Improved methods and compositions for synthetic biomarkers

Abstract

To provide new strategies and means for enabling highly sensitive and specific cancer detection.SOLUTION: Provided herein is a method comprising: (a) administering to a subject a composition, where the composition preferentially induces expression of a biomarker in diseased cells than in non-diseased cells in the subject so that the relative ratio of the biomarker expressed in the diseased cells to the biomarker expressed in non-diseased cells is larger than 1.0; (b) detecting the biomarker; and (c) determining the presence of diseased cells in the subject at an accuracy of at least 70% by using the biomarker detected in (b).SELECTED DRAWING: Figure 33A-C

Description

[Technical Field]

[0001] cross reference This application is filed on April 5, 2019, and is entitled "Improved Method for Synthetic Biomarkers." IMPROVED METHODS AND COMPOSITIONS "ONS FOR SYNTHETIC BIOMARKERS" U.S. Provisional Application No. No. 62 / 830,279 and the invention filed on December 31, 2019, are entitled "Synthetic Biomolecules" IMPROVED METHODS AND COMPOSITIONS FOR CARRYING OUT THE INVENTION COMPOSITIONS FOR SYNTHETIC BIOMARKERS) This application claims the benefit of U.S. Provisional Application No. 62 / 955,925, which is hereby incorporated by reference in its entirety. each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Cancer is a huge global health problem. The World Health Organization estimated that 1,800 people died in 2018 alone. It is estimated that 100,000 new cases of cancer were diagnosed and 9.6 million people died from cancer. Current treatments are likely to be more effective if detected early, so The time at which cancer is detected, both before cancer diagnosis and at the time of tumor recurrence, influences patient outcomes. Unfortunately, most cancers are detected relatively late and at high risk. These rates are expected to increase unless more effective detection strategies and treatments are developed. is expected to double by 2030. The staggering loss of life due to this terrible disease To stem the losses, a widely applicable method that can detect cancer at its earliest stage is needed. Effective measures are urgently needed.

[0003] Two current paradigms for improving cancer detection are endogenous factors entering or being released into the bloodstream. Cancer biomarkers (e.g., proteins, microRNAs, circulating tumor DNA, circulating tumors) blood-based assays for detecting tumor cells, as well as biomarker-targeted imaging Use probes to better visualize tumors that are undetectable by traditional anatomical imaging This includes the development of molecular imaging assays that visualize the disease. Summary of the Invention [Problem to be solved by the invention]

[0004] Blood assays are becoming increasingly popular as they facilitate affordable cancer screening programs. Although always attractive, blood biomarker concentrations are low (Nagrath et al., (2007) Nature 450:1235-1239), rapid in vivo and Ex vivo biomarker degradation (Haun et al., (2011)Sci. Translational Med. 3:71ra16), and in non-malignant tissues Highly variable background expression (Diamandis EP (2010) J. National Cancer Inst. 102:1462-1467) Current clinical biomarker assays often suffer from sensitivity and specificity issues due to their limited availability. before endogenous blood biomarker levels reach levels sufficient to indicate disease. It is estimated that the tumor can grow for 10 to 12 years and can exceed 2.5 cm in diameter (Hori & Gambhir (2011) Sci. Translational Med 3:109ra116). Of the thousands of potential blood biomarkers reported, Less than 1% of patients are used in clinical settings (7) and lack validated specificity and diagnostic value. However, the adoption of new blood biomarkers into clinical practice is declining (Haun et al. ., (2011) Sci. Translational Med. 3:71ra 16;Kern SE (2012) Cancer Res. 72:6097-61 01). Overall, the development of tools for detecting endogenous cancer blood biomarkers is a promising area. Although considerable effort has been made, there has been little success. New strategies and tools capable of cancer detection are urgently needed. [Means for solving the problem]

[0005] In some aspects, the present disclosure provides a method for (a) administering a composition to a subject, wherein the composition In patients with glaucoma, the expression of biomarkers in diseased cells was higher than that in non-diseased cells. Induction of marker expression allows for differentiation of biomarkers expressed in diseased versus non-diseased cells. (b) detecting a biomarker; (c) ) (b) Using the biomarkers detected in (b), subjects are diagnosed with a lesion with at least 70% accuracy. determining that the cell has

[0006] In some embodiments, the present disclosure provides a method for treating a subject having or suspected of having a disease. a method of treating a subject, the method comprising: overriding expression of a therapeutically effective agent by non-diseased cells in the subject; The present invention provides a composition that induces the expression of a therapeutically effective agent by diseased cells associated with the disease. When administered to the subject, the therapeutic effect expressed by the diseased cells relative to the non-diseased cells is a relative concentration of the therapeutically effective agent greater than 1.0, with a therapeutic efficacy of at least 10% as determined by a reduction in the cell population of the diseased cells. Methods of treating the subject are provided.

