to about 80 nucleotides, up to about 75 nucleotides, up to about 70 nucleotides, up to about 65 nucleotides, up to about 60 nucleotides, up to about 55 nucleotides, up to about 50 nucleotides, up to about 45 nucleotides, up to about 40 nucleotides, up to about 35 nucleotides, up to about 30 nucleotides, up to about 25 nucleotides, up to about 20 nucleotides, up to about 15 nucleotides, up to about 10 nucleotides).

[0097] It is considered that the transcriptional factor binding site can include an amount of nucleotides ranging from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the transcriptional binding factor can include from about 10 nucleotides to about 100 nucleotides (e.g., from about 15 nucleotides to about 95 nucleotides, from about 20 nucleotides to about 90 nucleotides, from about 25 nucleotides to about 85 nucleotides, from about 30 nucleotides to about 80 nucleotides, from about 35 nucleotides to about 75 nucleotides, from about 40 nucleotides to about 70 nucleotides, from about 45 nucleotides to about 65 nucleotides, from about 50 nucleotides to about 60 nucleotides, from about 10 nucleotides to about 55 nucleotides, from about 15 nucleotides to about 50 nucleotides, from about 20 nucleotides to about 45 nucleotides, from about 25 nucleotides to about 40 nucleotides, from about 30 nucleotides to about 35 nucleotides, from about 55 nucleotides to about 100 nucleotides, from about 60 nucleotides to about 95 nucleotides, from about 65 nucleotides to about 90 nucleotides, from about 70 nucleotides to about 85 nucleotides, from about 75 nucleotides to about 80 nucleotides).

[0098] In some aspects, the core promoter can be a minCMV core promoter or a YB core promoter.

[0099] In some aspects, the vector can further include two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more) core promoters. In some aspects, each core promoter can be the same. In other aspects, some or all of the core promoters may be different.

[0100] In some aspects, the portion of the construct including the transcriptional factor binding site and the core promoter can include a sequence at least 80% identical (e.g., at leastabout 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical) to any one of SEQ ID NOs: 6 and 8-19 or a fragment thereof. In some aspects, the portion of the construct including the transcriptional factor binding site and the core promoter can include any one of SEQ ID NOs: 6 and 8-19 or a fragment thereof.

[0101] In some aspects, the vector can further include one or more RelA binding sites. In some aspects, the RelA binding site can include a sequence at least 80% identical (e.g., at least about 81% identical, at least about 82% identical, at least about 83% identical, at least about 84% identical, at least about 85% identical, at least about 86% identical, at least about 87% identical, at least about 88% identical, at least about 89% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical) to SEQ ID NO: 3 or a fragment thereof. In some aspects, the RelA binding site can include SEQ ID NO: 3 or a fragment thereof.

[0102] In some aspects, the target gene can be at least about 50 nucleotides (e.g., at least about 60 nucleotides, at least about 70 nucleotides, at least about 80 nucleotides, at least about 90 nucleotides, at least about 100 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 130 nucleotides, at least about 140 nucleotides, at least about 150 nucleotides, at least about 160 nucleotides, at least about 170 nucleotides, at least about 180 nucleotides, at least about 190 nucleotides, at least about 200 nucleotides, at least about 210 nucleotides, at least about 220 nucleotides, at least about 230 nucleotides, at least about 240 nucleotides, at least about 250 nucleotides) away from the core promoter. In some aspects, the target gene can be up to 250 nucleotides (e.g., up to about 240 nucleotides, up to about 230 nucleotides, up to about 220 nucleotides, up to about 210 nucleotides, up to about 200 nucleotides, up to about 190 nucleotides, up to about 180 nucleotides, up to about 170 nucleotides, up to about 160 nucleotides, up to about 150 nucleotides, up to about 140 nucleotides, up to about 130 nucleotides, up to about 120 nucleotides, up to about 110 nucleotides, up to about 100 nucleotides, up to about 90 nucleotides, up to about 80 nucleotides,up to about 70 nucleotides, up to about 60 nucleotides, up to about 50 nucleotides) away from the core promoter.

[0103] It is considered that the target gene can be an amount of nucleotides ranging from any of the minimum values described above to any of the maximum values described above away from the core promoter. For example, in some aspects, the target gene can be from about 50 nucleotides to about 250 nucleotides (e.g., from about 60 nucleotides to about 240 nucleotides, from about 70 nucleotides to about 230 nucleotides, from about 80 nucleotides to about 220 nucleotides, from about 90 nucleotides to about 210 nucleotides, from about 100 nucleotides to about 200 nucleotides, from about 110 nucleotides to about 190 nucleotides, from about 120 nucleotides to about 180 nucleotides, from about 130 nucleotides to about 170 nucleotides, from about 140 nucleotides to about 160 nucleotides, from about 50 nucleotides to about 150 nucleotides, from about 60 nucleotides to about 140 nucleotides, from about 70 nucleotides to about 130 nucleotides, from about 80 nucleotides to about 120 nucleotides, from about 90 nucleotides to about 110 nucleotides, from about 150 nucleotides to about 250 nucleotides, from about 160 nucleotides to about 240 nucleotides, from about 170 nucleotides to about 230 nucleotides, from about 180 nucleotides to about 220 nucleotides, from about 190 nucleotides to about 210 nucleotides) away from the core promoter.

[0104] In some aspects, the target gene can encode a cytokine (e.g., IL-2, IL- 12, IL- 10), a chemokine, an antibody, a nanobody, a bispecific engager (BiTEs), a cytotoxic compound, a transcription factor for promoting T cell health, or an enzyme having antitumor or pro-T cell health activity.

[0105] In some aspects, the vector can further include two or more (e.g., three or more, four or more, five or more, six or more, seven or more, eight or more) core promoters and a target gene downstream of each core promoter. In some aspects, each core promoter can be the same. In other aspects, some or all of the core promoters may be different. In some aspects, each target gene can be the same. In other aspects, some or all of the target genes may be different.

[0106] In some aspects, the target gene may be expressed only in the presence of a high concentration of adenosine. In some aspects, the solid tumor can include a high concentration of adenosine, and the target gene may be expressed only within the solid tumor.

