Genetic circuit for DNA mutation detection and response

Abstract

The present disclosure generally relates to cancer therapies and, in some embodiments, to using genetic circuits designed for targeted cancer therapies. The genetic circuit, in some embodiments, utilizes a suitable vector capable of delivering DNA sequences comprising functional units comprising a transcriptional promoter, target sequences and a transcriptional terminator. Together, the genetic circuit, in some embodiments, may trigger apoptosis — a target protein activity where cell death occurs — in cancer cells, and protect non-cancer cells from apoptosis. In some embodiments, the genetic circuit may direct target protein activity in cancer cells, while not directing protein activity in non-cancer cells. However, in the presence of certain stimuli, the inhibition is relieved, which may lead to the induction of target protein activity in cancer cells. In addition, some embodiments are generally directed to vectors comprising a first sequence encoding at least a portion of Mdm2 and a promoter, and a second sequence encoding a promoter and a fusion protein comprising at least a portion of p53 and iCasp-9 (or another protein capable of achieving a therapeutic objective), optionally connected by a linker sequence capable of allowing each protein subunit of the fusion protein to be independently active but subject to the same proteolytic pathways as p53 in cancer and non-cancer cells. Other aspects are generally directed to methods of making or using such compositions, kits including such compositions, or the like.

Description

[0001] GENETIC CIRCUIT FOR DNA MUTATION DETECTION AND RESPONSE

[0002] REEATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 636,803, filed April 21, 2024, entitled “Genetic Circuit For DNA Mutation Detection and Response,” and of U.S. Provisional Patent Application Serial No. 63 / 766,799, filed March 4, 2025, entitled “Genetic Circuit For DNA Mutation Detection and Response.” Each of these is incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present disclosure generally relates to cancer therapies and, in some embodiments, to using genetic circuits designed for targeted cancer therapies.

[0006] BACKGROUND

[0007] Cancer is among the leading causes of death worldwide, accounting for almost 20 million new cases, and 9.7 million deaths per year. For example, small cell lung cancer (SCLC) is responsible for 15% of lung cancers (35,000 cases per year in the US), and represents a major clinical challenge due to rapid proliferation, early dissemination, metastases, acquired therapy resistance, and poor outcomes. This cancer is highly aggressive and lethal. Importantly, SCLC tumors have one of the highest mutational loads observed among cancers, and pathogenesis is driven by multiple aberrant pathways. The variety of mutations seen in SCLC tumors may confound attempts to understand mechanisms of primary and acquired chemotherapy resistance. Current therapies include radio- and chemotherapy. However, despite advances in the era of precision oncology, subjects with SCLC have poor prognosis, with median survival of only 7 to 12 months after diagnosis and a 5-year survival of 6-10%. Metastasis is common at diagnosis, with dissemination commonly occurring in the lymph nodes, brain, liver, and bones. At the cellular level, SCLC tumors have extremely high mutation rates (8.62 non-synonymous mutations per million base pairs). Accordingly, improvements in cancer therapies to treat SCLC and other tumors are still needed.

[0008] SUMMARY

[0009] The present disclosure generally relates to cancer therapies and, in some embodiments, to using genetic circuits designed for targeted cancer therapies. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles. One aspect is generally drawn to a composition. In one set of embodiments, the composition comprises a nucleic acid comprising a first sequence having at least 85% homology to an MDM2 cDNA, a second sequence having at least 85% homology to a p53 cDNA excluding silent mutations, and a third sequence encoding a target protein.

[0010] In another set of embodiments, the composition comprises a fusion protein, comprising a first portion having at least 85% identity to p53, and a second portion that is a target protein.

[0011] The composition, in yet another set of embodiments, comprises a cell expressing a fusion protein, where the fusion protein comprises a first portion having at least 85% homology to p53, and a second portion that is a target protein.

[0012] In still another set of embodiments, the composition comprises a nucleic acid comprising: a first sequence having at least 85% homology to an MDM2 gene; a first promoter sequence for the first sequence; a second sequence having at least 85% homology to a TP53 gene excluding silent mutations; a second promoter sequence for the second sequence; a poly-A sequence between the first sequence and the second sequence; a third sequence encoding a target protein; and a linker sequence between the second sequence and the third sequence.

[0013] The composition, in yet another set of embodiments, comprises an aqueous solution, comprising a first nucleic acid sequence having at least 85% homology to an MDM2 gene; a second nucleic acid sequence having at least 85% homology to a TP53 gene excluding silent mutations; and a third nucleic acid sequence encoding a target protein.

[0014] Another aspect is drawn to a method. According to one set of embodiments, the method comprises expressing, within a cell, a fusion protein comprising a first portion having at least 85% homology to p53, and a second portion that is a target protein; and expressing, within the cell, a non-native inhibitor of p53.

[0015] In another set of embodiments, the method comprises administering, to a cell, an agent able to alter activity of a p53-Mdm2 pathway within the cell; and controlling expression of a target protein by altering the activity of the p53-Mdm2 pathway.

[0016] Still another aspect is generally drawn to a genetic circuit for inducible apoptosis in cancer cells. In one set of embodiments, the genetic circuit comprises a bacterial plasmid DNA vector housing two main operons; the first operon regulated by a strong constitutive promoter driving the expression of the MDM2 gene; the second operon regulated by a medium-strength promoter driving the expression of a fusion protein consisting of the p53 gene linked to an executioner caspase (referred to as “Complex9” protein), via a linker sequence; the MDM2 gene within the first operon functioning to ubiquitinate and degrade the Complex9 protein, preventing apoptosis in non-cancerous cells; and upon detection of mutations in cancerous cells, proteins such as ARF and ATM are activated, leading to the inhibition of MDM2 and stabilization of p53, thereby relieving the inhibition and allowing for the activation of apoptosis (or other downstream functions depending on the specific gene if alternatives to iCasp-9 are selected for alternative purposes) specifically in cancer cells.

[0017] The genetic circuit, in another set of embodiments, comprises a bacterial plasmid DNA vector comprising: a first operon comprising an MDM2 gene and a first promoter configured to control expression of the MDM2 gene; and a second operon comprising a second promoter configured to control expression of a fusion protein.

[0018] In yet another set of embodiments, the genetic circuit comprises a nucleic acid vector housing two main genes; the first gene regulated by a promoter driving the expression of the Mdm2 protein; the second gene regulated by a promoter driving the expression of a fusion protein consisting of the p53 gene linked to an executioner caspase (referred to as “Complex- 9” protein), via a linker sequence; the MDM2 gene functioning to ubiquitinate and degrade the Complex-9 protein, preventing apoptosis in non-cancerous cells; and upon detection of mutations in cancerous cells, proteins such as ATM, ATR, CHK1, CHK2, and ARF are activated, leading to the inhibition of Mdm2 and stabilization of p53, thereby relieving the inhibition and allowing for the activation of apoptosis (or other downstream functions depending on the specific target protein if alternatives to iCasp-9 are selected for alternative purposes) specifically in cancer cells or cells which have mutations.

[0019] Still another set of embodiments is generally drawn to a genetic circuit, comprising a nucleic acid vector comprising: a first gene encoding an Mdm2 protein and a first promoter configured to control expression of the MDM2 gene; and a second gene comprising a second promoter configured to control expression of a fusion protein referred to as “Complex-9”. Several methods are disclosed herein of administering a subject with a compound for prevention, treatment, or diagnosis of a particular condition. It is to be understood that in each such aspect of the disclosure, the disclosure specifically includes, also, the compound for use in the treatment or prevention or diagnosis of that particular condition, as well as use of the compound for the manufacture of a medicament for the treatment or prevention or diagnosis of that particular condition.

[0020] In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, fusion proteins, or vectors for use in cancer therapies. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, fusion proteins, or vectors for use in cancer therapies.

[0021] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and not drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0024] Fig. 1 illustrates a genetic system in accordance with one embodiment;

[0025] Fig. 2 illustrates a linear map of an gene, in another embodiment;

[0026] Fig. 3 illustrates a linear map of an gene, in yet another embodiment;

[0027] Fig. 4 is a schematic illustrating various outcomes for cancer and non-cancer cells, in still another embodiment;

[0028] Fig. 5 illustrates certain embodiments of p53 regulation and signaling, in certain embodiments;

[0029] Fig. 6 illustrates certain endogenous and novel pathways for programmed cell death (apoptosis) initiated by Complex-9, in another embodiment;

[0030] Fig. 7 illustrates an example genetic circuit plasmid map, in still another embodiment;

[0031] Fig. 8 illustrates the design of an Mdm2 gene, according to one embodiment;

[0032] Fig. 9 illustrates the design of a Complex-9 operon (as discussed below), according to another embodiment;

[0033] Fig. 10 illustrates an example structure for a genetic sequence, in accordance with still another embodiment; and

[0034] Fig. 11 illustrates various p53 post-translational modifications, in other embodiments.

[0035] DETAILED DESCRIPTION

[0036] The present disclosure generally relates to cancer therapies and, in some embodiments, to using genetic circuits designed for targeted cancer therapies. The genetic circuit, in some embodiments, utilizes a suitable vector capable of delivering DNA sequences comprising functional units comprising a transcriptional promoter, target sequences and a transcriptional terminator. Together, the genetic circuit, in some embodiments, may trigger apoptosis — a target protein activity where cell death occurs — in cancer cells, and protect noncancer cells from apoptosis. In some embodiments, the genetic circuit may direct target protein activity in cancer cells, while not directing target protein activity in non-cancer cells. However, in the presence of certain stimuli, the inhibition is relieved, which may lead to the induction of target protein activity in cancer cells. In addition, some embodiments are generally directed to vectors comprising a first sequence encoding at least a portion of Mdm2 and a promoter, and a second sequence encoding a promoter and a fusion protein comprising at least a portion of p53 and an inducible caspase or another protein capable of achieving a therapeutic objective, optionally connected by a linker sequence capable of allowing each protein subunit of the fusion protein to be independently active but subject to the same proteolytic pathways as p53 in cancer and non-cancer cells. Other aspects are generally directed to methods of making or using such compositions, kits including such compositions, or the like.