[0007] In some aspects, the present disclosure provides a method for detecting a first polypeptide or nucleic acid biomarker. a first nucleic acid sequence encoding a second polypeptide or a second nucleic acid biomarker; and a second nucleic acid sequence, wherein the composition is the second polypeptide or nucleic acid biomarker is delivered to the cell via the first and second polypeptide or nucleic acid biomarkers. and a second nucleic acid is expressed in an amount reflective of delivery of the first polypeptide or nucleic acid biomarker. The present invention provides a composition in which the marker is differentially expressed in diseased and non-diseased cells.

[0008] In some aspects, the present disclosure provides a method for detecting diseased cells in a subject, the method comprising: administering to the subject a composition comprising a first polypeptide or nucleic acid vector a first nucleic acid sequence encoding a biomarker and a second polypeptide or a second nucleic acid biomarker; a second nucleic acid sequence encoding a marker; wherein the composition is in a cell (i) the cell exhibits expression of the first nucleic acid sequence in diseased cells in preference to expression in non-diseased cells; and configured to induce expression of a first nucleic acid sequence of the first polypeptide. (ii) the cells are a detectable biomarker or therapeutic agent; and (iii) the cells are a subset of diseased and non-diseased cells. induces equal expression of a second nucleic acid sequence in the cells, said second nucleic acid sequence being detectably and producing said second polypeptide which is not a biomarker or therapeutic agent, thereby The level of expression of the second polypeptide is used to assess the relative levels of the nucleic acid sequence in the cell. The present invention provides a method for providing a control for the detection of oxidative stress.

[0009] In some aspects, the disclosure provides a first nucleic acid sequence encoding a first polypeptide; A composition comprising a second nucleic acid sequence encoding a second polypeptide, the composition comprising: When the composition is in a cell, (i) the cell expresses the first nucleic acid sequence to (ii) the cell expresses a second nucleic acid sequence to produce the second polypeptide; (iii) producing the first polypeptide and the precursor peptide expressed by the cell; The second polypeptide is configured to bind to form a heterodimeric protein. The present invention provides a composition comprising:

[0010] In some aspects, the present disclosure provides a method for detecting or treating diseased cells, comprising: a first nucleic acid sequence encoding a polypeptide and a second nucleic acid sequence encoding a second polypeptide. and administering to said subject a composition comprising the sequence, wherein said first and second polypeptides are , which is selectively transcribed or translated in said diseased cells.

[0011] In some aspects, the present disclosure provides non-human endothelial cells that contain sequences encoding polypeptides or nucleic acid sequences. A composition comprising a naturally occurring recombinant gene construct, wherein the sequence is transfected into cells ex vivo. When introduced, said polypeptide is expressed in a plurality of different cell types isolated from a subject. a composition comprising a first promoter that selectively drives expression of a gene or nucleic acid biomarker sequence; Provide something.

[0012] In some embodiments, the present disclosure provides a method for detecting diseased or damaged cells ex vivo. The method comprises ex vivo transfection of a non-native recombinant gene construct into cells isolated from a subject. the non-native recombinant gene construct comprising a polypeptide or a nucleic acid sequence. a sequence encoding an acid biomarker sequence, the sequence being transduced into the cell; In this case, the polypeptide or nucleus is expressed in a plurality of different types of cells isolated from the subject. The method further includes a first promoter that selectively drives expression of a nucleic acid biomarker sequence. do.

[0013] In some aspects, the present disclosure provides a composition comprising a vector, wherein the vector comprises multiple a plurality of different promoters operably linked to a number of different nucleic acid sequences, each of said promoters A promoter drives expression of the plurality of nucleic acids to produce a plurality of polypeptides or nucleic acid biomolecules. generating marker sequences for individual polypeptide or nucleic acid biomarkers from the plurality of nucleic acid sequences; Compositions in which the level of the sequence is indicative of the stage of disease in the cell or tissue from which the cell originates. Provide something.

[0014] In some aspects, the disclosure includes administering to a subject a composition comprising the vector. a method for detecting a stage of a disease, wherein the vector is operable to bind to a plurality of different nucleic acids; a plurality of different promoters operably linked together, each of said promoters being capable of expressing said Driving expression of multiple nucleic acid sequences to produce multiple polypeptide or nucleic acid biomarker sequences. and the level of each polypeptide of said plurality of nucleic acid sequences is determined by said cell or the cell origin. A method for indicating the stage of disease in the tissue of origin is provided.

[0015] In some embodiments, the present disclosure provides a reporter comprising the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:2. - Approximately 10% in normal cells compared to diseased cells when compared to recombinant nucleic acids containing the gene A composition comprising a genetically engineered nucleic acid encoding an expressible reporter gene that exhibits expression of less than 5%. Provide something.

[0016] In some aspects, the present disclosure provides a method for the preparation of a reporter gene encoding the above-described expressible reporter gene. Methods are provided that include administering to a subject a composition that includes a genetically engineered nucleic acid.

[0017] In some embodiments, the present disclosure provides a method for detecting an increase in the incidence of a diseased cell by about 10% or less in normal cells compared to diseased cells. reporter gene that shows expression and contains one or more miRNA binding sequences in the 3' untranslated region of the reporter gene. The present invention provides a composition comprising a recombinant nucleic acid comprising a nucleic acid sequence encoding a target gene.