[0107] In some aspects, the high concentration of adenosine can be at least about 10 pM (e.g., at least about 15 pM, at least about 20 pM, at least about 25 pM, at least about 30 pM, at least about 35 pM, at least about 40 pM, at least about 45 pM, at least about 50 pM, at least about 55 pM, at least about 60 pM, at least about 65 pM, at least about 70 pM, at least about75 pM, at least about 80 pM, at least about 85 pM, at least about 90 pM, at least about 95 pM, at least about 100 pM). In some aspects, the high concentration of adenosine can be up to about 100 pM (e.g., up to about 95 pM, up to about 90 pM, up to about 85 pM, up to about 80 pM, up to about 75 pM, up to about 70 pM, up to about 65 pM, up to about 60 pM, up to about 55 pM, up to about 50 pM, up to about 45 pM, up to about 40 pM, up to about 35 pM, up to about 30 pM, up to about 25 pM, up to about 20 pM, up to about 15 pM, up to about 10 pM).

[0108] It is considered that the high concentration of adenosine can range from any of the minimum values described above to any of the maximum values described above. For example, in some aspects, the high concentration of adenosine can be from about 10 pM to about 100 pM (e.g., from about 15 pM to about 95 pM, from about 20 pM to about 90 pM, from about 25 pM to about 85 pM, from about 30 pM to about 80 pM, from about 35 pM to about 75 pM, from about 40 pM to about 70 pM, from about 45 pM to about 65 pM, from about 50 pM to about 60 pM, from about 10 pM to about 55 pM, from about 15 pM to about 50 pM, from about 20 pM to about 45 pM, from about 25 pM to about 40 pM, from about 30 pM to about 35 pM, from about 55 pM to about 100 pM, from about 60 pM to about 95 pM, from about 65 pM to about 90 pM, from about 70 pM to about 85 pM, from about 75 pM to about 80 pM).

[0109] In some aspects, the vector can include any of the disclosed vectors.

[0110] In some aspects, the vector can be transfected into an engineered cell, and the method can include administering to the subject a therapeutically effective amount of said engineered cell.

[0111] In some aspects, the engineered cell can be a T cell. In some aspects, the engineered cell can be a CAR-T cell. In some embodiments, T cells can be derived from one or more suitable mammals, for example, one or more human donors. T cells can be obtained from a number of sources, including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as sedimentation, e.g., FICOLL™ separation.

[0112] In some examples, T cells can be isolated from a mixture of immune cells (e.g., those described herein) to produce an isolated T cell population. For example, after isolation of peripheral blood mononuclear cells (PBMC), both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T cell subpopulations either before or after activation, expansion, and / or genetic modification.

[0113] A specific subpopulation of T cells, expressing one or more of the following cell surface markers: TCRab, CD3, CD4, CD8, CD27 CD28, CD38, CD45RA, CD45RO, CD62L, CD127, CD122, CD95, CD197, CCR7, KLRG1, MCH-I proteins and / or MCH-II proteins, can be further isolated by positive or negative selection techniques. In some embodiments, a specific subpopulation of T cells, expressing one or more of the markers selected from the group listed above, can be used. In some embodiments, subpopulations of T cells may be isolated by positive or negative selection prior to genetic engineering and / or post genetic engineering.

[0114] An isolated population of T cells may express one or more of the T cell markers, including, but not limited to a CD3+, CD4+, CD8+, or a combination thereof. In some embodiments, the T cells are isolated from a donor, or subject, and first activated and stimulated to proliferate in vitro prior to undergoing gene editing (engineering).

[0115] In some instances, the T cell population comprises primary T cells isolated from one or more human donors. Such T cells are terminally differentiated, not transformed, depend on cytokines and / or growth factors for growth, and / or have stable genomes.

[0116] Alternatively, the T cells may be derived from stem cells (e.g., HSCs or iPSCs) via in vitro differentiation.

[0117] T cells from a suitable source can be subjected to one or more rounds of stimulation, activation and / or expansion. T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041. In some embodiments, T cells can be activated and expanded for about 1 day to about 4 days, about 1 day to about 3 days, about 1 day to about 2 days, about 2 days to about 3 days, about 2 days to about 4 days, about 3 days to about 4 days, or about 1 day, about 2 days, about 3 days, or about 4 days prior to introduction of the genome editing compositions into the T cells.

[0118] In some embodiments, T cells are activated and expanded for about 4 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours prior to introduction of the gene editing compositions into the T cells. In some embodiments, T cells are activated at the same time that genome editing compositions are introduced into the T cells. In some instances, the T cell population can be expanded and / or activated after the genetic editing as disclosed herein. T cell populations or isolated T cells generated by any of the gene editing methods described herein are also within the scope of the present disclosure.

[0119] Any of the genetically engineered T cells described herein can be prepared using conventional gene editing methods or those described herein to edit one or more of the target genes disclosed herein (targeted editing). Targeted editing can be achieved either through a nuclease-independent approach, or through a nuclease-dependent approach. In the nuclease- independent targeted editing approach, homologous recombination is guided by homologous sequences flanking an exogenous polynucleotide to be introduced into an endogenous sequence through the enzymatic machinery of the host cell. The exogenous polynucleotide may introduce deletions, insertions or replacement of nucleotides in the endogenous sequence.

[0120] Alternatively, the nuclease-dependent approach can achieve targeted editing with higher frequency through the specific introduction of double strand breaks (DSBs) by specific rare-cutting nucleases (e.g., endonucleases). Such nuclease-dependent targeted editing also utilizes DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor template containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material.

[0121] In some embodiments, gene disruption may occur by deletion of a genomic sequence using two guide RNAs. Methods of using CRISPR-Cas gene editing technology to create a genomic deletion in a cell (e.g., to knock out a gene in a cell) are known (Bauer D E et al. Vis. Exp. 2015; 95:e52118).

[0122] Available endonucleases capable of introducing specific and targeted DSBs include, but not limited to, zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas9 nuclease (CRISPR / Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Additionally, DICE (dual integrase cassette exchange) system utilizing phiC31 and Bxbl integrases may also be used for targeted integration. Some exemplary approaches are disclosed in detail below.

[0123] In the methods described herein, a solid tumor can be treated. In some aspects, the solid tumor can be cancerous. In some aspects, the solid tumor can be bladder cancer, nonsmall cell lung cancer, cervical cancer, anal cancer, pancreatic cancer, squamous cell carcinoma including head and neck cancer, renal cell carcinoma, melanoma, ovarian cancer, small cell lung cancer, glioblastoma, gastrointestinal stromal tumor, breast cancer, lung cancer, colorectal cancer, thyroid cancer, bone sarcoma, stomach cancer, oral cavity cancer,oropharyngeal cancer, gastric cancer, kidney cancer, liver cancer, prostate cancer, colorectal cancer, esophageal cancer, testicular cancer, gynecological cancer, thyroid cancer, colon cancer, or brain cancer.EXAMPLESExample 1: Materials and Methods

[0124] Cell Lines: HEK293T cells were maintained in GlutaMAX supplemented with 10%FBS from Gibco. Jurkat, MAD109, and 4T1 cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS. All cell lines used in this study were tested for mycoplasma and verified negative.