[0037] One aspect of the present disclosure is now described, directed to a cancer therapy that uses a genetic circuit capable of selectively inducing apoptosis in cancer cells while sparing non-cancer ones. Such therapies may be used to treat small cell lung cancer (SCLC), skin cancer, or other cancers, such as any of, but not limited to, those described herein. In some cases, the cancer cells contain a mutated version of p53. p53 may activate the transcription of genes involved in DNA repair, cell cycle arrest, senescence, and apoptosis, which regulate cell growth and cancer development. Mutations in p53 can lead to certain types of cancers, as a majority of tumors are thought to contain mutated versions of this gene.

[0038] Certain embodiments are thus generally directed to systems, such as genetic circuits, that exploit the cellular response to DNA damage, which target the interaction between a tumor suppressor, p53 and an E3 ubiquitin ligase, Mdm2. In non-cancer cells, Mdm2 promotes degradation of p53, while in some cancer cells, weakened Mdm2 interactions may stabilize p53 by reducing proteolysis promoted by Mdm2. Once stabilized, p53 may direct cellular programs for DNA repair, cell cycle arrest, senescence, and apoptosis. These systems may also promote in some embodiments the stabilization of a fusion protein comprising p53 and another protein, referred to as a target protein, such that the fusion protein has the same fate as p53 in a cancer cell. In some embodiments, the target protein initiates caspase- mediated apoptosis in a cancer cell, but not in a non-cancer cell, e.g., because the fusion protein is unstable. This may be useful, for example, for treating SCLC or other types of cancer. Based on the natural interaction between p53 and Mdm2, certain embodiments are generally directed to a genetic circuit comprising two genes: one encoding Mdm2 and another encoding a fusion protein consisting of p53 and a target protein joined by a linker. Expression of Mdm2 and the fusion protein is directed by distinct promoters with different strengths. Promoter strength refers to the efficiency and effectiveness of a promoter in initiating transcription in cells. A strong promoter leads to high levels of gene expression, while a weak promoter results in lower expression levels. The Mdm2 promoter may have a higher or lower strength than the fusion protein promoter.

[0039] Accordingly, certain embodiments may be based on the cellular response to DNA damage, particularly involving proteins encoded by other genes like ARF, ATM, ATR, CHK1, and CHK2, which may modulate the interactions between p53 and Mdm2. These proteins can directly or indirectly stabilize or interact with p53 or a fusion protein containing p53 and a target protein. For example, some of these proteins can phosphorylate p53, which may prevent subsequent ubiquitination and proteolysis and therefore stabilize the p53 protein. It is believed that compositions such as those described herein may be used to target cancer cells.

[0040] There may be additional tightly regulated interactions between p53, the tumor suppressor, and Mdm2, its repressor. These interactions may be mediated by cellular proteins, such as ARF, ATM, ATR, CHK1, CHK2, and others, to either directly or indirectly stabilize p53 in response to DNA mutations, oncogene activation, and other cellular stress signals. Once stabilized, p53 initiates programs leading to DNA repair, cell cycle arrest, senescence, and apoptosis. See, e.g., Fig. 5.

[0041] For example, it is believed that upon activation, ARF (ADP Ribosylation Factor) binds to the Mdm2 acidic domain, inhibiting p53 ubiquitination, and indirectly promoting p53 accumulation and activity. ATM (Ataxia Telangiectasia Mutated), upon activation, phosphorylates (adds phosphate molecule to) Mdm2, which may inhibit Mdm2's ability to ubiquitinate p53. This may prevent p53 destruction and / or promote p53’s ability to act as a transcription factor. ATM also may activate CHK2 by phosphorylation. ATR (Ataxia Telangiectasia and Rad3 -related), in response to DNA damage, may phosphorylate Mdm2, which may reduce its affinity for p53 and favor stabilization. It also may activate CHK1 by phosphorylation. CHK1 (Checkpoint Kinase 1) may be phosphorylated by active ATR. The active CHK1 may phosphorylate and stabilize p53. CHK2 (Checkpoint Kinase 2) may be phosphorylated by ATM. Activated CHK2 may play a role in regulating the cell cycle and / or DNA repair processes. It also may be able to phosphorylate and stabilize p53. Non-limiting examples of possible post-translational modifications of p53 that may be present in various embodiments are shown in Fig. 11.

[0042] Fig. 5 illustrates p53 regulation and signaling. Certain embodiments are thus based on interactions between p53and Mdm2. Without wishing to be bound by any theory, it is believed that in non-cancer, unstressed cells, p53 stimulates the production of Mdm2, and is rapidly degraded by it. In response to stress, p53 and Mdm2 are post-translationally modified by proteins such as ATM, ATR, CHK1, and / or CHK2, etc., which may stabilize and / or activate p53 directly or indirectly. Activated p53 is a sequence-specific transcriptional activator that induces a variety of cellular outcomes: cell cycle arrest, DNA repair, senescence and apoptosis.

[0043] The p53 gene may be mutated (e.g., missense, gain-of-function, silent, or other), truncated, or deleted in about half of all cancers, while the p53 signaling pathway is at least disrupted in many others. Many tumors contain modified p53 that promotes tumor growth and spread, or mutations that interfere with wild type p53. These mutated versions of p53 are referred to as “gain-of-function” and “dominant negative” mutants (dnP53), respectively, where the latter is a subset of the former.

[0044] Thus, certain embodiments may be used to restore the antitumor activity of p53 to a cancer cell, which may cause the tumor cells to receive a strong apoptotic signal that is resistant to gain-of-function mutants, including dnP53. This may be achieved, as a nonlimiting example, as described below.

[0045] Referring now to Fig. 6, a fusion protein between wildtype p53 and an inducible Caspase 9 (iCasp-9) may be expressed from a constitutive promoter upon delivery of the plasmid into the tumor. These may be connected via linker. iCasp-9 is a genetically modified version of a lethal initiator caspase. Caspases are a family of protease enzymes that cleave target proteins only after an aspartic acid residue. Caspases play an important role in apoptosis, which plays a crucial role in tumor suppression. Once the fusion protein is stabilized the p53 portion may build a tetramer capable of transactivating pro-apoptotic genes, including BCL2 Associated X (BAX) and BCL2 Antagonist / Killer 1 (BAK). This is referred to as the endogenous pathway of apoptosis initiation. In addition to the endogenous pathway, the iCasp-9 portion of the fusion protein may trigger apoptosis upon chemical induction with its cognate drug, referred to as a novel pathway of apoptosis initiation, see Fig. 6.

[0046] The plasmid may also encode Mdm2. In non-cancer cells, Mdm2 may destabilize p53 through its ubiquitination activity, preventing the expression of the fusion protein and the activation of apoptosis. In cancer cells, however, stabilized p53 and the fusion protein may cause apoptosis to be initiated via an endogenous pathway (e.g., through BAX and BAK), and / or through a second novel pathway involving drug-activated iCasp-9 dimerization.

[0047] Accordingly, certain aspects of the present disclosure pertain to cancer therapies, and / or to technologies for cancer therapy. For example, some aspects are generally directed to plasmids, or other entities, which can be administered to cells within a subject, e.g., to treat a subject having or at risk of cancer. The subject may be human, or a non-human animal. Examples of subjects include, but are not limited to, a mammal such as a cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, cat, a primate (e.g., a monkey, a chimpanzee, etc.), or the like. In some cases, the subject is a non-mammal such as a bird, an amphibian, or a fish.

[0048] In some embodiments, the subject may have cancer. For example, the subject may have a solid tumor or a hematological malignancy. Other non-limiting examples of cancer include skin cancer, biliary tract cancer, bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, endometrial cancer, esophageal cancer, gastric cancer, hematological neoplasms, multiple myeloma, liver cancer, lung cancer, lymphomas, neuroblastomas, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, sarcomas, testicular cancer, stromal tumors, thyroid cancer, renal cancer, or the like. Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancer.

[0049] However, it should be understood that other aspects are directed to indications other than cancer. For example, certain aspects are generally directed to combinations of Mdm2 and a fusion protein consisting of p53 and a target protein, which can be used to treat a variety of indications, e.g., as discussed herein. In some cases, the Mdm2 portion may be partial, e.g., a sequence, such as an amino acid or a nucleic acid sequence, that has 85% homology to Mdm2 or its sequence. In some cases, the p53 may be partial, e.g., a sequence, such as an amino acid or a nucleic acid sequence, that has 85% homology to p53 or its sequence. The target protein may also be any of a variety of possible proteins. As a nonlimiting example, in some embodiments, the sequences may be present within plasmids, referred to as a genetic circuit. In one set of embodiments, for instance, the genetic circuit that is created may be composed of two operons, con: an “Mdm2” operon, and a fusion protein operon. An operon may be a cluster of genes on a DNA strand that are transcribed together as a single unit.

[0050] The fusion protein gene may encode at least a portion of p53 and a target protein, or portion thereof, such as a caspase (for example, iCasp-9 or others such as described herein). A fusion protein of p53 and a caspase (optionally connected by a linker) may be referred to herein as a “Complex-9” protein, e.g., to avoid confusion. However, other fusion proteins, e.g., using other caspases or other suicide genes, etc., are also possible in other embodiments. Non-limiting examples of suicide genes include alternative caspases, genes that are involved in the p53 apoptotic pathway, or the like. Additional non-limiting examples of suicide genes include transfection of herpes simplex virus thymidine kinase (HSV-TK) along with administration of Ganciclovir (GSV), Diphtheria toxin, or the like.

[0051] In accordance with some embodiments, the Mdm2 gene may be responsible for degradation of the fusion protein (e.g., Complex-9 or other fusion proteins), for example, through ubiquitination starting at the p53 portion, which may allow the destruction of the iCasp-9 portion of the fusion protein (or other such portions in other types of fusion proteins). The Mdm2 gene may be regulated under the expression of a promoter. The promoter may be a CMV (cytomegalovirus) promoter, or other promoter, such as any of those described herein. Non-limiting examples include Human cytomegalovirus immediate early enhancer / promoter (SV40), Human ubiquitin C promoter (UBC), EF1A Human eukaryotic translation elongation factor 1 al promoter (EF1A), Human phosphoglycerate kinase 1 promoter (hPGK), CMV early enhancer fused to modified chicken P-actin promoter (CAG) Gene.). The promoter may be capable of driving robust gene expression in mammalian cells in certain embodiments. In some cases, the promoter is of enough strength to control fusion protein expression in non-cancer cells.