[0018] In some embodiments, the present disclosure provides a method for detecting a decrease in the level of erythrocyte proliferation in normal cells by about 10% or more compared to the diseased cells. and a method for detecting diseased cells, the method comprising administering to a subject a composition that exhibits expression of: do.

[0019] In some embodiments, the present disclosure provides a method for activating a DNA sequence encoding a synthetic biomarker. a plasmid DNA comprising a linear vector containing a double-stranded nucleic acid comprising a promoter operably linked to the Compositions showing significantly longer expression of synthetic biomarkers relative to A or minicircle DNA wherein the forward and reverse strands of the double-stranded nucleic acid are covalently linked at each of their ends. whereby the promoter is expressed in diseased cells relative to non-diseased cells. Compositions are provided in which the relative concentration of the synthetic biomarker is greater than 1.0.

[0020] In some embodiments, the present disclosure provides a method for producing a plasmid DNA or a minicircle DNA as described above. administering to the subject a composition that exhibits significantly longer expression of the synthetic biomarker in comparison with the previous A method for identifying diseased cells, comprising detecting the synthetic biomarker, The expression of the synthetic biomarker in diseased cells of the subject is preferentially higher than that of the diseased cells of the subject. expressed in the diseased cells relative to the non-diseased cells, thereby A method is provided in which the relative concentration of the synthetic biomarker is greater than 1.0.

[0021] In some embodiments, the present disclosure provides a method for the preparation of a nucleic acid molecule operable to encode a DNA sequence encoding a therapeutically effective agent. a plasmid DNA comprising a linear vector comprising a double-stranded nucleic acid comprising a promoter linked to or a composition that shows significantly longer expression of a synthetic biomarker relative to minicircle DNA. wherein the forward and reverse strands of the double-stranded nucleic acid are covalently linked at their respective ends. wherein the promoter preferentially regulates expression of the synthetic biomarker in non-diseased cells. Inducing expression of a therapeutically effective agent in diseased cells, thereby inhibiting expression of a therapeutically effective agent in said non-diseased cells. a composition in which the relative concentration of said therapeutically effective agent expressed in said diseased cells is greater than 1.0; Provide something.

[0022] In some aspects, the present disclosure provides a method for administering the above-described compositions to a subject and administering the synthetic biologics 1. A method for treating diseased cells comprising detecting a marker, wherein the synthetic biomarker is expression of said synthetic biomarker in diseased cells in said subject preferentially over expression in non-diseased cells and a synthetic biomarker expressed in said diseased cells relative to said non-diseased cells. provides a method for making the relative concentration of β-β greater than 1.0.

[0023] In some aspects, the present disclosure provides non-viral vectors that express synthetic biomarkers. wherein said synthetic biomarker is about 1% higher in normal organ cells than in diseased cells. A composition exhibiting 0% or less expression is provided.

[0024] In some embodiments, the present disclosure provides a method for detecting the expression of a biomarker preferentially in non-diseased cells. Inducing the expression of a biomarker in diseased cells, thereby are engineered particles that mimic one or many functions of macrophages, and are delivered to said non-pathological cells. The manipulation of the relative concentration ratio of the biomarker expressed in the diseased cells to the Provides working particles.

[0025] In some aspects, the present disclosure provides at least one vector, said at least A single vector may contain multiple different promoters operably linked to multiple different nucleic acid sequences. the promoter drives expression of multiple nucleic acid sequences in a cell to produce multiple and generating a polypeptide or nucleic acid biomarker sequence of the present invention, wherein the promoter is preferential expression of said plurality of polypeptide or nucleic acid biomarker sequences in diseased cells and inducing expression of said plurality of polypeptide or nucleic acid biomarker sequences in diseased cells. and wherein the plurality of polypeptides or polypeptides expressed in the diseased cells relative to the non-diseased cells are Vectors are provided that result in a relative ratio of nucleic acid biomarker sequences greater than 1.0.

[0026] In some aspects, the present disclosure provides a method for detecting a disease in a subject, comprising: at least one comprising a plurality of different promoters operably linked to different nucleic acids; administering to a subject a composition comprising said plurality of polypeptide or nucleic acid biomolecules; Detecting the marker sequences to obtain an expression profile; and determining the expression profile based on the diseased cells. detecting the disease by detecting the profile.

[0027] In some aspects, the present disclosure provides a method for detecting the presence or absence of a disease in a subject, comprising: contacting one or more cells of the present invention with a genetic construct ex vivo, wherein the genetic construct is a disease-activating promoter operably linked to a code molecule, The gene encoding the barcode molecule drives expression of the barcode molecule in cells affected by the disease. quantifying the expression level of said barcode molecule; and and detecting the presence or absence of said disease.