[0125] Plasmid, and promoter construction: GFuc-P2A-eGFP sequences were employed. Base, golden gate compatible pcDNA3 vectors containing the minCMV and YB core promoters with Esp3I cut sites and the GEuc-P2A-eGFP gene were constructed using Gibson Assembly. From there, either 6xCRE promoter repeats were ordered from Twist Biosciences as double-stranded gene fragments bearing golden gate assembly cut sites / overhangs, or 3x CRE promoter repeats (and their respective reverse complements) bearing the proper golden gate assembly overhangs were ordered as single stranded DNA fragments from Eurofins Genomics and annealed prior to Golden Gate Assembly. A complete list of promoter DNA sequences can be found within SEQUENCE TABLE 1. The minCMV core promoter is shown in bold, the YB-TATA core promoter in italics, the RelA binding sequence in underline, the palindromic cAMP Response element (CRE) is shown in bold underline, and the half-CRE motif is shown in double underline. The COX2 promoter was PCR amplified from HEK293T genomic DNA using primers jrc305 / 306 including within SEQUENCE TABLE 2. For stable cell line experiments, the promoter-GLuc-P2A-eGFP genes were PCR amplified and inserted into pLEGO-B backbones that were digested with Xbal and Xhol.

[0126] Using standard electroporation, lentiviral backbones were transformed into NEB® Stable E. coli, and all pcDNA backbones were transformed into NEB® 10(3 E. coli (Greene et al. 2012). All plasmids were sequence confirmed via Sanger sequencing prior to use.

[0127] FBS adenosine degradation experiments: Heat deactivations of FBS were carried out in a water bath set to 56°C for the indicated time. For adenosine degradation analysis within growth media, media was supplemented with ImM of adenosine and the indicated FBS and cultured in a 96-well U-bottom plate. Media was sampled at the indicated time points and read on a spectrophotometer for absorbance at 260nm. Percent of adenosine remaining was calculated by normalizing to the initial reading.

[0128] Transient promoter induction experiments'. For transient transfection promoter screening, HEK293T cells were transfected by reverse transfection with the indicated promoter construct (pcDNA3 backbones). Briefly, 400 ng of plasmid DNA was combined with 0.6pL of TransIT-LTl (Mirus) and 18pL of OptiMEM (Gibco). After incubation for 10 minutes, 50,000 cells were added to each well of a 96-well TC plate. After cell attachment, the cells were treated as indicated (e.g., adenosine, forskolin, etc.) for 8 hours, and supernatants were harvested for analysis for GLuc expression by the Pierce Gaussian Luciferase kit (ThermoFisher) according to the manufacturer’s instructions or the cells were trypsinized for analysis by flow cytometry.

[0129] Lentivirus production'. For lentiviral preparation, 4 x 106HEK293T cells were seeded in a 10cm dish. The next day, the cells were transfected with 5pg of transfer vector, Ipg of pMD2.G (Addgene #12259), and 4pg of psPAX2 (Addgene #12260) with TransIT-LTl according to the manufacturer’s instructions. Two days later, supernatants were harvested, spun down at lOOOxg for 5 minutes, passed through a 0.45pm PES filters (VWR), and then finally precipitated with PEG-zt™ viral precipitation solution (SBI, LV825A-1) according to the manufacturer’s instructions. A day later, the precipitated virus was resuspended in ice-cold PBS and stored at -80°C until use.

[0130] Promoter kinetics experiment'. For experiments using stably engineered cell lines, cells were transduced with lentivirus as described and sorted for tagBFP+ expression prior to use on a BD FACS Melody. Promoter induction experiments and sampling with these sorted cell lines followed the same protocol as transient transfection experiments without the transfection step. Overall, cells were induced with the indicated compound and at the specified time point, harvested for analysis by flow cytometry to ascertain eGFP expression. For inductions containing adenosine, media was replenished every 12 hours to account for cellular consumption. To demonstrate promoter shut-off potential, 12 hours before analysis, media was replenished with 250pM of adenosine and 3pM of recombinant human adenosine deaminase (ADA1).Example 2: Synthetic CREB-promoter design and optimization of CRE sequence number for YB core promoter

[0131] Experimental Results'. Based on these design criteria, synthetic promoters were constructed harboring between three and eighteen full palindromic CRE elements, with six nucleotides between each element and a G nucleotide placed at the 3’ end of each element (FIG. IB) (Tinti et al. 1997). Replicates of 3X CRE tandem repeats were inserted upstream of the YB core promoter, which promotes minimal background transcription (Ede et al. 2016) (Miller et al. 2021). As a positive control for adeno sine-mediated CREB activation, the nativecyclooxygenase-2 (C0X2) promoter was used (Pouliot et al. 2002) (Cadieux et al. 2005) (Schroer et al. 2002). To measure inducible gene expression, all synthetic promoters were inserted on the pcDNA3 vector backbone upstream of a Gaussian luciferase (GLuc)-P2A-eGFP construct that allows measurement of both bulk culture and individual cell expression levels. Promisingly, upon reverse transfection into HEK293T cells, all the synthetic promoter constructs enabled inducible GLuc expression after an eight hour incubation with forskolin (FIG. 1C). It was found that the 12X-CRE_YB promoter mediated the highest observed expression level, and nearly the highest observed fold-induction (6.2x fold-induction for 12x- CRE_YB vs. 6.5x for 9x-CRE_YB), in response to forskolin, while the YB core promoter without any response elements did not induce gene expression, showing that the CRE sequence elements are mediating increased gene expression. Synthetic promoters harboring more than twelve CRE sequence elements had reduced expression, possibly because the additional CRE sequences are too far away from the YB core promoter’s TATA box (Table 1). For instance, the additional three CRE elements within 15x-CRE_YB are at least 171 nucleotides from the YB core promoter’s TATA box, while the typical distance between functional CRE elements and an endogenous promoter’s TATA box is -100 nucleotides (Mayr et al. 2001) (Tinti et al. 1997). As pCREB or other transcriptional co-activators may have a resource budget, pCREB binding the distal (and less effective) CRE sequence elements could reduce the availability of pCREB proteins to bind the proximal and more effective CRE elements, dampening promoter activity. Promisingly, the 12x-CRE_YB promoter afforded twice as much protein expression than the native COX2 promoter, which has previously been characterized as CREB -responsive, with much less background activity (FIG. 1C) (Pouliot et al. 2002) (Cadiuex et al. 2005) (Schroer et al. 2002). The COX2 promoter exhibited a -1.25X increase in expression upon treatment with forskolin. Therefore, engineered synthetic promoters appear to be better able to mediate controllable induction of gene expression in response to CREB -activation than native promoters. In sum, functional CREB-activated promoters were designed that outperform a native promoter in terms of strength, background expression, and fold induction.