[0052] The fusion protein (e.g., Complex-9 or others) may be under the control of a second promoter, such as an Human eukaryotic translation elongation factor 1 al short form(EFS) promoter, including any of those described herein. Other non-limiting examples of such promoters include RSV (Rous sarcoma virus), UBS or EF1A. The promoter may be a promoter capable of driving gene expression in mammalian cells, in certain embodiments. In some cases, this promoter may be a promoter of titratable strength (e.g., inducible), and / or the promoter may have a strength that is ample for target protein expression.

[0053] In addition, in some embodiments, a linker may connect components of the fusion protein. The linker may be any linker that is appropriate for folding and function of each portion of the fusion protein. In some cases, the linker may be a 3xGGGGS linker (e.g., GGGGSGGGGSGGGGS) (SEQ ID NO: 1) or a 3xGS linker (e.g., GSGSGS) (SEQ ID NO: 2). In some embodiments, the linker may be a glycine- serine (GS) linker (e.g., (GGGGS)n, where n may be 1, 2, 3, 4, etc.), (EAAAK)n, where n may be 1, 2, 3, 4, etc., (XP)n, where X may be A, K, or E, and n may be 1, 2, 3, 4, etc., or (PGPG)n, where n may be 1, 2, 3, 4, etc. As another example, the linker may be a cleavable linker, such as a cleavable disulfide linker (for example, LEAGCKNFFPRSFTSCGSLE (SEQ ID NO: 5), an enzymatically cleavable peptide (e.g., Furin, etc.), or the like. The linker may also be a naturally-derived linker or a fusion alpha-helix, etc. Still other non-limiting examples include (G)n, where n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., KESGSVSSEQLAQFRSLD (SEQ ID NO: 6), EGKSSGSGSESKST (SEQ ID NO: 7), etc.

[0054] Without wishing to be bound by any theory, it is believed that, due to the gene’s close proximity and the enzymatic nature of Mdm2, the Mdm2 may partially or completely prevent protein expression of the fusion protein, e.g., Complex-9, in non-cancer cells. However, in cancer cells (e.g., featuring mutagenesis), a variety of genes are often able to detect DNA damage and respond accordingly. These may include proteins such as the ARF and ATM proteins (however, there are many more). These proteins (e.g., ARF) may play an important role. The ARF protein, for example, may directly bind to and inhibit the Mdm2 protein by sequestering it to the nucleus, indirectly allowing for the accumulation and stabilization of p53, for example, such that after interacting with ARF, Mdm2 may no longer be able to degrade the protein. In some cases, the plasmid may encode a fusion protein (e.g., Complex- 9), which may also encode an apoptosis-initiating caspase, or another suicide gene. In some cases, this may allow apoptotic function to occur in mutated or cancer cells (e.g., at the delivery site or elsewhere in a subject). In some cases, such synthetic genetic pathways may allow distinct, low cytotoxicity options that target mutated cells indiscriminately but with low off-target effects in certain cases.

[0055] Fig. 10 illustrates a non-limiting example of a genetic circuit, in accordance with certain aspects of the present disclosure. This figure shows a sequence 5 having a first sequence 10, a second sequence 20, and a third sequence 30. First sequence 10 may include a sequence having at least some homology to Mdm2, second sequence 20 may include a sequence having at least some homology to p53, and third sequence 30 may be a target protein that is desired to expressed, e.g., within a cell, as controlled by the genetic circuit. The homology of the first and second sequences may each independently be, for example, at least 75%, at least 85%, at least 90%, or other homologies such as any of those described herein. In some cases, the sequences may be wild-type sequences, e.g., having 100% homology. The target protein may be, for example, a naturally occurring protein, a portion thereof, a protein that has been modified in some way (e.g., mutated, truncated, etc.), for example, such that it is not naturally occurring, etc.

[0056] One or more of the first sequence 10 and second sequence 20 may be controlled by a promoter, e.g., first promotor 15 and second promoter 25. The first and second promoters may be the same or different. In some cases, the first promoter has higher strength than the second promoter. Non-limiting examples of promoters include EFS1, EF1A, UBC, or other promoters such as any of those described herein.

[0057] In some embodiments, one or more linker sequences may optionally be present, for example, linker 41 between first sequence 10 and second sequence 20, and / or linker 42 between second sequence 20 and third sequence 30.. Other potential linker sequences include, but are not limited to, 3xGGGGS (SEQ ID NO: 1), 3xGS (SEQ ID NO: 2), or others such as any of those described herein.

[0058] In addition, it should be understood that Fig. 10 is described by way of example only, and in other embodiments, the sequences may be present in a different order, other sequences may also be present, or the like. For example, the first, second, and third sequences may be present in any order such as second-first-third, second-third-first, first-third-second, third- second-first, or third-first-second.

[0059] Thus, while the disclosed technology may have embodiments in many different forms, e.g., as shown in the drawings, and as herein described in detail, e.g., in several specific embodiments, it should be understood that the present disclosure is to be considered as an exemplification of the principles of the technology and is not intended to limit the technology to the embodiments illustrated. The terminology used herein is for the purpose of describing particular embodiments and is not intended to limit the technology.

[0060] Cancer presents itself as a debilitating disease often leading to death. There is a need for a less cytotoxic and more efficient method of treating various types. Accordingly, in some aspects, the present disclosure is related to various embodiments of systems and methods that may combine one or more genes with existing cellular processes (e.g., the phosphorylation of p53 by proteins such as ATM, ATR, CHK1, and CHK2). In some cases, these may be used to create a negative feedback loop which can be controlled by proteins in response to DNA mutations, and which may ultimately lead in certain cases to apoptosis in cancer cells.

[0061] Fig. 1 illustrates a genetic system in accordance with one embodiment of the present disclosure. However, it should be understood that this is by way of example only. In this nonlimiting example, the vector map of Fig. 1 depicts a mammalian transient expression vector which holds multiple components. The components shown in Fig. 1 also include exemplary details in the plasmids, consistent with plasmids used for synthetic biology, molecular biology, genetic engineering, and other related fields. These exemplary fields include (but are not limited to): the gene for enzyme P-lactamase, which inactivates ampicillin (AmpR), pUC origin of replication (ori), SV40 late poly- A, and the Kozak motif. It should be understood, however, that the present disclosure is not so limited, and in other embodiments, some or all of these exemplary fields may not be present.

[0062] The iCasp-9 protein may be linked to p53 (forming the “Complex-9” fusion protein in this particular example), e.g., for initiating apoptosis. However, it should be understood that this is presented to act as a placeholder for various cancer therapy treatments, and / or different use cases running under similar circuits. In some cases, the genetic circuit may be used to detect mutations and / or activate different downstream open reading frames (ORFs). For instance, possible downstream replacement ORFs within the feedback loop may include, but are not limited to, alternative suicide genes (alternative caspases, genes that are involved in the p53 apoptotic pathway, or others), DNA repair genes (DNA polymerases lambda ( ) and mu (p)), Cas-9 systems, etc.), or genes that activate or suppress senescence. Other genes, e.g., suicide genes or other genes, are also possible in yet other embodiments.

[0063] Fig. 2 illustrates a linear map of one example gene shown in Fig. 1. This gene contains a promoter, such as the CMV promoter, followed by the Mdm2 ORF. However, the promoter region upstream of the ORF is not limited to the CMV (this is just an example of a promoter that could be used interchangeably with other promoters). The promoter region can be any promoter. For example, the promoter may be any promoter having a strength that is able to make the circuit appropriately target mutated or cancer cells. For example, the promoter may be strong enough in certain cases to prevent partial or full functionality of the Complex-9 protein (or other fusion protein) synthesized in non-mutated cells, such that the signals from other proteins (ATM, ATR, ARF, CHK1, CHK2, and more) are able to bind to and move Mdm2 to the nucleus, or phosphorylate p53.

[0064] Fig. 3 also illustrates a linear map of another example gene shown in Fig. 1. This gene contains an EFS promoter, followed by the TP53 (p53) ORF, a 3xGGGGS linker, and the iCasp-9 ORF. This gene is used to form the “Complex-9” protein, which is hypothesized to be degraded by the Mdm2 protein, e.g., through ubiquitination. It should be understood, however, that these promoters and this structure are by way of example only, and in other embodiments, other genes are also possible.

[0065] Certain embodiments are generally directed to plasmids, e.g., plasmids that are capable of transiently delivering genes and genetic elements to mammalian cells. In one set of embodiments, the plasmid may carry Complex-9 and Mdm2, which may be controlled under constitutive, inducible, or tissue- specific promoters of varying strengths. Fig. 7 shows an example genetic circuit plasmid map (linear). As non-limiting examples, the plasmid may express Mdm2 and Complex-9 under constitutive promoters of varying strengths. For example, some versions may carry genes for mCherry fluorescent reporter to evaluate p53 stability in cancer and non-cancer cells, Enhanced Green Fluorescent Protein (EGFP) to optimize transfection conditions, or a nuclear export signal (NES), etc. In some cases, the plasmids may carry a bacterial origin of replication (pUC ori), or a gene coding the enzyme P-lactamase, which inactivates ampicillin (AmpR) to allow production in Escherichia coli. Other elements may also be added to the plasmid in yet other embodiments.

[0066] Caspases may be activated when they form a complex with other cellular proteins. For example, inducible iCasp-9 may be activated when it forms a dimer (two molecules of iCasp- 9), e.g., in the presence of a Chemical Inducer of Dimerization (CID) such as AP1903, AP20187, or rapamycin and their analogs.

[0067] Fig. 5 illustrates p53 regulation and signaling. The disclosed technology is based on a negative feedback loop consisting of the tumor suppressor, p53 and an E3 ubiquitin ligase, Mdm2. In non-cancer, unstressed cells, p53 stimulates the production of Mdm2, and is rapidly turned over by it. In response to stress, p53 and Mdm2 are post-translationally modified by ATM, ATR, CHK1 and 2, which stabilize and activate p53. Active p53 is a sequence-specific transcriptional activator that induces a variety of cellular outcomes including cell cycle arrest, DNA repair, senescence and apoptosis.

[0068] Fig. 6 shows certain endogenous and novel apoptosis pathways initiated by Complex- 9, as a non-limiting example. In a cancer cell, Complex-9 may initiate two pathways leading to apoptosis. The p53 moiety may stimulate pro-apoptotic genes BAX and BAK, resulting in the activation of full-length Caspase 9 in an apoptosome, which may trigger apoptosis through the executioner Caspase 3 protease. This pathway requires active p53 and is inhibited by dnP53. A second pathway may be activated by the iCasp-9 moiety, e.g., activated through its drug binding domain (FKBP12-F36V). In the presence of the cognate drug (the Chemical Inducer of Dimerization, or CID), iCasp-9 may dimerize and trigger apoptosis, e.g., through Caspase 3. Unlike the other pathway stimulated by active p53, the novel pathway may not be inhibited by dnP53.