[0028] By attributing dedicated labels to unique members of a larger group, barcodes A code is a more complex mixture of larger and more numerous members (e.g., expressed in the same cell). Identifying and quantifying its members within the context of multiple promoter-reporter constructs (multiple promoter-reporter constructs) This provides an opportunity to develop new gene expression models (e.g., reporter expression under the control of specific cancer-specific promoters). presently available), offers the opportunity to isolate single members from such complex mixtures. In the case of barcodes based on DNA, hybridization of the barcode based on base pair complementarity is performed. The capture event can be used to capture and isolate a mixture or otherwise reduce the complexity of the mixture. In the case of peptide-based barcodes, immunocapture or ligand and The capture event uses unique characteristics, including receptor interactions, to capture and separate the mixture. The complexity can be reduced by separating the

[0029] In some aspects, the present disclosure provides a method for generating a disease profile for a subject, comprising: contacting one or more cells of said subject with a plurality of genetic constructs; a plurality of disease-activated promoters each operably linked to a plurality of barcode molecules; wherein the disease-activated promoter activates the corresponding gene in cells affected by the disease. driving expression of barcode molecules comprising the plurality of barcode molecules; and determining the expression level of the plurality of barcode molecules. quantitating to generate said profile.

[0030] Additional aspects and advantages of the present disclosure are shown and described with reference to exemplary embodiments thereof only. It will be readily apparent to those skilled in the art from the following detailed description. The present disclosure is capable of other and different embodiments, the details of some of which may all be found in the present disclosure. Therefore, the drawings and description are not to be construed as limiting the scope of the present invention. The description should be viewed as illustrative in nature and not restrictive. There is no.

[0031] Incorporation by Reference All publications, patents, and patent applications mentioned herein are to be interpreted as though each individual publication were incorporated by reference. , patents, or patent applications are specifically and individually indicated to be incorporated by reference. To the same extent, they are incorporated herein by reference.Publications and Patents Incorporated by Reference or patent application conflicts with the disclosure contained in the specification, the specification shall The above-mentioned provisions take precedence over and / or supersede the above-mentioned provisions. [Brief explanation of the drawings]