[0132] Discussion'. To allow for tumor- specific activation, an ideal pCREB / adenosine- inducible promoter would have tight regulation (low expression when ‘OFF’) and mediate robust gene expression when activated (high strength when ‘ON,’ resulting in several foldinduction). Studies of endogenous human CREB-activated promoters were used, in addition to a preliminary experiment (FIG. 2), to develop design criteria for an optimized synthetic adenosine-induced promoter (Mayr et al. 2001) (Gonzalez et al. 1989) (Tinti et al. 1997) (Yamamoto et al. 1988) (Fink et al. 1988) (Craig et al. 2001). These design criteria focused on(1) CRE sequence element placement in relation to the gene of interest, (2) the optimal nucleotide sequence to be used for the CRE sequence element itself, and (3) the number of CRE sequence elements required to robustly induce gene expression.

[0133] For instance, limiting the total promoter size could be a design criteria, as CRE sequences more than 200 base pairs from the transcriptional start site (TSS) may not function. Notably, two-thirds of native CREs are located within -50 to -150 base pairs of TSSs and exhibit maximal activity when placed about lOObp from the TATA box (Mayr et al. 2001) (Tinti et al. 1997). In addition to TSS proximity, the specific CRE and adjacent nucleotide sequences may also impact synthetic promoter function. The CRE sequence is palindromic, and it has been shown that both half-CRE motifs and the full palindromic motif are functionally bound by CREB. However, the full palindromic motif is bound stronger and mediates superior activation than the half-site, suggesting that the full ‘TGACGTCA’ CRE sequence element should be included in synthetic promoters (Yamamoto et al. 1988) (Fink et al. 1988) (Craig et al. 2001). In certain promoters with multiple CRE sequences in tandem, including the somatostatin, phosphoenolpyruvate, and tyrosine aminotransferase (TAT) promoters, there is a distinct nucleotide sequence (5’-GCCCC-3’) between neighboring CRE sequence elements (Craig et al. 2001). Saturation mutagenesis has shown that the only nucleotide of this sequence that affects CREB responsiveness is the 5 ’guanine nucleotide, which is adjacent to the 3’ end of the CRE sequence element relative to the direction of transcription (Tinti et al. 1997). The impact includes multiple CRE sequence elements in engineered promoters has yet to be thoroughly investigated. However, initial experiments were performed that showed that synthetic promoters containing less than three CRE sequence elements did not induce reporter gene expression in response to an adenylate cyclase-stimulating compound, forskolin, that is known to strongly activate CREB -responsive promoters (FIG. 2) (Seamon et al. 1981).Example 3: Tandem CREs activate gene expression with an alternative core promoter

[0134] The minCMV core promoter, which is slightly larger than the YB core, has been shown to exhibit greater strength when incorporated into synthetic promoters, though often with higher background activity (Ede et al. 2016). To determine if tandem repeats of CRE sequences proximal to the minCMV core promoter could also enable adenosine / CREB inducible gene expression, promoters were created with 6, 9, or 12 CRE sequence elements upstream of the minCMV core, using similar design constraints as above. It was hypothesized that synthetic promoters containing more than 12 CRE sequence elements would have reduced function, as distal elements would be too far from the TATA box (>170bp) for effective function (TABLE 1) (Mayr et al. 2001) (Tinti et al. 1997). It was found that the 9x-CRE_minCMV promoter had the highest observed gene expression level and fold-induction when induced with 20pM forskolin, and as expected, the 12x-CRE_minCMV synthetic promoter with more distal CRE sequence elements afforded lower expression (TABLE 1, FIG. ID). Compared to 12x-CRE_YB, the 9x-CRE_minCMV promoter enabled roughly double the level of protein expression, though with higher background expression. Still, fold-induction was still comparable (5-fold compared to 6.5-fold) for both promoters. As observed with the YB core promoter without CRE elements, the minCMV core promoter drove minimal gene expression by itself and did not exhibit forskolin-induced gene activation responsiveness. Therefore, it was demonstrated that tandem repeats of CRE sequence elements mediate inducible gene expression when proximal to other core promoters, and stronger CREB- activated promoters can be created using minCMV compared to the YB core (FIGS. 1C-1D).TABLE 1Example 4: Synthetic CREB promoters are induced by adenosine

[0135] It was next confirmed that the CRE sequence element-containing synthetic promoters could be induced by extracellular adenosine, testing for reporter protein expressionafter eight hours of induction. As expected, the minCMV and YB core promoters did not activate gene expression in response to adenosine stimulation and had low background expression levels (FIGS. 3A-3C). Both minCMV and YB-based CRE-containing synthetic promoters induced significant eGFP expression in response to incubation with 0.750mM adenosine, with the 12x-CRE_YB promoter exhibiting 32-fold induction and the 9x- CRE_minCMV exhibiting 24-fold induction (FIGS. 3A-3B). It was further determined that the relative optimum adenosine level to add to media to induce expression from the 12x- CRE_YB promoter was -ImM, which resulted in a -13-fold activation, higher than ~6-fold activation induced by forskolin (FIG. 3C). The optimal adenosine concentration was unchanged by the amount of response elements in a YB-based promoter (e.g., 6x-CRE vs. 12x- CRE). The difference in fold-activation due to adenosine stimulation from the 12x-CRE_YB as measured by eGFP median fluorescent intensity (MFI) using flow cytometry and as measured in bulk supernatant via GLuc activity indicated more robust expression changes could be detected at the single cell level than in bulk. The C0X2 native adenosine-responsive promoter again had high background expression and limited adenosine-induced expression. In sum, the synthetic CRE-containing promoters are fully functional and strongly activated by adenosine and greatly outperform an endogenous CREB -responsive promoter.Example 5: FBS in cell culture media degrades adenosine, altering promoter responsiveness

[0136] It has been shown that HEK293T cells likely express the lower affinity adenosine receptor, A2BR (ECSO = 23.5|lM), and not the higher affinity A2AR, (ECSO = 0.73pM)26 2s. Still, considering adenosine’s affinity for A2BR, maximal promoter responsiveness was hypothesized to occur at micromolar concentrations instead of -ImM (FIGS. 3A-3C). Therefore, the possibility was considered that a component in the media could be degrading supplemented adenosine, increasing required adenosine levels for maximal promoter activation. In this vein, adenosine deaminase activity, mediated by the bovine adenosine deaminase I, can be detected in cattle serum, so it was hypothesized that fetal bovine serum (FBS) might also be able to degrade adenosine (Tanabe et al. 1993) (Altug et al. 2008).