[0069] Thus, certain embodiments are generally directed to plasmids, e.g., plasmids that are capable of transiently delivering genes and genetic elements to mammalian cells. In one set of embodiments, the plasmid may carry Complex-9 and Mdm2, which may be controlled under constitutive, inducible, or tissue- specific promoters of varying strengths (e.g., Fig. 7). This figure shows an example genetic circuit plasmid map (linear). As non-limiting examples, the plasmid may express Mdm2 and Complex-9 under constitutive promoters of varying strengths. For example, some versions may carry genes for mCherry fluorescent reporter to evaluate p53 stability in healthy and cancer cells, EGFP to optimize transfection conditions, or a nuclear export signal (NES), etc. In some cases, the plasmids may carry the pUC ori, and the P-lactamase gene to allow production in Escherichia coli. Other elements may also be added to the plasmid in yet other embodiments.

[0070] A variety of techniques may be used to deliver the plasmids to cells in various embodiments. Non-limiting examples include transfection, electroporation, or viral transduction. In some cases, the plasmid may be fabricated into microneedle patches containing liposomes for delivery into cells on or below the skin surface.

[0071] Certain aspects are generally directed to combinations of Mdm2, p53, and a target protein, as well as methods of making or using such combinations, e.g., to treat a variety of indications such as is discussed herein. In some cases, for example, the combination may be administered to a subject, e.g., as a protein, nucleic acid (e.g., for use in gene therapy), or the like.

[0072] For example, various embodiments are generally directed to certain combinations of Mdm2, p53, and a target protein, and / or portions of one or more of these. In some cases, the p53 and the target protein (or portions of either or both of these) may be present as a fusion protein, optionally connected by a linker. These may be connected in any suitable order. In some cases, the p53 and / or the target protein may be expressed with MDM2, e.g., within a cell. In certain embodiments, proteins and / or portions thereof may be present. In addition, certain embodiments such as discussed herein are directed to nucleic acid sequences (e.g., DNA or RNA) that encode such proteins or portions thereof. For example, some embodiments may be directed to a nucleic acid that encodes a first sequence encoding Mdm2 (or portions thereof), p53 (or a portions thereof), and a target protein (or a portions thereof). These may be present within the nucleic acid in any suitable order, e.g., Mdm2-p53-target, Mdm2-target-p53, p53-target-Mdm2, p53-Mdm2-target, target-Mdm2-p53, target-p53- Mdm2, etc. In addition, in some cases, one or more of these may contain linkers such as those discussed herein, e.g., separating one or more of these regions. In addition, in some embodiments, these may be present on more than on nucleic acid (e.g., a first nucleic acid encoding Mdm2 and a second nucleic acid encoding p53-target, etc.). Optionally, the MDM2 and fusion protein genes may each contain a promoter, and the promoters may be the same or different.

[0073] In one set of embodiments, the composition may contain Mdm2 or a portion thereof. The structure of MDM2 is shown in SEQ ID NO: 8 (nucleic acid sequence) and SEQ ID NO: 9 (amino acid sequence). As discussed herein, Mdm2 is a protein that is encoded by the MDM2 gene, and Mdm2 is a negative regulator of p53. In some cases, a portion of Mdm2 may be present. For example, a sequence (e.g., a nucleic acid or an amino acid sequence) directed to MDM2, such as discussed herein, may exhibit a homology to MDM2 (e.g., human MDM2) of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some cases, e.g., in a nucleic acid sequence, silent mutations which do not affect the encoding of the amino acids (e.g., replacing CUU with CUC, both of which are codons for leucine) are excluded from such homology. In addition, in some cases, a promoter may also be present, e.g., as discussed herein.

[0074] In some cases, the Mdm2 portion may be any portion of at least 30 or more consecutive amino acids of SEQ ID NO: 9. For example, the Mdm2 portion may be portion having at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, at least 360, or at least 380 consecutive amino acids of SEQ ID NO: 9. In some cases, the portion may have no more than 390, no more than 380, no more than 360, no more than 340, no more than 320, no more than 300, no more than 280, no more than 260, no more than 240, no more than 220, no more than 200, no more than 180, no more than 160, no more than 140, no more than 120, no more than 100, no more than 80, no more than 60, no more than 50, or no more than 40 consecutive amino acids of SEQ ID NO: 9.

[0075] Combinations of any of these ranges are also possible in other embodiments. For example, the Mdm2 portion may have between 30 and 300, between 100 and 200, between 50 and 80, between 200 and 400, etc. consecutive amino acids of SEQ ID NO: 9.

[0076] In one set of embodiments, the composition may contain p53, or a portion thereof. The structure of p53 is shown in SEQ ID NO: 3 (nucleic acid sequence) and SEQ ID NO: 4 (amino acid sequence). p53 is a protein that is encoded by the TP53 gene. In some cases, a portion of p53 may be present. For example, a sequence (e.g., a nucleic acid or an amino acid sequence) directed to p53, such as discussed herein, may exhibit a homology to p53 (e.g., human p53) of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some cases, e.g., in a nucleic acid sequence, silent mutations which do not affect the encoding of the amino acids (e.g., replacing CUU with CUC, both of which are codons for leucine) are excluded from such homology. In addition, in some cases, a promoter may also be present, e.g., as discussed herein.

[0077] In some cases, the p53 portion may be any portion of at least 30 or more consecutive amino acids of SEQ ID NO: 4. For example, the p53 may be portion having at least 40, at least 50, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 220, at least 240, at least 260, at least 280, at least 300, at least 320, at least 340, at least 360, or at least 380 consecutive amino acids of SEQ ID NO: 4. In some cases, the portion may have no more than 390, no more than 380, no more than 360, no more than 340, no more than 320, no more than 300, no more than 280, no more than 260, no more than 240, no more than 220, no more than 200, no more than 180, no more than 160, no more than 140, no more than 120, no more than 100, no more than 80, no more than 60, no more than 50, or no more than 40 consecutive amino acids of SEQ ID NO: 4. Combinations of any of these ranges are also possible in other embodiments. For example, the p53 portion may have between 30 and 300, between 100 and 200, between 50 and 80, between 200 and 400, etc. consecutive amino acids of SEQ ID NO: 4.

[0078] Certain regions or domains within the primary amino acid sequence of p53 are shown in Fig. 11. These domains have been referred to as the amino-terminal transactivation domain (TAD) (residues 1-61), the proline-rich domain (PRD) (residues 62-92), the DNA-binding domain (DBD) (residues 94-292), the tetramerization domain (OD) (residues 326-353), or the carboxy-terminal regulatory domain (CTD) (residues 353-390). In some embodiments, the p53 portion of the fusion protein may include one or more of these regions.

[0079] In addition, in some cases, the p53 or portion thereof may be part of a fusion protein. For example, a fusion protein may include p53 (or portion thereof), and a target protein (or portion thereof), e.g., as described herein. In some cases, the fusion protein may comprise p53 (or portion thereof), and a target protein (or portion thereof), where the fusion protein is co-expressed with Mdm2, e.g., such that the p53 sequence is stabilized and functional in a p53-stabilizing environment, and the target protein is functional in this stabilized state.

[0080] One set of embodiments is generally directed to a variety of promoters, which can be used as promoters for MDM2 and / or promoters for TP53. In some cases, the promoter for TP53 is of different strength than the promoter for MDM2. Non-limiting examples of promoters that can be used include CMV (human cytomegalovirus immediate early enhancer / promoter), CMV+intron (human cytomegalovirus immediate early enhancer / promoter fused with the splicing signal from the human beta- globin intron), EFl A (human eukaryotic translation elongation factor 1 alpha 1 promoter), EFS (human eukaryotic translation elongation factor 1 alpha 1 short form promoter), CAG (CMV early enhancer fused to modified chicken beta- actin promoter), CBA (CMV early enhancer fused to chicken beta-actin promoter and SV40 intron), CBh (a modified mini-CBA promoter), SFFV (spleen focus-forming virus promoter), MSCV (murine embryonic stem cell virus promoter), MNDU3 (U3 region from the myleproliferative sarcoma virus long terminal repeat (LTR) with the negative control region removed), SV40 (simian virus 40 promoter), mPGK (mouse phosphoglycerate kinase 1 promoter), hPGK (human phosphoglycerate kinase 1 promoter), UBC (human ubiquitin C promoter), Nanog promoter, Nes (rat nestin intron 2 enhancer fused to mouse Hsp68 minimal promoter), Tubala (Rat alpha- 1 A tubulin promoter), Camk2a (mouse alpha-calcium-calmodulin dependent kinase II promoter) long or short version , SYN1 (human synapsin I promoter), MIR218 (human motor-neuron specific microRNA-218 promoter), Hb9 (mouse Hb9 enhancer fused to mouse Hsp68 minimal promoter), Th (mouse tyrosine hydroxylase promoter), Thyl (mouse thymus cell antigen 1 promoter), NSE (rat neuron- specific enolase promoter), GFAP (human glial fibrillary acidic protein promoter) long or short version , Ibal (mouse ionized calium binding adapter molecule 1 promoter), ProA (mouse Gnat2 promoter)! or 7, hRHO (human rhodopsin promoter), hBESTl (human bestorphin 1 promoter), Grm6 (mouse metabotropic glutamate receptor 6 fused to a basal SV40 promoter) , Prnp (mouse prion protein promoter), Cnp (mouse 2’,3’-cyclic nucleotides’ -phosphodiesterase promoter), K14 (human keratin 14 promoter), K19 (mouse keratin 19 promoter), BK5 (bovine keratin 5 promoter), mTyr (mouse tyrosine promoter), cTnT (chicken cardiac troponin T promoter), alpha-MHC (mouse alpha-cardiac myosin heavy chain promoter) long or short version , Myog (mouse myogenin promoter), ACTA1 (human skeletal muscle actin alpha- 1 promoter), MHCK7 (murine muscle creatine kinase promoter and enhancer region of alpha-myosin heavy-chain), CK8e (muscle specific creatine kinase promoter), SPc5-12 (synthetic promoter c5-12), SM22a (mouse transgelin promoter), EnSM22a (chimeric vascular smooth muscle-specific enhancer / promoter), Runx2 (mouse runt related transcription factor 2 promoter), OC (human osteocalcin promoter), Collal (rat collagen type I alpha- 1 promoter), Col2al (mouse collagen type II alpha- 1 promoter), aP2 (rat adipocyte P2 promoter), Adipoq (mouse adiponectin promoter), Tiel (mouse tyrosine kinase with immunoglobulin-like and EGF-like domains 1 promoter), Cd 144 (mouse cadherin 5 promoter), CD68 (human CD68 promoter) short or long version, CD 11b (human integrin subunit alpha M promoter), Afp (mouse alpha- fetoprotein enhancer II fused with human beta globin promoter), Alb (mouse albumin promoter), TBG (human thyroxine- binding globulin promoter), MMTV (mouse mammary tumor virus long terminal repeat ), Wap (mouse whey acidic protein promoter), HIP (human insulin promoter), Pdxl (mouse pancreatic and duodenal homeobox 1 promoter), Ins2 (rat insulin 2 promoter), Hcn4 (hyperpolarization-activated cyclic nucleotide-gated K+ 4 promoter), NPHS2 (human podocin promoter), SPB (human surfactant protein B promoter), CD 144 (human cadherin 5 promoter), TERT (human telomerase reverse transcriptase promoter), CEA (carcinoembryonic antigen promoter), OC (osteocalcin promoter) TRE (tetracyclineresponsive element promoter), TRE3G (tetracycline-responsive element promoter), etc.