[0032] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be provided by the following description of illustrative embodiments, in which the principles of the present invention are utilized. The following detailed description and accompanying drawings ("FIGURE" or "FIG") illustrate the present invention. This is obtained by referencing the [Figure 1] Figure 1 is a schematic diagram illustrating the blood-based tumor-activatable minicircle (MC) approach for cancer detection. (A) Tumor-activatable MCs driven by a tumor-specific promoter and encoding a secretable reporter protein are complexed with a nontargeting transfection agent (TA). These nanocomplexes are delivered systemically (via the tail vein). (B) Although MCs transfect many tissues, reporter protein production occurs almost exclusively within tumor cells, and the expressed reporter is secreted into the bloodstream (BS). Minimal protein expression should occur in tumor-free subjects due to promoter leakage. (C) Blood sampling and detection of the secreted reporter in plasma allows for differentiation of tumor-bearing (reporter-positive) from tumor-free (reporter-negative) subjects. [Figure 2] Figures 2A-2B show the design and construction of tumor-activatable vectors. Figure 2A shows the vector maps of both the survivin promoter (pSurv)-driven parental plasmid (PP; top) and MC (bottom). These constructs encoded the reporter protein secreted embryonic alkaline phosphatase (SEAP). PP and MC have identical transcription units (pSurv-SEAP-WPRE-polyA), but MC lacks the prokaryotic backbone (light gray). WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE)) is also present. Figure 2B shows agarose gel electrophoresis confirming the ability to generate both PP (7.9 kb) and MC (4.1 kb). [Figure 3]FIG. 3 is a schematic map of the minicircle vector construct MC-pSurv-SEAP-WPRE-SV40 polyA-Pause. [Figure 4] FIG. 4 is a schematic map of the minicircle vector construct MC-pSurv-Luc2-WPRE-SV40 polyA-Pause. [Figure 5] FIG. 5 is a graph showing a comparison of tumor-specific plasmid (PP-SEAP) and minicircle (MC-SEAP) in MeWo human melanoma cancer cells. [Figure 6] FIG. 6 is a graph showing a comparison of tumor-specific plasmid (PP-SEAP) and minicircle (MC-SEAP) in SK-MEL-28 human melanoma cancer cells. [Figure 7] Figure 7 shows the SEAP assay standard curve for blood-based cancer detection after systemic administration of tumor-specific SEAP minicircles. Standard curve analysis of the SEAP assay revealed that RLU values ​​above approximately 104 are within the linear range of detectable SEAP levels in plasma. [Figure 8] Figure 8 is a graph showing that intratumoral administration of tumor-activatable MCs results in detectable blood reporter activity. Nude mice bearing subcutaneous human melanoma xenografts were intratumorally (IT) administered tumor-activatable MCs expressing SEAP (n = 4; MC IT) or 5% glucose (n = 3; Mock). A group of control mice also received intramuscular (IM) injections of MCs (n = 3; MC IM). Plasma SEAP measurements before and up to 2 weeks after MC administration revealed elevated SEAP levels from days 3 to 14 only in MC IT mice (*p < 0.05; **p < 0.01; ***p < 0.001). Data are presented as mean ± SD. [Figure 9] FIG. 9 shows representative mouse blood SEAP activity after systemic administration of tumor-specific SEAP minicircles. [Figure 10]Figure 10 is a graph showing blood-based cancer detection after systemic administration of tumor-specific SEAP minicircles. From 3 to 14 days after injection of p-survivin-SEAP MC, significantly higher SEAP activity was detected in blood samples from tumor-bearing mice than in control mice (p<0.05). No significant differences were observed between control mice administered MC or 5% glucose. Error bars represent SD. [Figure 11] FIG. 11 is a series of digital images showing molecular genetic imaging cancer detection 3 days after systemic administration of tumor-specific FLUC minicircles. [Figure 12] FIG. 12 is a graph showing molecular genetic imaging cancer detection after systemic administration of tumor-specific FLUC minicircles. [Figure 13] FIG. 13 shows the nucleic acid sequence of minicircle MC-pSurv-SEAP-WPRE-pA. [Figure 14] FIG. 14 shows the nucleic acid sequence of minicircle MC-pSurv-Luc2-WPRE-pA. [Figure 15] 15 is a graph showing a comparison of transfection of constructs of the present disclosure in cultured cancer cells. Transfection of MeWo human melanoma cells with equal masses of MC (n=3) and PP (n=3) using equal volumes of transfection agent results in significantly higher SEAP concentrations in MC-containing media from day 3 to day 8 (**p<0.01; ***p<0.001). Data are presented as mean ± SD. [Figure 16]Figures 16A-16D show systemic delivery of tumor-activatable MCs, enabling confirmation of tumor-bearing subjects. Figures 16A-16C show the development of human melanoma tumors after intravenous cell administration in nude mice (n = 7) monitored using bioluminescence imaging (BLI) (left image). Representative BLI images show tumor growth primarily within the lungs, with individual mice exhibiting extensive tumor burden within 3 days prior to MC administration. The BLI scales in Figures 16A and 16B are the same, while those in Figure 16C are an order of magnitude lower. Tumor-activatable MCs were administered systemically, and SEAP levels were measured before administration (day 0) and up to 14 days after administration (right graph). Various SEAP concentrations were detected in tumor-bearing mice over the 14-day period. Figure 16D shows healthy (tumor-free) mice administered either MCs (control + MC; n = 7) or 5% glucose carrier only (control - MC; n = 5). No statistically significant differences in plasma SEAP levels were detected between these two groups. Importantly, significantly higher plasma SEAP concentrations were detected in tumor-bearing mice treated with MC between days 3 and 14 compared to both control groups in all mice, regardless of tumor burden (#*p<0.05; ##**p<0.01). Data are presented as mean ± SEM. [Figure 