[0137] To test the hypothesis, either (1) complete cell culture media, i.e., with 10% FBS or (2) cell culture media lacking FBS, was supplemented with ImM adenosine, and it was analyzed how adenosine concentration changed over time in each media type (FIG. 4A). It was found that cell culture media containing 10% FBS degraded more than half of the adenosine in just 12 hours and nearly all adenosine within 24 hours, while cell culture media without FBSdid not degrade adenosine over the course of the full 24 hours. It was further shown that heat deactivating the FBS could eliminated its ability to mediate adenosine degradation, but surprisingly, neither a 30-minute nor a six-hour, high temperature (56°C) incubation was inadequate to eliminate adenosine degradation. Instead, a 24-hour heat-deactivation / incubation was used to prevent FBS-mediated adenosine degradation (FIG. 4A). After discerning that the prior analyses of promoter induction may have been impacted by concurrent FBS-mediated adenosine depletion, the adenosine-responsiveness of the 12x-CRE_YB and 9x- CRE_minCMV synthetic promoters was retested in media that lacked adenosine degradation ability. Both promoters were strongly induced by much lower adenosine levels, with near full induction of 12x-CRE_YB at <50pM and of 9x-CRE_minCMV at <100pM when measuring eGFP (FIGS. 4B-4D). Therefore, HEK293T cells expressing the synthetic promoters are responsive to physiologically relevant, micromolar adenosine concentrations, but only when background adenosine degradation is eliminated from cell culture media.Example 6: Combinatorial additions of cAMP-stimulating compounds amplify synthetic promoter induction

[0138] Because compounds like prostaglandin E2 (PGE2) and norepinephrine (Norep) also stimulate the cAMP / PKA pathway, the engineered adenosine / CREB-responsive promoters could be activated by these compounds (Jennings et al. 2021). As a first pass, the ability of the 12x-CRE_YB and 9x-CRE_minCMV promoters to be induced by PGE2 and Norep independently was tested. It was shown that both PGE2 and Norep could stimulate the synthetic promoters, with PGE2 having a 3.6-fold maximal induction at 50pM for 9x-CRE_minCMV and Norep having a 1.8-fold maximal induction at 25|lM for the 12x-CRE_YB promoter (FIGS. 5A-5B). In a direct comparison, fold induction of the 9x-CRE_minCMV promoter induced by a PGE2 and Norep was lower than that of adenosine (FIG. 5C), but the ability of other cAMP-producing compounds to induce expression of the synthetic promoters might imply that they can combine to enhance overall promoter strength, or that their combination might reduce the level of any individual component needed for maximal induction. In this vein, adenosine, PGE2, and Norep can all promote the synthesis of each other, meaning that they are rarely found in complete isolation (Jennings et al. 2021).

[0139] Therefore, it was tested how well combinatorial additions of each cAMP stimulating compound could stimulate gene expression from the synthetic promoters. The triple combination of the maximal, half maximal, or quarter maximal concentrations of adenosine, Norep, or PGE2 was tested, and the resulting promoter induction was compared to thatmediated by adenosine or forskolin only (FIG. 5D), where for instance, the quarter maximal concentration is the inducer level that stimulated -25% of the maximal gene expression attained by the inducer at any concentration. Maximal additions of each compound in combination exhibited the highest observed promoter induction, and notably, quarter-maximal additions of each compound (e.g., 25|lM compared to 250pM for adenosine) outperformed both maximal adenosine induction and forskolin induction (FIG. 5D). Taken together, these results suggest that combinations of adenosine, PGE2, and norepinephrine can amplify CREB- responsive promoter activation and act in a synergistic manner towards enabling high gene expression levels at low inducer concentration.Example 7: Synthetic promoters display favorable on and off kinetics and enable higher gene expression than a strong, endogenous promoter

[0140] To further compare the synthetic promoters to a strong, endogenous promoter, and to determine their kinetics of induction, HEK293T cells were stably transduced with constructs encoding the synthetic 9x-CRE_minCMV promoter, the minCMV core promoter, or the constitutively active endogenous murine PGK promoter, in which each promoter drove expression of the eGFP reporter. As a marker for transduction, a SFFV promoter driving expression of tagBFP was also included. After sorting BFP positive cells, the synthetic 9x- CRE_minCMV promoter was compared, in terms of maximum protein expression, to the PGK promoter. Promisingly, forskolin-induced activation of the 9x-CRE_minCMV yielded nearly 2.5-fold higher eGFP expression compared to PGK promoter, confirming that the synthetic promoters mediate high levels of gene expression that can surpass commonly used constitutive native promoter (FIG. 6A). The PGK promoter generate slightly higher eGFP expression when incubated with forskolin, but was slightly repressed by adenosine-only or adenosine / PGE2 / Norep stimulation, while the minCMV core promoter was not impacted by an inducer (FIG. 6A and FIG. 7).

[0141] Next, to test the speed of promoter induction, i.e., time to achieve maximal eGFP expression for each promoter, eGFP expression was measured after 3, 6, 9, 12, 24, and 48 hours post inducer addition. Forskolin (20|lM), adenosine (250|lM), and the triple combination of quarter maximal PGE2 (4.17p.M), Norep (6.125|lM), and adenosine (25|lM) were tested as inducers. In addition, to determining if inducer removal (or degradation) could result in a quenching of the promoter activity, after the 48-hour induction test, enough recombinant adenosine deaminase 1 was added to the adenosine-only induced cells to degrade the 250pM adenosine within minutes, and then the cells were tested for eGFP expression 12 hours later.