[0081] As non-limiting examples, in some embodiments, the MDM2 promoter may be EF1A, EFS, or UBC. In some embodiments, the TP53 promoter may be EF1A, EFS, or UBC. These may be independently selected, i.e., both promoters may be the same or different (for example, both promoters may be EFl A, EFS, or UBC). For example, the first and second promoters may be EF1A / EF1A, EF1A / EFS, EF1A / UBC, UBC / EF1A, UBC / EFS, UBC / UBC, EFS / EF1A, EFS / UBC, or EFS / EFS.

[0082] The target protein may be any of a wide variety of proteins in accordance with one set of embodiments, and / or a portion thereof. In general, the target protein may be any protein, or portion thereof, that can be expressed, e.g., when controlled in a genetic circuit controlled by Mdm2 and p53, as discussed herein. In some cases, for example, the target protein is a caspase, or a suicide gene, e.g., a gene that, when expressed, results in cellular death by apoptosis or another process. For example, the target protein may be an initiator or executioner caspase such as iCasp-9, iCasp-8, iCasp-6, or the like. As other examples, the target protein may be a fusion protein composed of an antigen that may be expressed in cancer cells, where the antigen may be targeted by the immune system. One example of such a target protein is influenza hemagglutinin. Another example is a fusion partner that encodes for a gene for the replication for an oncolytic (cancer killing) virus, e.g., an engineered Talimogene laherparepvec (T-VEC) herpes virus with knocked out thymidine kinase (TK), which would make it dependent on TK from the host cell. In some cases, the cancer cells may be engineered to produce viral TK. As still another example, the target protein may include Cas9, which may be used to knock out a gene that allows for cell survival / replication, e.g., which may be knocked out in the cancer cells. Yet another example of a target protein is a cellular protein that induces senescence, e.g., in a permanent state of cell cycle arrest. As another example, the target protein may be a gene encoding resistance to an exogenous drug or compound. This may be used, for example, to screen or select for cancer cell lines, i.e., by identifying cells resistant to the drug. As yet another example, the target protein may be a fluorescent or chemiluminescent biomolecule, which could be used to distinguish between cancer and non-cancer cells. In some cases, this could be useful for identifying the locations of cancer or tumors within a human or animal subject, e.g., for surgical removal.

[0083] One non-limiting example of a target protein is FKBP / Casp8, which is FK506- binding protein 12 (with F36V mutation) fused with human caspase-8 protein deleted for amino acids 1 through 215, and linked to the N-myristoylation signal from Src kinase. Another example is FK50612 / Casp9, which is FK506-binding protein 12 (with F36V mutation) fused with human Caspase-9 protein deleted for amino acids 1 through 134, and linked to a HA-tag corresponding to amino acids 98-10 of the human influenza hemagglutinin (HA) molecule. Yet another example is deltaTK, which is a truncated thymidine kinase that can convert GCV (ganciclovir) into a toxic metabolite that inhibits DNA synthesis. Still another example is E. coli cytosine deaminase (encoded by the gene, CodA), which catalyzes 5-FC (5-fluorocytosine) into 5-FU (toxic 5-fluorouracil). Another example is DTA (Diphtheria toxin A), which is capable of inducing apoptosis. Yet another example is DTR or simian Diphtheria toxin receptor, which allows for Diphtheria toxin to bind to a cell surface and induce apoptosis. Still another example is CYP2B1 (Cytochrome P4502B1), which converts CPA (cyclophosphamide) and IFO (ifosfamide), which may lead to active inhibitors which can lead to cell death by apoptosis and ROS (reactive oxygen species) generation. Yet another example is NTR (nitroreductase) from Escherichia coli, in which CB1954 (5-[aziridin-l-yl]-2,4-dinitrobenzamide, a DNA alkylating agent) is converted to a toxic metabolite. Still another example is CPG2 (Carboxypeptidase G2), which is able to convert certain pro-drugs (e.g., nitrogen mustard based drugs) such as CMDA (4-([2- chloroethyl] [2-mesyloxy ethyl] amino)benzyol- L -glutamic acid) or ZD2767P into toxic forms. Other examples are BAX and BAK, which are proteins for inducing MOMP (mitochondrial outer membrane permeabilization), which is a step in apoptosis. Another example is CE (carboxylesterase), which may convert irinotecan into SN38, inhibiting topoisomerase I, leading to DNA synthesis issues and cell death. Still another example is HRP (Horseradish peroxidase), which can convert the non-toxic prodrug IAA (indole-3- acetic acid) into cytotoxic metabolites within cells. Another example is PNP (purine nucleoside phosphorylase)) / , which can catalyze purine ribonucleoside prodrugs such as MEP (6-methylpurine 2-deoxyriboside) and fludarabine into toxic forms within cells. Another example are certain chemical compounds, such as PAC-1 and PAC-la, which are capable of converting procaspase-3 into active caspase-3, which can initiate an apoptotic cascade.

[0084] In some cases, the target protein may be an antigen. For example, the antigen may be one that causes an immune response. Thus, for example, cells such as cancer cells may be caused to express the antigen, e.g., as discussed herein, and the antigen may make such cancer cells susceptible to being attacked and removed by the immune system. Non-limiting examples of such antigenic target proteins include Influenza hemagglutinin (HA), Hepatitis B surface protein (HBsAg), human Papillomavirus LI protein, Tetanus toxoid (TT), Diphtheria toxoid (DT), calreticulin (CALR), or HER2 growth factor receptor, which may be recognized by monoclonal antibodies. For example, trastuzumab a monoclonal antibody that binds to the human epidermal growth factor receptor 2 (HER2) protein, could direct an immune response against such cells.

[0085] In some embodiments, the target protein may be replication protein, e.g., involved in oncolytic viruses. For example, the target protein may be thymidine kinase (TK), which can be targeted due to its function in DNA synthesis.

[0086] In certain embodiments, the target protein may be a CRISPR or Cas9 protein. These include hCas9, Cas9 (D10A mutation), dCas9 (D10A + H840A mutations), dCas9 / VP64 (dCas9 fused with V64 transcriptional activator), dCas9 / VPR (dCas9 fused with VP64-p65- Rta (VPR) activator), dCas9 / KRAB (dCas9 fused with a KRAB transcriptional silencer), dCas9 / KRAB / MeCP2 (dCas9 with KRAB-MeCP2 bipartite repressor domain), dCas9- 10xGCN4_v4 (dCas9 with SunTagl0x_V4), SpCas9-HFl, eSPCas9, BE3 (base editor with rAPOBEC, Cas9 (D10A), and UGI), Casl2a (Cpfl), Cas9 / mSA (Cas9 fused with monomeric streptavidin), SaCas9, CjCas9, or the like.

[0087] The target protein may be a cell cycle or senescence protein in some cases. For example, the target protein may be CDKN1A (p21) or p27Kipl.

[0088] The target protein may be a drug selection marker in some cases. For example, the target protein may be Neo (neomycin resistance gene), Puro (puromycin resistance gene), Hygro (hygromycin resistance gene), Bsd (blasticidin resistance gene), Bleo (bleomycin resistance gene), or the like.

[0089] The target protein may be a reporter protein, e.g., a fluorescent or a chemiluminescent reporter. In some cases, the fluorescent reporter may be a pH-sensitive fluorescent reporter. Examples of pH-sensitive fluorescent reporters include, but are not limited to, pHluorin2, Superecliptic-Phluorin, or mt-mKeima, etc. Examples of chemiluminescent reporters include, but are not limited to, luciferase, Luc2 (humanized Firefly luciferase), MetLuc (Metridia luciferase), Rluc (Renilla luciferase), hRluc (humanized Renilla luciferase), NLuc (Nano luciferase), Aequorin, SEAP (human secreted Embryonic Alkaline Phosphatase), or the like. Examples of fluorescent reporters include, but are not limited to, EGFP (Enhanced Green Fluorescent Protein), mGreenLantern, sfGFP (Superfolder Green Fluorescent protein), emGFP (Emerald Green Fluorescent protein), TurboGFP or maxGFP; hrGFP (humanized recombinant Green Fluorescent protein), d2EGFP, ZsGreenl, mNeonGreen, Venus, EYFP (enhanced Yellow Fluorescent protein), YPet, Cerulean, CyPet, AmCyan, EBFP, TagBFP or mTagBFP, TagBFP2 or mTagBFP2, Electral, dTomatao, tdTomato, DsRed_Express2, TurboRFP, mRFPl, mCherry, mScarlet, mApple, or the like. In addition, provided herein in accordance with various aspects are certain agents for the prevention and / or treatment of proliferative diseases (e.g., cancer) in humans and other mammals. Such cancers may include uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs. Such effects may be countered, at least in part, by the application of the compositions discussed herein, in accordance with certain embodiments. In some embodiments, the cancer is a lung cancer (e.g., SCLC).