17]Figures 17A-17C demonstrate that tumor-activatable MCs can reliably identify tumor-bearing subjects and measure tumor burden. Figure 17A: Area under the curve (AUC) analysis of plasma SEAP measurements over a 2-week period revealed significant differences between tumor-bearing mice (n=7) treated with MC compared with healthy mice treated with either MC (n=7) or 5% glucose (n=5) (*p<0.05; **p<0.01). Data are expressed as mean SD. Figure 17B: Receiver operating characteristic (ROC) analysis revealed the remarkable ability of the tumor-activatable MC system to distinguish tumor-bearing subjects from healthy subjects by measuring and calculating plasma SEAP AUC. Figure 17C: Correlation analysis of SEAP AUC measurements and lung tumor burden (measured by BLI lung mean radiance). A significant positive correlation was observed between these two measurements across six mice, demonstrating the ability of our tool to assess tumor burden when tumors are present. One mouse was excluded from the analysis because it had tumors in both lungs and multiple metastases outside the lung (BLI measurements were taken from just inside the lung, explaining the overall low BLI signal in this mouse). This mouse had a higher SEAP AUC level than expected based on its lung tumor burden. [Figure 18]Figures 18A-18D show a comparison of promoter activity in vivo in healthy (tumor-free) mice. Mice were systemically injected with a plasmid (30 μg; PGL4.2 backbone; complexed with PEI (N / P = 6)) expressing the bioluminescence imaging (BLI) reporter gene codon-optimized firefly luciferase (Luc2) driven by pCMV (n = 3), pSurv (n = 5), or pPEG (n = 3). Mock-injected mice were administered 5% glucose (n = 3). To assess transfection efficiency, each mouse was also co-injected with a plasmid (3 μg; 1 / 10 the mass of the Luc2 plasmid) expressing the BLI reporter gene humanized Renilla luciferase (hRluc) driven by pCMV. Figure 18A shows a representative BLI image 48 hours after injection. The image scale of the pCMV mice is two orders of magnitude higher than all other mice. BLI signals, primarily in the lungs, were observed in all mice administered the Luc2 plasmid. Figure 18B shows a mouse-wide control region analysis performed on BLI images, revealing significantly higher (*p<0.05; approximately 100-fold) BLI signals in mice administered the pCMV-Luc2 plasmid compared with all other mice (*p<0.05). Significantly higher (*p<0.05) BLI signals were also observed in pPEG mice compared with mock-injected mice. Although qualitatively higher BLI signals were evident in pSurv mice compared with mock-injected mice, quantitative measurements revealed only a trend toward higher BLI signals (p=0.16). Thus, in this mouse strain, Luc2 expression in normal tissues was lowest in tumor-specific pSurv mice. Figure 18C shows that 48 hours after plasmid injection, ex vivo analysis of Luc2 activity across multiple tissues revealed significantly higher (*p<0.05) expression in pCMV compared to all other groups. pPEG showed significantly higher (*p<0.05) Luc2 activity in the heart, lung, and spleen compared to mock-injected animals.In pSurv, there was significantly higher Luc2 activity in the spleen (*p<0.05) and a trend toward higher activity in the lung (p=0.13). Figure 18D shows that the only tissue showing hRluc activity above background was the lung (values ​​presented are normalized to the average background value from mock-injected mice). For this reason, Luc2 values ​​determined from both imaging (Figure 18B) and ex vivo tissue analysis (Figure 18C) were not normalized by hRluc values. No significant differences in hRluc levels in the lung were observed among the three promoter mouse groups. Therefore, the differences in Luc2 measurements among the three groups are unlikely to be related to differences in transfection efficiency and may be related to differences in promoter activity. Data are presented as mean ± SD. [Figure 19] Figures 19A-19C show a comparison of tumor-specific promoter activity in primary human fibroblasts and human cancer cell lines. Primary human fibroblasts, MDA-MB-231 cells (human breast cancer), and MeWo cells (human melanoma) were transfected with pPEG- or pSurv-driven plasmids (1 μg) expressing Luc2 and cotransfected with a promoterless plasmid (50 ng) expressing hRluc to normalize transfection efficiency. No differences in Rluc transfection efficiency were observed in any of the three cell types. The pPEG-driven plasmid resulted in significantly higher Luc2 activity than pSurv in fibroblasts (*p<0.05). The pSurv-driven plasmid resulted in significantly higher Luc2 activity in MeWo cells (***p<0.001) and comparable activity in MDA-MB-231 cells. Data are presented as mean ± SD. [Figure 20]Figure 20 shows a comparison of tumor-activatable PP and MC in cultured SK-MEL-28 melanoma cells. SK-MEL-28 human melanoma cells were transfected with equal masses of tumor-activatable MC (n=3) and PP (n=3) and equal volumes of the transfection agent PEI. Significantly higher SEAP activity was observed in the medium of MC-transfected cells from day 2 to day 7 (**p<0.01; ***p<0.001). Data are presented as mean ± SD. [Figure 21] Figures 21A and 21B show a comparison of transgene expression between MC and PP mice driven by a strong constitutive promoter in healthy (tumor-free) mice. Figure 21A shows mice receiving systemic administration of either MC (n = 4) or PP (n = 5) (40 μg; N / P = 8), which express hRluc driven by the strong constitutive EF1 promoter after complexation with PEI. BLI imaging was performed on days 1, 2, 3, 5, and 7 using the substrate coelenterazine. Representative images show higher BLI signals in MC-treated mice at all time points examined. For comparison, signals from mice receiving a 5% glucose injection are shown (the signal in the liver is from oxidized coelenterazine). Figure 21B shows a region-of-interest analysis across the lung region, demonstrating significantly higher BLI signals in MC vs. PP mice on days 1, 2, and 5 (*p < 0.05; **p < 0.01). Data are presented