[0142] Regardless of the stimulating compound, the 9x-CRE_minCMV promoter exhibited favorable on / off kinetics. Interestingly, the PGE2 + Norep + adenosine inducer combination reached maximal induction fastest, after only 12 hours, but had the lowest observed level of induced gene expression (FIG. 6B). Forskolin resulted in the highest observed level of induced gene expression seen within 24 hours, though eGFP expression was substantially decreased by 48 hours, potentially due to overly persistent PKA activation. Using adenosine as the only inducer continuously increased gene expression of the course of 48 hours, reaching 47-fold induction, and the addition of adenosine deaminase 1 resulted in a decrease in eGFP levels within 12 hours, suggesting that removal of adenosine could turn off the synthetic promoters (FIG. 6B). In sum, the synthetic 9x-CRE_minCMV promoter exhibits rapid and strong induction of gene expression in response to a variety of adenosine associated stimulation, and its maximal expression level surpasses that of an often-used strong native promoter, pPGK. The 9x-CRE_minCMV also turns off when adenosine is eliminated, losing approximately 20% of eGFP signal in just 12 hours, despite the day long half-life of eGFP (Corish et al. 1999).Example 8: Enhancing the strength of CREB -activated synthetic promoters through rational introduction binding sites for CREB partners

[0143] The 12x-CRE-YB promoter is weaker at full induction that the 9x-CRE_minCMV promoter, despite harboring more CRE elements in a useable proximity to its TATA box. Interestingly, a target of PKA other than CREB is the p65 subunit of NKKB (REEA), which has been shown to recruit a CREB interaction partner, CREB binding protein (CBP) (Zhong et al. 1998). This interaction can functionally increase gene expression induced by CREB, likely through its lysine acetyltransferase domain, which has been hypothesized to increase the strength of the full CMV promoter (FIG. 8) (He et al. 2004) (Dutta et al. 2016). Indeed, moderate (~1.5x) adenosine-mediated induction of the CMV promoter was observed (FIG. 9) suggesting incorporating RELA response elements could further increase the strength of the CREB -activated promoters.

[0144] Therefore, the same consensus RELA sites (GGGACTTTCC) found within the CMV were interspersed on the 5’ end, 3 ’end, and in the middle of the two 6xCREs in the 12xYB promoter (FIG. 8) and tested the resulting promoter’s adenosine responsiveness. It was found that the 3x-RELA-12x-CRE_YB promoter was between 1.24x-1.35x stronger than the 12xYB alone at each tested adenosine concentration without increased gene expression in the promoter’s off state - OpM adenosine added (FIG. 8). While the 3x-RELA-12x-CRE_YB wasstill not as strong as the 9x-CRE_minCMV, it did still show superior fold-induction due to its lower OFF state.Example 9: Confirming synthetic promoter function in additional cell lines

[0145] Finally, to confirm that the promoters retained function in cells lines apart from HEK293T, it was demonstrated that both the 12x-CRE_YB and 9x-CRE_minCMV promoters were strongly induced in both a second human cell line (Jurkat immortalized T cells), as well as two murine cells lines, the 4T1 mammary carcinoma and the Madison 109 lung cancer cell line (FIG. 10) (Schneider et al. 1977) (DuPre et al. 2007) (Schultz et al. 1977). As before, the 12x-CRE_YB promoter generally afforded lower maximal gene expression levels but higher fold- induction, due to its lower off- state, than the 9x-CRE-minCMV promoter.

[0146] FIG. 11 and FIG. 12 depict the results of additional in vitro and in vivo experiments, respectively.

[0147] The following patents, applications and publications as listed below and throughout this document are hereby incorporated by reference in their entirety herein.Reference List[1] Gamboa, E. et al. Synthetic Immunity by Remote Control. Theranostics 2020, 10 (8), 3652-3667.[2] Cazier, A. P. et al. Advances in Promoter Engineering: Novel Applications and Predefined Transcriptional Control. Biotechnology Journal 2021, 16 (10), 2100239.[3] Ede, C. et al. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells. ACS Synth. Biol. 2016, 5 (5), 395-404.[4] Miller, I. C. et al. Remote Control of Mammalian Cells with Heat-Triggered Gene Switches and Photothermal Pulse Trains. ACS Synth. Biol. 2018, 7 (4), 1167-1173.[5] Miller, I. C. et al. Enhanced Intratumoural Activity of CAR T Cells Engineered to Produce Immunomodulators under Photothermal Control. Nat Biomed Eng 2021, 5 (11), 1348— 1359.[6] Yamada, M. et al. Light Control of the Tet Gene Expression System in Mammalian Cells. Cell Reports 2018, 25 (2), 487-500.e6.[7] Shen, Y. et al. Challenges for Therapeutic Applications of Opsin-Based Optogenetic Tools in Humans. Frontiers in Neural Circuits 2020, 14.[8] Zhang, D. et al. Drug Concentration Asymmetry in Tissues and Plasma for Small Molecule-Related Therapeutic Modalities. Drug Metab Dispos 2019, 47 (10), 1122-1135.[9] Sun, D. et al. Why 90% of Clinical Drug Development Fails and How to Improve It? Acta Pharmaceutica Sinica B 2022, 12 (7), 3049-3062.

[0010] Zhao, Z. et al. Targeting Strategies for Tissue-Specific Drug Delivery. Cell 2020, 181 (1), 151-167.

[0011] Jennings, M. R. et al. Immunosuppressive Metabolites in Tumoral Immune Evasion: Redundancies, Clinical Efforts, and Pathways Forward. J Immunother Cancer 2021, 9 (10), e003013.

[0012] Sheth, S. et al. Adenosine Receptors: Expression, Function and Regulation. Int J Mol Sci 2014, 15 (2), 2024-2052.

[0013] Oberprieler, N. G. et al. High-Resolution Mapping of Prostaglandin E2-Dependent Signaling Networks Identifies a Constitutively Active PKA Signaling Node in CD8+CD45RO+ T Cells. Blood 2010, 116 (13), 2253-2265.

[0014] Mayr, B. et al. Transcriptional Regulation by the Phosphorylation-Dependent Factor CREB. Nat Rev Mol Cell Biol 2001, 2 (8), 599-609.

[0015] Gonzalez, G. A. et al. Cyclic AMP Stimulates Somatostatin Gene Transcription by Phosphorylation of CREB at Serine 133. Cell 1989, 59 (4), 675-680.

[0016] Kalinski, P. Regulation of Immune Responses by Prostaglandin E2. J Immunol 2012, 188 (1), 21-28.

[0017] Dror, R. O. et al. Activation Mechanism of the B2-Adrenergic Receptor. Proceedings of the National Academy of Sciences 2011, 108 (46), 18684-18689.

[0018] Tinti, C. et al. Structure / Function Relationship of the CAMP Response Element in Tyrosine Hydroxylase Gene Transcription. Journal of Biological Chemistry 1997, 272 (31), 19158-19164.

[0019] Yamamoto, K. K. et al. Phosphorylation-Induced Binding and Transcriptional Efficacy of Nuclear Factor CREB. Nature 1988, 334 (6182), 494-498.