[0090] In some embodiments an effective amount of the agent is administered to a subject (e.g., a mammalian or non-mammalian subject, such as a human or a non-human subject) to treat a cancer or multiple cancers. The agent may be administered by any route that results in a therapeutically effective outcome, including but not limited to intravenous, intraperitoneal, dermal, intradermal, intramuscular, intranasal, and / or subcutaneous administration. In some cases, the agent is administered intratumorally. An “effective amount” of an agent is based at least in part, on the tissue and / or cell type targeted, the means of administration, and / or characteristic of the agent. Other determinants include the body weight, age, height, sex and general health of the subject. Typically, an effective amount of an agent treats cancer. As used herein, “treat” to either therapeutic treatment or prophylactic or preventative or diagnostic measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein (e.g., cancer). Thus, in some embodiments, treating may include directly affecting or curing, suppressing, inhibiting, preventing, reducing the severity of, delaying the onset of, reducing symptoms associated with the disease, disorder or condition, or a combination thereof. In some embodiments, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.

[0091] Provided herein in certain embodiments are pharmaceutical compositions comprising the agent with a carrier (e.g., a pharmaceutically acceptable carrier), inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A “pharmaceutically acceptable carrier,” after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be “acceptable” also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences. The agent may be formulated or administered alone or in conjunction with one or more other components. For example, a pharmaceutical composition may further comprise other components, such as chemotherapeutics, immune checkpoint inhibitors, adjuvants, or combinations thereof.

[0092] Relative amounts of the agent, the pharmaceutically acceptable excipient, and / or any additional components in a pharmaceutical composition will differ. Pharmaceutical composition will depend upon the condition, age, weight, height or the subject, and additionally upon the route by which the composition will be administered. As an example, the pharmaceutical composition may comprise less than 100%, e.g., between 1 and 10%, between 5-50%, between 10-90%, at least 75% weight for weight (w / w) active ingredient. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the agents described herein.

[0093] As a non-limiting example, subject doses of the compounds described herein for delivery typically range from about 0.1 microgram to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time there between. More typically doses range from about 10 micrograms to 5 milligrams per administration, and most typically from about 100 micrograms to 1 milligrams, with 2 to 4 administrations being spaced days or weeks apart. More typically, doses range from 1 microgram to 10 milligrams per administration, and most typically 10 micrograms to 1 milligrams, with daily or weekly administrations. Subject doses of the compounds described herein for parenteral delivery for the purpose of treating cancers may be typically 5 to 10,000 times higher than the effective dose, and more typically 10 to 1,000 times higher, and most typically 20 to 100 times higher. More typically parenteral doses for these purposes range from about 10 micrograms to 5 mg per administration, and most typically from about 100 micrograms to 1 milligrams, with 2 to 4 administrations being spaced days or weeks apart. In some embodiments, however, parenteral doses for these purposes may be used in a range of 5 to 10,000 times higher than the typical doses described above. The compositions of the present disclosure may be administered in multiple doses over an extended period of time. For any compound described herein the therapeutically effective amount can be initially determined from animal models. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well- known in the art is well within the capabilities of the ordinarily skilled artisan.

[0094] Administration of a composition of the disclosure may be accomplished by any medically acceptable method which allows the composition to reach its target. The particular mode selected will depend of course, upon factors such as those previously described, for example, the particular composition, the severity of the state of the subject being treated, the dosage required for therapeutic efficacy, etc. As used herein, a “medically acceptable” mode of treatment is a mode able to produce effective levels of the composition within the subject without causing clinically unacceptable adverse effects.

[0095] The pharmaceutical composition may be formulated to be administered to the subject via any medically acceptable method. The administration may be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated. For example, the composition may be formulated to be administered orally, vaginally, rectally, buccally, pulmonary, topically, nasally, transdermally, through parenteral injection or implantation, via surgical administration, or any other method of administration where access to the target by the composition of the disclosure is achieved. Examples of parenteral modalities that can be used with the disclosure include intravenous, intradermal, subcutaneous, intracavity, intramuscular, intraperitoneal, epidural, or intrathecal. Examples of implantation modalities include any implantable or injectable drug delivery system. Oral administration may be preferred in some embodiments because of the convenience to the subject as well as the dosing schedule. Compositions suitable for oral administration may be presented as discrete units such as hard or soft capsules, pills, cachettes, tablets, troches, or lozenges, each containing a predetermined amount of the active compound. Other oral compositions suitable for use with the disclosure include solutions or suspensions in aqueous or non-aqueous liquids such as a syrup, an elixir, or an emulsion. In some embodiments, the composition may be used to fortify a food or a beverage.

[0096] In some embodiments, the compositions of the disclosure are formulated for administered by inhalation. For administration by inhalation, the compositions for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0097] Other delivery systems suitable for use with the present disclosure include timerelease, delayed release, sustained release, or controlled release delivery systems. Such systems may avoid repeated administrations of the composition in many cases, increasing convenience to the subject. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include, for example, polymer-based systems such as polylactic and / or polyglycolic acids, polyanhydrides, polycaprolactones and / or combinations of these; non-polymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides; hydrogel release systems; liposome-based systems; phospholipid based- systems; silastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; or partially fused implants. The formulation may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems. In some embodiments, the system may allow sustained or controlled release of the composition to occur, for example, through control of the diffusion or erosion / degradation rate of the formulation containing the composition. In addition, a pumpbased hardware delivery system may be used to deliver one or more embodiments of the disclosure.

[0098] The present disclosure also provides any of the above-mentioned compositions in kits, optionally including instructions for use of the composition for the treatment of a cancer. That is, the kit can include a description of use of the composition for participation in any biological or chemical mechanism disclosed herein. The kits can further include a description of activity of the condition in treating the pathology, as opposed to the symptoms of the condition. That is, the kit can include a description of use of the compositions as discussed herein. The kit also can include instructions for use of a combination of two or more compositions of the disclosure, or instruction for use of a combination of a composition of the disclosure and one or more other compounds indicated for treatment of the cancer. Instructions also may be provided for administering the composition by any suitable technique as previously described, for example, orally, intravenously, pump or implantable delivery device, or via another known route of drug delivery.

[0099] The kits described herein may also contain one or more containers, which may contain the composition and other ingredients as previously described herein. The kits also may contain instructions for mixing, diluting, and / or administrating the compositions of the disclosure in some cases. The kits also can include other containers with one or more solvents, surfactants, preservative and / or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting or administering the components in a sample or to a subject in need of such treatment.

[0100] The compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders. When the composition provided is a dry powder, the composition may be reconstituted by the addition of a suitable solvent, which may also be provided. In embodiments where liquid forms of the composition are used, the liquid form may be concentrated or ready to use. The solvent will depend on the composition and the mode of use or administration. Suitable solvents for drug compositions are well known, for example as previously described, and are available in the literature. The solvent will depend on the composition and the mode of use or administration.

[0101] U.S. Provisional Patent Application Serial No. 63 / 636,803, filed April 21, 2024, entitled “Genetic Circuit for DNA Mutation Detection and Response,” by Paoletti, is incorporated herein by reference in its entirety. In addition, U.S. Provisional Patent Application Serial No. 63 / 766,799, filed March 4, 2025, entitled “Genetic Circuit For DNA Mutation Detection and Response,” by Paoletti, is incorporated herein by reference in its entirety.

[0102] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0103] EXAMPLE 1

[0104] This example illustrates a genetic circuit for cancer therapy, in accordance with one embodiment. The underlying principles of the genetic circuit for cancer therapy, in this example, are rooted in the interplay between the proteins p53 and Mdm2, and how this is regulated by various other cellular proteins to prevent p53 degradation in response to DNA damage. p53 is a tumor suppressor gene that plays a pivotal role in regulating cell growth and cancer development. It acts as a transcription factor, activating genes involved in critical cell functions (cell cycle arrest, senescence, DNA repair, and apoptosis) in response to cellular stressors like DNA damage or oncogene activation (Fig. 5).

[0105] Mdm2 is a negative regulator of p53. It ubiquitinates p53, targeting it for destruction by protein enzymes. In a non-cancer cell, Mdm2 maintains p53 at low levels, suppressing the apoptosis signal. This tightly-regulated interaction is important to the cellular response to cancer. But the interaction is modulated by other cellular proteins which sense DNA damage through a variety of mechanisms and stabilize p53 (Fig. 5). These include ARF, ATM, ATR, CHK1 and CHK2. In cancer cells, an array of proteins detect DNA damage and, upon activation, may interact with MDM2 and p53 to stabilize p53 initiating apoptosis.

[0106] The genetic circuit uses the cellular response mechanisms to DNA damage, particularly those involving proteins in MDM2-p53 signaling pathways. These pathways lead to apoptosis (cellular suicide), a powerful and conserved function for eliminating cancer cells from the organism.

[0107] The genetic circuit in this example comprises two genes: human MDM2 and a novel gene, “Complex-9,” which is a fusion of human TP53 and an inducible version of human Caspase-9, iCasp-9. In non-cancer cells, Complex-9 is destroyed by Mdm2 ubiquitination. However, in cancer cells, Complex-9 delivers two independent apoptosis signals, first via activation of p53 responding genes, and second via the inducible caspase-mediated apoptosis pathway, referred to as the endogenous and novel pathways respectively (Fig. 6).

[0108] The cellular version of caspase is activated when it forms a complex with other cellular proteins. Inducible iCasp-9 is activated when it forms a dimer (two molecules of iCasp-9) in the presence of a chemical inducer of dimerization (CID) (Fig. 6).

[0109] It is believed, based on the connection between p53 and Mdm2, that the genetic circuit in this example effectively targets mutated cells, aided by differential promoter strengths, and DNA damage signaling proteins (ARF, ATM, ATR, CHK1, and CHK2). The genetic circuit uses a single MDM2 gene, a gene encoding the Complex-9 protein and EGFP, a marker of Complex-9 expression (Fig. 7).

[0110] MDM2: Controlled by a constitutive promoter, the encoded protein, Mdm2 facilitates the degradation of the iCasp-9 portion of Complex-9 and prevents the expression of Complex-9 in non-cancer cells, minimizing off-target effects.

[0111] Complex-9: Controlled by another constitutive promoter, this gene encodes a fusion of p53 linked to iCasp-9. The destruction of Complex-9 by Mdm2 is used to induce apoptosis selectively in cancer cells. A second gene, EGFP, if present, may be expressed from the same promoter (by the addition of an internal ribosome entry site or IRES) to monitor expression of Complex-9 (Fig. 9).