as mean ± SD. [Figure 22] Figure 22A shows a standard curve analysis of the plasma SEAP assay. Triplicate samples were measured at 10-fold dilutions of SEAP in 25 IA plasma. SEAP activity was linear over five orders of magnitude, demonstrating a detection limit of approximately 3 x 10 µg (0.3 pg) in 25 µL of plasma. Figure 22B shows SEAP measurements across the entire linear range were reproducible, with coefficient of variation (%CV) measurements less than 4%. [Figure 23]Figure 23 shows tumor burden before and after MC administration. Bioluminescence imaging (BLI) images (left) of two representative mice (top and bottom) before and 2 weeks after MC administration and the corresponding ex vivo images (right) of the lungs at the time of sacrifice (2 weeks after MC administration) are shown. The values ​​below each BLI image represent the average radiance of the region of interest depicted in the lung. There is a difference in the image scale between the two mice. Both mice showed an approximately 4.5-fold increase in BLI signal over the 2 weeks after MC administration, indicating continued tumor growth. At the time of sacrifice, tumors within the lungs were melanotic, and multiple tumor foci were observed throughout the lungs in both mice (white arrows). Based on the BLI signal changes, the total tumor burden at the time of MC administration (2 weeks before sacrifice) was approximately 4.5-fold lower than that seen in the ex vivo images provided here. [Figure 24] Figure 24 shows the results of the experiment in Example 11, in which FLuc-expressing cells were doped into normal PBMCs, demonstrating that the detection limit of such a detection method is at least 3-10 diseased cells per 5 million normal PBMCs. [Figure 25] Figure 25 shows the results of the experiment in Example 12, in which the cancer-activating DNA construct distinguished between tumor-bearing and healthy mice: After intravenous administration of live SEAP DNA nanoplasmid, whole blood was collected by submandibular gland exsanguination and processed to plasma. SEAP assays were performed on 20 μL aliquots. Cohort size was n=5. [Figure 26]Figures 26A–26F show the in vivo efficacy and in vitro cytotoxicity of polymer / DNA complexes. Figure 26A shows an experiment in which 40 μg of DNA encoding CMV-luciferase was formulated into a polymer formulation or complexed with JetPEI and then administered intravenously to Balb / C mice. Four days after transfection, the animals were then injected with D-luciferin, after which the animals were sacrificed and their lungs harvested for ex vivo BLI analysis. For cytotoxicity assessment (Figures 26B, 26C, 26D, 26E, and 26F), polyplexes containing 250 ng of CMV-Luc DNA were added to each well of a 96-well plate, and 10,000 cells were plated per well the day before transfection. Each formulation was tested in triplicate. After 48 hours, cell morphology was recorded microscopically, and cell viability was measured using an MTT assay. Figure 26B shows blank cells. Figure 26C shows in vivo delivery of constructs using JetPEI. Figure 26D shows delivery of DNA using high molecular weight amine-terminated poly(β-amino ester) C32-122. Figure 26E shows delivery of DNA using high molecular weight amine-terminated poly(β-amino ester) C32-145. Figure 26F shows cell viability results by MTT assay. [Figure 27] Figure 27 shows that protamine compacts DNA polyplex size. 62.5 μg of DNA was concentrated by thoroughly mixing 130 μg of protamine in a 1:1 volume ratio in 50 mM sodium acetate buffer (pH = 5.0). The DNA / protamine complex was then diluted to 1.5 mL with 50 mM sodium acetate buffer (pH = 5.0). Protamine:DNA LNPs were assembled using a NanoAssemblr (Precision NanoSystems) at a total flow rate of 12 mL / min. The as-prepared particles were dialyzed against 1x PBS for at least 18 hours and then sized using a Zetasizer. [Figure 28]Figure 28 shows an example of using luciferase to determine the biodistribution of a delivery formulation. A) In vivo bioluminescence imaging (BLI) of a mouse injected via the tail vein with 40 mg of CMV luciferase vector formulated with JetPEI. Four days after administration, the mouse was anesthetized, administered D-luciferin substrate, and imaged on an AMI-HT (Spectral Instruments Imaging). B) After in vivo imaging, the mouse was sacrificed and organs were harvested for ex vivo BLI. [Figure 29] Figures 29A and 29B show the sensitivity and specificity of Ad-survivin-FLuc in ex vivo assays in canine PBMCs and cells derived from canine tumors. Naive, untransduced cells derived from various subtypes of canine malignancies, including osteosarcoma, melanoma, and hemangiosarcoma, were spiked into 5e5 canine PBMCs and transduced with Ad-survivin-FLuc at an MOI of 0.3. A) The analysis demonstrates robustness for single-cell detection of A17 osteosarcoma cells or B) for detection across cells derived from multiple tumor types. [Figure 30] Figures 30A-C demonstrate the sensitivity and specificity of Ad-survivin-FLuc in ex vivo assays. A) H1299 cells engineered to constitutively express firefly luciferase protein were spiked into 56 normal human PBMCs, and the entire sample was processed and analyzed for luciferase expression. B) Untransduced, naive H1299 cells were spiked into human PBMCs, which were then transduced with a recombinant adenovirus (Ad-survivin-FLuc) carrying an expression cassette for the human survivin promoter driving the expression of firefly luciferase. After 48 hours of growth, the samples were processed and analyzed for luciferase expression. C) Samples containing only human PBMCs transduced with either Ad-survivin-FLuc or Ad-CMV-FLuc, the latter under the control of a strong constitutive promoter. After 2 days of incubation, FLuc expression was quantified using a luminescence assay. [Figure 31]Figure 31 shows that Ad-survivin-FLuc distinguishes between cancer and normal cells in an ex vivo assay on human PBMCs. Commercially available samples of human PBMCs from normal healthy volunteers and cancer patients were counted, and