[0020] Fink, J. S. et al. The CGTCA Sequence Motif Is Essential for Biological Activity of the Vasoactive Intestinal Peptide Gene CAMP-Regulated Enhancer. Proc. Natl. Acad. Sci. U.S.A. 1988, 85 (18), 6662-6666.

[0021] Craig, J. C. et al. Consensus and Variant CAMP-Regulated Enhancers Have Distinct CREB-Binding Properties. Journal of Biological Chemistry 2001, 276 (15), 11719-11728.

[0022] Seamon, K. B. et al. Forskolin: Unique Diterpene Activator of Adenylate Cyclase in Membranes and in Intact Cells. Proc Natl Acad Sci U S A 1981, 78 (6), 3363-3367.

[0023] Pouliot, M. et al. Adenosine Up-Regulates Cyclooxygenase-2 in Human Granulocytes: Impact on the Balance of Eicosanoid Generation. The Journal of Immunology 2002, 169 (9), 5279-5286.

[0024] Cadieux, J.-S. et al. Potentiation of Neutrophil Cyclooxygenase-2 by Adenosine: An Early Anti-Inflammatory Signal. Journal of Cell Science 2005, 118 (7), 1437-1447.

[0025] Schroer, K. et al. Obligatory Role of Cyclic Adenosine Monophosphate Response Element in Cyclooxygenase-2 Promoter Induction and Feedback Regulation by Inflammatory Mediators. Circulation 2002, 105 (23), 2760-2765.

[0026] Fredholm, B . B . et al. Comparison of the Potency of Adenosine as an Agonist at Human Adenosine Receptors Expressed in Chinese Hamster Ovary Cells 11 Abbreviations: CAMP, Cyclic Adenosine 3 ',5 '-Monophosphate; CHO, Chinese Hamster Ovary; NBMPR, Nitrobenzylthioinosine; and NECA, 5'-N-Ethyl Carboxamido Adenosine. Biochemical Pharmacology 2001, 61 (4), 443-448.

[0027] Goulding, J. et al. Characterisation of Endogenous A2A and A2B Receptor-Mediated Cyclic AMP Responses in HEK 293 Cells Using the GloSensorTM Biosensor: Evidence for an Allosteric Mechanism of Action for the A2B-Selective Antagonist PSB 603. Biochemical Pharmacology 2018, 147, 55-66.

[0028] Cooper, J. et al. An Endogenous A2B Adenosine Receptor Coupled to Cyclic AMP Generation in Human Embryonic Kidney (HEK 293) Cells. British Journal of Pharmacology 1997, 122 (3), 546-550.

[0029] Tanabe, T. Adenosine Deaminase Activities in the Sera and Tissues of Animals and their Clinical Significance. Japanese Journal of Veterinary Research 1993, 41 (1), 52-52.

[0030] Altug, N. et al. Determination of Adenosine Deaminase Activity in Cattle Naturally Infected with Theileria Annulata. Trop Anim Health Prod 2008, 40 (6), 449-456.

[0031] Corish, P. et al. Attenuation of Green Fluorescent Protein Half-Life in Mammalian Cells. Protein Engineering, Design and Selection 1999, 12 (12), 1035-1040.

[0032] Zhong, H. et al. Phosphorylation of NF-KB P65 by PKA Stimulates Transcriptional Activity by Promoting a Novel Bivalent Interaction with the Coactivator CBP / P300. Molecular Cell 1998, 1 (5), 661-671.

[0033] He, B. et al. Synergistic Activation of the CMV Promoter by NF-KB P50 and PKG. Biochemical and Biophysical Research Communications 2004, 321 (1), 13-20.

[0034] Dutta, R. et al. CBP / P300 Acetyltransferase Activity in Hematologic Malignancies. Mol Genet Metab 2016, 119 (1-2), 37-43.

[0035] Schneider, U. et al. Characterization of EBV-Genome Negative “Null” and “T” Cell Lines Derived from Children with Acute Lymphoblastic Leukemia and Leukemic Transformed Non-Hodgkin Lymphoma. International Journal of Cancer 1977, 19 (5), 621-626.

[0036] DuPre, S. A. et al. The Mouse Mammary Carcinoma 4T1: Characterization of the Cellular Landscape of Primary Tumours and Metastatic Tumour Loci. Int J Exp Pathol 2007, 88 (5), 351-360.

[0037] Schultz, R. et al. Establishment and Characterization of a Cell Line Derived from a Spontaneous Murine Lung Carcinoma (M109). In vitro 1977, 13 (4), 223-231.

[0038] Stagg, J. et al. Extracellular Adenosine Triphosphate and Adenosine in Cancer. Oncogene 2010, 29 (39), 5346-5358.

[0039] Gaudreau, P.-O. et al. CD73-Adenosine Reduces Immune Responses and Survival in Ovarian Cancer Patients. Oncoimmunology 2016, 5 (5), el 127496.

[0040] Tardif, V. et al. Adenosine Deaminase- 1 Delineates Human Lollicular Helper T Cell Lunction and Is Altered with HIV. Nat Commun 2019, 10 (1), 823.

[0041] Ohta, A. A Metabolic Immune Checkpoint: Adenosine in Tumor Microenvironment. Pront. Immunol. 2016, 7.

[0042] Ma, M. T. et al. Catalytically Active Holo Homo Sapiens Adenosine Deaminase I Adopts a Closed Conformation. Acta Cryst D 2022, 78 (1), 91-103.

[0043] Giuffrida, L. et al. CRISPR / Cas9 Mediated Deletion of the Adenosine A2A Receptor Enhances CAR T Cell Efficacy. Nat Commun 2021, 12 (1), 3236.

[0044] Qu, Y. et al. Adenosine Deaminase 1 Overexpression Enhances the Antitumor Efficacy of Chimeric Antigen Receptor- Engineered T Cells. Hum Gene Ther 2021.

[0045] Buchbinder, E. I. et al. Therapy with High-Dose Interleukin-2 (HD IL-2) in Metastatic Melanoma and Renal Cell Carcinoma Following PD1 or PDL1 Inhibition. Journal for ImmunoTherapy of Cancer 2019, 7 (1), 49.

[0046] Nguyen, L. T. et al. Phase II Clinical Trial of Adoptive Cell Therapy for Patients with Metastatic Melanoma with Autologous Tumor-Infiltrating Lymphocytes and Low-Dose Interleukin-2. Cancer Immunol Immunother 2019, 68 (5), 773-785.

[0047] Triplett, T. A. et al. Reversal of Indoleamine 2,3-Dioxygenase-Mediated Cancer Immune Suppression by Systemic Kynurenine Depletion with a Therapeutic Enzyme. Nature Biotechnology 2018, 36 (8), 758-764.