[0112] Promoters: Different combinations of promoters (EFS, UBS, EFl A, etc.) may be used to minimize toxicity in non-cancer cells and maximize toxicity in cancer cells.

[0113] Accordingly, in some cases, the genetic circuit includes:

[0114]

[0115] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0116] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0117] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0118] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0119] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0120] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0121] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0122] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0123] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0124] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMSWhat is claimed is:

1. A composition, comprising: a nucleic acid comprising a first sequence having at least 85% homology to an MDM2 cDNA, a second sequence having at least 85% homology to a p53 cDNA excluding silent mutations, and a third sequence encoding a target protein.

2. The composition of claim 1, wherein in a 5’ to 3’ direction, the first sequence occurs before the second sequence and the second sequence occurs before the third sequence.

3. The composition of claim 1, wherein in a 5’ to 3’ direction, the first sequence occurs before the third sequence and the third sequence occurs before the second sequence.

4. The composition of claim 1, wherein in a 5’ to 3’ direction, the second sequence occurs before the third sequence and the third sequence occurs before the first sequence.

5. The composition of claim 1, wherein in a 5’ to 3’ direction, the third sequence occurs before the second sequence and the second sequence occurs before the first sequence.

6. The composition of any one of claims 1-5, wherein the nucleic acid further comprises a first promoter for the first sequence.

7. The composition of claim 6, wherein the first promoter is a CMV promoter.

8. The composition of claim 6, wherein the first promoter is a SV40 promoter.

9. The composition of claim 6, wherein the first promoter is EF1A.

10. The composition of claim 6, wherein the first promoter is EFS.

11. The composition of claim 6, wherein the first promoter is UBC.

12. The composition of any one of claims 6-11, wherein the nucleic acid further comprises a second promoter for the second sequence.

13. The composition of claim 12, wherein the second promoter is an elongation factor 1- alpha (EF) promoter.

14. The composition of claim 12, wherein the second promoter is a RSV promoter.

15. The composition of claim 12, wherein the second promoter is EF1A.

16. The composition of claim 12, wherein the second promoter is EFS.

17. The composition of claim 12, wherein the second promoter is UBC.

18. The composition of any one of claims 12-17, wherein the first promoter has higher strength than the second promoter.

19. The composition of any one of claims 12-17, wherein the second promoter has higher strength than the first promoter.

20. The composition of claim 12, wherein the first promoter is EF1A and the second promoter is EF1A.

21. The composition of claim 12, wherein the first promoter is EFS and the second promoter is EF1A.

22. The composition of claim 12, wherein the first promoter is EF1A and the second promoter is EFS.

23. The composition of claim 12, wherein the first promoter is EFS and the second promoter is EFS.

24. The composition of claim 12, wherein the first promoter is UBC and the second promoter is EFS.

25. The composition of claim 12, wherein the first promoter is EFS and the second promoter is UBC.

26. The composition of claim 12, wherein the first promoter is UBC and the second promoter is UBC.

27. The composition of claim 12, wherein the first promoter is EF1A and the second promoter is UBC.

28. The composition of claim 12, wherein the first promoter is UBC and the second promoter is EF1A.

29. The composition of any one of claims 1-28, wherein the nucleic acid further comprises a poly-A sequence between the first sequence and the second sequence.

30. The composition of any one of claims 1-29, wherein the nucleic acid further comprises a linker sequence between the second sequence and the third sequence.

31. The composition of claim 30, wherein the linker sequence comprises three tandem iterations of (GGGGS) from the amino terminus to the carboxy terminus.

32. The composition of any one of claims 1-31, further comprising a poly-A sequence adjacent to the third sequence.

33. The composition of any one of claims 1-32, wherein the nucleic acid further comprises a fourth sequence encoding an origin site.

34. The composition of claim 33, wherein the origin site is a bacterial origin of replication (ori).

35. The composition of any one of claims 1-34, wherein the nucleic acid further comprises a fifth sequence encoding P-lactamase, or another gene suitable for selection of E. coli containing a plasmid of interest.

36. The composition of claim 35, wherein the gene suitable for selection is AmpR.

37. The composition of any one of claims 1-36, wherein the second sequence and the third sequence are expressible as a fusion protein.

38. The composition of any one of claims 1-37, wherein the first sequence, the second sequence, and the third sequence are expressible as a fusion protein.

39. The composition of any one of claims 1-38, wherein the third sequence encodes iCasp-9.

40. The composition of any one of claims 1-38, wherein the third sequence encodes a suicide gene.

41. The composition of any one of claims 1-38, wherein the third sequence encodes a caspase.

42. The composition of any one of claims 1-38, wherein the third sequence encodes a gene in a p53 apoptotic pathway.

43. The composition of any one of claims 1-38, wherein the third sequence encodes a DNA repair gene.

44. The composition of any one of claims 1-38, wherein the third sequence encodes DNA polymerase lambda.

45. The composition of any one of claims 1-38, wherein the third sequence encodes DNA polymerase mu.

46. The composition of any one of claims 1-38, wherein the third sequence encodes a gene in a Cas-9 mediated repair mechanism.

47. The composition of any one of claims 1-38, wherein the third sequence encodes cyclin-dependent kinase 1 (CDK1).

48. The composition of any one of claims 1-38, wherein the third sequence encodes cyclin-dependent kinase 2 (CDK2).

49. The composition of any one of claims 1-38, wherein the third sequence encodes influenza hemagglutinin.

50. The composition of any one of claims 1-38, wherein the third sequence encodes a thymidine kinase.

51. The composition of any one of claims 1-38, wherein the third sequence encodes a fluorescent protein.

52. The composition of any one of claims 1-38, wherein the third sequence encodes a bioluminescent enzyme.

53. The composition of any one of claims 1-52, wherein the composition is a plasmid.

54. The composition of any one of claims 1-52, wherein the composition is a viral vector.

55. The composition of any one of claims 1-52, wherein the composition is DNA.

56. The composition of any one of claims 1-52, wherein the composition is RNA.

57. The composition of any one of claims 1-52, wherein the composition is messengerRNA.

58. The composition of any one of claims 1-57, wherein the composition further comprises a pharmaceutically acceptable carrier.

59. A composition, comprising: a fusion protein, comprising a first portion having at least 85% identity to p53, and a second portion that is a target protein.

60. The composition of claim 59, wherein the second portion comprises at least 30 sequential amino acids of SEQ ID NO: 3.

61. The composition of any one of claims 59 or 60, wherein the composition further comprises an inhibitor of p53.

62. The composition of any one of claims 59-61, wherein the composition further comprises a protein having at least 85% homology to MDM2.

63. A composition, comprising: a cell expressing a fusion protein, wherein the fusion protein comprises a first portion having at least 85% homology to p53, and a second portion that is a target protein.

64. The composition of claim 63, wherein the cell further comprises a protein having at least 85% homology to Mdm2.

65. A composition, comprising: a nucleic acid comprising: a first sequence having at least 85% homology to an MDM2 gene; a first promoter sequence for the first sequence; a second sequence having at least 85% homology to a TP53 gene excluding silent mutations; a second promoter sequence for the second sequence; a poly-A sequence between the first sequence and the second sequence; a third sequence encoding a target protein; and a linker sequence between the second sequence and the third sequence.

66. The composition of claim 65, wherein the nucleic acid further comprises a fourth sequence encoding an origin site.

67. The composition of claim 66, wherein the nucleic acid further comprises a fifth sequence encoding a selection gene.

68. A method, comprising: administering the composition of any preceding claim to a subject.

69. The method of claim 68, wherein the subject is human.

70. The method of claim 68, wherein the subject is a non-human animal.

71. The method of any one of claims 68-70, wherein the subject has or is at risk for cancer.

72. The method of any one of claims 68-71, comprising administering the composition intravenously.

73. The method of any one of claims 68-71, comprising administering the composition intratumorally.

74. The method of any one of claims 68-71, comprising administering the composition intraperitoneally .

75. A method, comprising: expressing, within a cell, a fusion protein comprising a first portion having at least 85% homology to p53, and a second portion that is a target protein; and expressing, within the cell, a non-native inhibitor of p53.

76. The method of claim 75, wherein the first portion comprises at least 30 sequential amino acids of SEQ ID NO: 3.

77. The method of any one of claims 75 or 76, wherein the non-native inhibitor of p53 is not produced by the cell.

78. A method, comprising: administering, to a cell, an agent able to alter activity of a p53-Mdm2 pathway within the cell; andcontrolling expression of a target protein by altering the activity of the p53- Mdm2 pathway.

79. A composition, comprising: an aqueous solution, comprising a first nucleic acid sequence having at least 85% homology to an MDM2 gene; a second nucleic acid sequence having at least 85% homology to a TP53 gene excluding silent mutations; and a third nucleic acid sequence encoding a target protein.

80. The composition of claim 79, wherein the first nucleic acid sequence and the second nucleic acid sequence are present on a common nucleic acid.

81. The composition of any one of claims 79 or 80, wherein the first nucleic acid sequence and the second nucleic acid sequence are present on a contiguous portion of a nucleic acid.

82. The composition of any one of claims 79-81, wherein the first nucleic acid sequence and the third nucleic acid sequence are present on a common nucleic acid.

83. The composition of any one of claims 79-82, wherein the second nucleic acid sequence and the third nucleic acid sequence are present on a common nucleic acid.

84. The composition of claim 79, wherein the first nucleic acid sequence and the second nucleic acid sequence are not present on a common nucleic acid.

85. The composition of claim 79, wherein the first nucleic acid sequence and the third nucleic acid sequence are not present on a common nucleic acid.

86. The composition of claim 79, wherein the second nucleic acid sequence and the third nucleic acid sequence are not present on a common nucleic acid.

87. A genetic circuit for inducible apoptosis in cancer cells, comprising: a bacterial plasmid DNA vector housing two main operons; the first operon regulated by a strong constitutive promoter driving theexpression of the MDM2 gene; the second operon regulated by a medium- strength promoter driving the expression of a fusion protein consisting of the p53 gene linked to an executioner caspase (referred to as “Complex9” protein), via a linker sequence; the MDM2 gene within the first operon functioning to ubiquitinate and degrade the Complex9 protein, preventing apoptosis in non-cancerous cells; and upon detection of mutations in cancerous cells, proteins such as ARF and ATM are activated, leading to the inhibition of MDM2 and stabilization of p53, thereby relieving the inhibition and allowing for the activation of apoptosis (or other downstream functions depending on the specific gene if alternatives to iCasp-9 are selected for alternative purposes) specifically in cancer cells.