[0048] He, H. et al. Conditioned CAR-T Cells by Hypoxia-Inducible Transcription Amplification (HiTA) System Significantly Enhances Systemic Safety and Retains Antitumor Efficacy. J Immunother Cancer 2021, 9 (10), e002755.

[0049] Y ang, Q. et al. Superior Antitumor Immunotherapy Efficacy of Kynureninase Modified CAR-T Cells through Targeting Kynurenine Metabolism. Oncoimmunology 2022, 11 (1), 2055703.

[0050] Green, M. R. et al. Molecular Cloning: A Laboratory Manual, 4th ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y, 2012.

[0051] Durai, L. et al. A Novel Reporter System for Cyclic AMP Mediated Gene Expression in Mammalian Cells Based on Synthetic Transgene Expression System. European Journal of Pharmacology 2019, 855, 56-64.

[0052] Shan, Q. et al. Optimization of a CAMP Response Element Signal Pathway Reporter System. Journal of Neuroscience Methods 2010, 191 (1), 21-25.SEQUENCE TABLE 1SEQUENCE TABLE 2

Claims

What is claimed is:

1. A construct comprising a transcriptional factor binding site and a core promoter, wherein the transcriptional factor binding site comprises two or more cAMP response element (CRE) motifs.

2. The construct of claim 1, wherein the transcriptional factor binding site comprises four or more CRE motifs.

3. The construct of any one of claims 1-2, wherein each CRE motif comprises a sequence at least about 80% identical to SEQ ID NO: 4 or a fragment thereof.

4. The construct of any one of claims 1-3, wherein each CRE motif comprises a sequence at least about 90% identical to SEQ ID NO: 4 or a fragment thereof.

5. The construct of any one of claims 1-4, wherein each CRE motif comprises SEQ ID NO: 4 or a fragment thereof.

6. The construct of any one of claims 1-5, wherein each CRE motif is the same.

7. The construct of any one of claims 1-5, wherein some or all of the CRE motifs are different.

8. The construct of any one of claims 1-7, wherein the transcriptional factor binding site further comprises a spacer between adjacent CRE motifs.

9. The construct of claim 8, wherein each spacer independently comprises from about 10 nucleotides to about 20 nucleotides.

10. The construct of any one of claims 1-9, wherein the transcriptional factor binding site comprises up to about 100 nucleotides.

11. The construct of any one of claims 1-10, wherein the core promoter is a minimal cytomegalovirus (minCMV) core promoter, a Y-box (YB) core promoter, or a minimal thymidine kinase (miniTK) core promoter.

12. The construct of any one of claims 1-11, further comprising two or more core promoters.

13. The construct of any one of claims 1-12, comprising a sequence at least about 80% identical to any one of SEQ ID NOs: 6 and 8-19 or a fragment thereof.

14. The construct of any one of claims 1-13, comprising a sequence at least about 90% identical to any one of SEQ ID NOs: 6 and 8-19 or a fragment thereof.

15. The construct of any one of claims 1-14, comprising any one of SEQ ID NOs: 6 and 8- 19 or a fragment thereof.

16. The construct of any one of claims 1-15, further comprising one or more RelA binding sites.

17. The construct of claim 16, wherein the RelA binding site comprises a sequence at least about 80% identical to SEQ ID NO: 3 or a fragment thereof.

18. The construct of any one of claims 16-17, wherein the RelA binding site comprises a sequence at least about 90% identical to SEQ ID NO: 3 or a fragment thereof.

19. The construct of any one of claims 16-18, wherein the RelA binding site comprises SEQ ID NO: 3 or a fragment thereof.

20. A vector comprising the construct of any one of claims 1-19 and a target gene downstream of the core promoter.

21. The vector of claim 20, wherein the target gene is up to about 250 nucleotides away from the core promoter.

22. The vector of any one of claims 20-21, wherein the target gene encodes a cytokine, a chemokine, an antibody, a nanobody, a bispecific engager (BiTEs), a cytotoxic compound, a transcription factor for promoting T cell health, or an enzyme having antitumor or pro-T cell health activity.

23. The vector of any one of claims 20-22, wherein the construct further comprises two or more core promoters, and wherein the vector further comprises a target gene downstream of each core promoter.

24. The vector of claim 23, wherein each target gene is different.

25. The vector of any one of claims 20-24, wherein the target gene is expressed only in the presence of a high concentration of adenosine.

26. The vector of claim 25, wherein the high concentration of adenosine is from about 10 pM to about 100 pM.

27. An engineered cell comprising the vector of any one of claims 20-26.

28. The engineered cell of claim 27, wherein the engineered cell is a T cell.

29. The engineered cell of any one of claims 27-28, wherein the engineered cell is a CAR- T cell.

30. A method of treating a solid tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of a vector comprising a transcriptional factor binding site, a core promoter, and a target gene downstream of the core promoter, wherein the transcriptional factor binding site comprises two or more cAMP response element (CRE) motifs.

31. The method of claim 30, wherein the transcriptional factor binding site comprises four or more CRE motifs.

32. The method of any one of claims 30-31 , wherein each CRE motif comprises a sequence at least about 80% identical to SEQ ID NO: 4 or a fragment thereof.

33. The method of any one of claims 30-32, wherein each CRE motif comprises a sequence at least about 90% identical to SEQ ID NO: 4 or a fragment thereof.

34. The method of any one of claims 30-33, wherein each CRE motif comprises SEQ ID NO: 4 or a fragment thereof.

35. The method of any one of claims 30-34, wherein the target gene encodes a cytokine, a chemokine, an antibody, a nanobody, a bispecific engager (BiTEs), a cytotoxic compound, a transcription factor for promoting T cell health, or an enzyme having antitumor or pro-T cell health activity.

36. The method of any one of claims 30-35, wherein the target gene is expressed only in the presence of a high concentration of adenosine.

37. The method of claim 36, wherein the solid tumor comprises a high concentration of adenosine, and wherein the target gene is expressed only within the solid tumor.

38. The method of claim 37, wherein the high concentration of adenosine is from about 10 pM to about 100 pM.

39. The method of any one of claims 30-38, wherein the vector comprises the vector of any one of claims 20-27.

40. The method of any one of claims 30-39, wherein the vector is transfected into an engineered cell, and wherein the method comprises administering to the subject a therapeutically effective amount of said engineered cell.

41. The method of claim 40, wherein the engineered cell is a T cell.

42. The method of any one of claims 40-41, wherein the engineered cell is a CAR-T cell.

43. The method of any one of claims 30-42, wherein the solid tumor is cancerous.

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