88. The genetic circuit of claim 87, wherein said strong constitutive promoter is selected from the group consisting of the cytomegalovirus (CMV) promoter, SV40 promoter, and any other promoter capable of driving robust gene expression in mammalian cells.

89. The genetic circuit of any one of claims 87 or 88, wherein said medium- strength promoter is selected from the group consisting of the elongation factor 1 -alpha (EFS) promoter, Rous sarcoma virus (RSV) promoter, and any other promoter capable of driving moderate gene expression in mammalian cells.

90. The genetic circuit of claims 87-89, wherein said linker sequence comprises a 3xGGGGs linker.

91. A method for inducing apoptosis in cancer cells using the genetic circuit of any one of claims 87-90, comprising: introducing said genetic circuit into cancer cells; allowing for the expression of the Complex9 protein; and relieving the inhibition of MDM2 in response to mutations in cancerous cells, leading to the activation of apoptosis specifically in said cancer cells.

92. The method of claim 91, wherein said introducing step is performed via transfection, electroporation, or viral transduction.

93. A pharmaceutical composition for cancer therapy, comprising: the genetic circuit of any one of claims 87-92; and one or more pharmaceutically acceptable carriers or excipients.

94. The pharmaceutical composition of claim 93, further comprising one or more additional therapeutic agents for combination therapy.

95. A method for treating cancer in a subject, comprising administering to said subject the pharmaceutical composition of any one of claims 93 or 94.

96. The method of claim 95, wherein said cancer comprises solid tumors or hematological malignancies.

97. The method of any one of claims 95 or 96, wherein said administration is performed via a route selected from the group consisting of intravenous, intratumoral, and intraperitoneal administration, or others such as dermal application, etc.

98. A method for screening potential therapeutic agents for cancer therapy, comprising: introducing the genetic circuit of any one of claims 87-97 into cancer cells; treating said cancer cells with candidate therapeutic agents; and assessing an efficacy of said candidate therapeutic agents by measuring the activation of apoptosis in said cancer cells.

99. A genetic circuit for inducible apoptosis in cancer cells, comprising: a bacterial plasmid DNA vector comprising: a first operon comprising an MDM2 gene and a first promoter configured to control expression of the MDM2 gene; and a second operon comprising a second promoter configured to control expression of a fusion protein.

100. The genetic circuit of claim 99, wherein the fusion protein comprises a p53 gene and an executioner caspase.

101. The genetic circuit of claim 100, wherein the p53 and the executioner caspase are linked by a linker sequence.

102. The genetic circuit of claim 101, wherein the linker sequence comprises a (GGGGS)3 linker.

103. The genetic circuit of any one of claims 99-102, wherein the first promoter is a cytomegalovirus (CMV) promoter.

104. The genetic circuit of any one of claims 99-102, wherein the first promoter is an SV40 promoter.

105. The genetic circuit of any one of claims 99-104, wherein the second promotor is an elongation factor 1 -alpha (EFS) promoter.

106. The genetic circuit of any one of claims 99-104, wherein the second promotor is a Rous sarcoma virus (RSV) promoter.

107. A method for inducing apoptosis in cancer cells using the genetic circuit of any one of claims 99-106, comprising administering the genetic circuit to cancer cells.

108. A method for inducing apoptosis in cancer cells using the genetic circuit of any one of claims 99-107, comprising: administering the genetic circuit into cancer cells; and allowing a Complex9 protein to be expressed.

109. The method of claim 108, wherein the administering is performed via transfection.

110. The method of claim 108, wherein the administering is performed via electroporation.

111. The method of claim 108, wherein the administering is performed via viral transduction.

112. A pharmaceutical composition for cancer therapy, comprising: the genetic circuit of any one of claims 99-111; and one or more pharmaceutically acceptable carriers or excipients.

113. A method for treating cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 112.

114. The method of claim 113, wherein the cancer comprises a solid tumor.

115. The method of any one of claims 113 or 114, wherein the cancer comprises a hematological malignancy.

116. The method of any one of claims 113-115, wherein the cancer comprises a skin cancer.

117. The method of any one of claims 113-116, wherein the administering comprises intravenous administrating.

118. The method of any one of claims 113-117, wherein the administering comprises intratumoral administrating.

119. The method of any one of claims 113-118, wherein the administering comprises intraperitoneal administrating.

120. The method of any one of claims 113-119, wherein the administering comprises dermal administrating.

121. A method for screening therapeutic agents for cancer therapy, comprising: administering the genetic circuit of any one of claims 99-106 into cancer cells; treating said cancer cells with candidate therapeutic agents; and assessing an efficacy of said candidate therapeutic agents by determining apoptosis in the cancer cells.

122. A genetic circuit for inducible apoptosis in cancer cells, comprising: a nucleic acid vector housing two main genes; the first gene regulated by a promoter driving the expression of the Mdm2 protein; the second gene regulated by a promoter driving the expression of a fusion protein consisting of the p53 gene linked to an executioner caspase (referred to as “Complex-9” protein), via a linker sequence; the MDM2 gene functioning to ubiquitinate and degrade the Complex- 9 protein, preventing apoptosis in non-cancerous cells; and upon detection of mutations in cancerous cells, proteins such as ATM, ATR, CHK1, CHK2, and ARF are activated, leading to the inhibition of Mdm2 and stabilization of p53, thereby relieving the inhibition and allowing for the activation of apoptosis (or other downstream functions depending on the specific target protein if alternatives to iCasp-9 are selected for alternative purposes) specifically in cancer cells or cells which have mutations.

123. The genetic circuit of claim 122, wherein said strong constitutive promoter is selected from the group consisting of the cytomegalovirus (CMV) promoter, SV40 promoter, and any other promoter capable of driving robust gene expression in mammalian cells.

124. The genetic circuit of any one of claims 122 or 123, wherein said medium- strength promoter is selected from the group consisting of the elongation factor 1 -alpha (EFS) promoter, Rous sarcoma virus (RSV) promoter, and any other promoter capable of driving moderate gene expression in mammalian cells.

125. The genetic circuit of any one of claims 122-124, wherein said linker sequence comprises a 3xGGGGS linker, or another suitable linker sequence as described herein.

126. A method for inducing apoptosis in cancer cells using the genetic circuit of any one of claims 122-125, comprising: introducing said genetic circuit into cancer cells; allowing for the expression of the Complex-9 protein; andrelieving the inhibition of Mdm2 in response to mutations in cancer cells, leading to the activation of apoptosis specifically in said cancer cells.

127. The method of claim 126, wherein said introducing step is performed via transfection, electroporation, or viral transduction.

128. A pharmaceutical composition for cancer therapy, comprising: the genetic circuit of any one of claim 122-127; and one or more pharmaceutically acceptable carriers or excipients.

129. The pharmaceutical composition of claim 128, further comprising one or more additional therapeutic agents for combination therapy.

130. A method for treating cancer in a subject, comprising administering to said subject the pharmaceutical composition of any one of claims 128 or 129.

131. The method of claim 130, wherein said cancer comprises solid tumors or hematological malignancies.

132. The method of claim 130, wherein said administration is performed via a route selected from the group consisting of intravenous, intratumoral, and intraperitoneal administration, or others such as dermal application, etc.

133. A method for screening potential therapeutic agents for cancer therapy, comprising: introducing the genetic circuit of any one of claims 122-132 into cancer cells; treating said cancer cells with candidate therapeutic agents; and assessing an efficacy of said candidate therapeutic agents by measuring the activation of apoptosis in said cancer cells.

134. A genetic circuit for inducible apoptosis in cancer cells, comprising: a nucleic acid vector comprising: a first gene encoding an Mdm2 protein and a first promoter configured to control expression of the MDM2 gene; anda second gene comprising a second promoter configured to control expression of a fusion protein referred to as “Complex-9”.

135. The genetic circuit of claim 134, wherein the fusion protein comprises a p53 gene and an executioner caspase referred to as “Complex-9”.

136. The genetic circuit of any one of claims 134 or 135, wherein the p53 and the executioner caspase are linked by a linker sequence.

137. The genetic circuit of claim 136, wherein the linker sequence comprises a (GGGGS)3 linker, or another suitable linker sequence as described herein.

138. The genetic circuit of any one of claims 134-137, wherein the first promoter is a cytomegalovirus (CMV) promoter.

139. The genetic circuit of any one of claims 134-137, wherein the first promoter is an SV40 promoter.

140. The genetic circuit of any one of claims 134-139, wherein the second promoter is an elongation factor 1 -alpha (EFS) promoter.

141. The genetic circuit of any one of claims 134-139, wherein the second promoter is a Rous sarcoma virus (RSV) promoter.

142. A method for inducing apoptosis in cancer cells using the genetic circuit of claim any one of claims 134-141, comprising administering the genetic circuit to cancer cells.

143. A method for inducing apoptosis in cancer cells using the genetic circuit of claim any one of claims 134-141, comprising: administering the genetic circuit into cancer cells; and allowing the Complex-9 protein to be expressed.

144. The method of any one of claims 134-143, wherein the administering is performed via transfection.

145. The method of any one of claims 1342-144, wherein the administering is performed via electroporation.

146. The method of any one of claims 134-145, wherein the administering is performed via viral transduction.

147. A pharmaceutical composition for cancer therapy, comprising: the genetic circuit of any one of claims 134-141; and one or more pharmaceutically acceptable carriers or excipients.

148. A method for treating cancer in a subject, comprising administering to the subject the pharmaceutical composition of claim 147.

149. The method of claim 148, wherein the cancer comprises a solid tumor.

150. The method of any one of claims 148 or 149, wherein the cancer comprises a hematological malignancy.

151. The method of any one of claims 148-150, wherein the cancer comprises a skin cancer.

152. The method of any one of claims 148-151, wherein the administering comprises intravenous administration.

153. The method of any one of claims 148-152, wherein the administering comprises intratumoral administration.

154. The method of any one of claims 148-153, wherein the administering comprises intraperitoneal administration.

155. The method of any one of claims 148-154, wherein the administering comprises dermal administration.

156. A method for screening therapeutic agents for cancer therapy, comprising: administering the genetic circuit of any one of claims 134-141 into cancer cells; treating said cancer cells with candidate therapeutic agents; and assessing an efficacy of said candidate therapeutic agents by determining apoptosis in the cancer cells.

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