then equal numbers of cells were transduced with Ad-survivin-FLuc at consistent MOIs. The samples were then split into triplicates. After 3 days of incubation, the cells were lysed and analyzed for luciferase activity. Data are calculated as the mean and standard deviation of triplicate sample measurements. P values ​​were calculated by Student's t-test compared to normal PBMCs. [Figure 32] Figures 32A and 32B show the diagnostic performance of survivin-activated luciferase expression in discriminatory healthy canine individuals and canine lymphoma cancer patients. (A) Comparison of fold change in luminescence expression between healthy canine individuals (n=31) and canine lymphoma cancer patients (n=17). (B) Diagnostic predictive ability of survivin-activated luciferase activity to distinguish canine lymphoma cancer subjects from healthy canine subjects. [Figure 33]Figures 33A, 33B, 33C, 33D, 33E, and 33F show the activation of various promoter-reporter constructs in specific cell lines of various tissue origins, with the gene labels indicating the source of the promoter used in the construct. Figure 33A shows various promoter-reporter constructs in cell lines of liver origin (e.g., HepG2 and Hep3B), demonstrating that CXCR4, TRIP13, MCM10, COL10A1, BIRC5, and BIRC5-501 are particularly activated in liver cancer. Figure 33B shows the activation of various promoter-reporter constructs in immortalized cell lines of ovarian origin (e.g., SKOV3 and OVCAR), demonstrating that COL10A1, MMP13, UBE2C, MUC1, CEP55, CEACAM5, KIF20A, FAM111B, and CST1 are particularly activated in ovarian cancer. Figure 33C shows the activation of various promoter-reporter constructs in immortalized cell lines of pancreatic origin (e.g., ASPC1, BXPC3, and PANC1), demonstrating that BIRC5, ABCC4, MMP13, CXCR4, UBE2C, MUC1, CDKN3, MCM10, CDC20, CEP55, CEACAM5, KIF20A, CST1, and FAM111B are particularly activated in pancreatic cancer. Figure 33D shows the activation of various promoter-reporter constructs in breast-derived cell lines (e.g., MDA-MB-231), demonstrating that BIRC5, MCM10, MMP1, DTL, CEP55, KIF4A, RGS13, KIF20A, UBE2T, CENPF, CST1, TOP2A, FAM111B, and MMP13 are particularly activated in breast cancer. Figure 33E shows the activation of various promoter-reporter constructs in cell lines of lung origin (e.g., A549, H460, and H1299), demonstrating that MCM10, AFP, MMP1, CEP55, CEACAM5, RGS13, KIF20A, CST1, FAM111B, and MMP13 are particularly activated in lung cancer.Figure 33F shows a comparison of the same promoter-reporter construct as 33E but in a non-transformed breast cancer line, demonstrating that genes other than MMP1 shown to be activated in 33E may be particularly useful in distinguishing breast cancer from normal tissue. [Figure 34] Figures 34A, 34B, and 34C show activation of a panel of promoter-reporter constructs in melanoma, osteosarcoma, and angiosarcoma cancer cell lines, where the gene labels indicate the promoters used in the constructs. Figure 34A shows activation of panel members in cell lines of melanoma origin (e.g., M2, M3, M4, M5, and CMGD), where BIRC5, BIRC5-501, CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are specifically activated in melanoma cancer. Figure 34B shows activation of panel members in cell lines of osteosarcoma origin (e.g., OS17, OS29, OS40, and OS484), demonstrating that BIRC5, BIRC5-501, CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are specifically activated in osteosarcoma. Figure 34C shows activation of panel members in cell lines of angiosarcoma origin, demonstrating that BIRC5, BIRC5-501, MMP13, CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are specifically activated in angiosarcoma cancer. [Figure 35]Figures 35A, 35B, and 35C show the design of a multiplex detection assay using multiple different cancer-specific promoters and linked barcodes. Figure 35A shows the design of a multiplex construct, in which various cancer-specific promoters (non-descriptively designated as Px, Py, and Pz) are used to drive expression of orthogonal reporters created by fusion of a signal peptide to luciferase with intervening nucleic acid barcode sequences unique to the promoter used to drive the construct (barcodes A, B, and C for promoters Px, Py, and Pz, respectively). Figure 35B shows the relative expression of each promoter construct when transfected individually in equimolar amounts into H1299 cells, and Figure 35C shows the relative expression of each promoter construct when transfected in combination in equimolar amounts into H1299 cells, demonstrating that co-transfection of multiple reporter-promoter constructs into the same cells does not appreciably alter the expression pattern of a given promoter in a given cell line, demonstrating that the multiplex format is a viable format for generating "profiles" of promoter activation in a single cell type. [Figure 36]Figures 36A, 36B, and 36C illustrate the design of a multiplex detection assay using multiple different peptide epitopes to detect reporters driven from separate promoters. Figure 36A illustrates the design of a multiplex construct in which different copies of the CMV promoter drive expression of orthogonal reporters created by fusing a signal peptide to luciferase with an intervening epitope peptide (e.g., FLAG, HA, V5, or HSV peptide epitope) barcode unique to the promoter used to drive the construct. (36A shows the CMV promoter used, but ultimately multiple distinct promoters such as Px, Py, and Pz in Figure 35 are envisioned.) Figure 36B illustrates how multiple epitope barcodes can be used with epitope-specific capture antibodies (e.g., anti-FLAG, anti-HA, anti-V5, or anti-HSV) to separate secreted reporter constructs and obtain independent measurements of activity for each promoter. Figure 36C shows an example using FLAG / HA / V5 / HSV barcoded luciferase constructs co-transfected into cells, demonstrating that each peptide epitope-tagged luciferase construct can be separated and used to independently read out promoter activation in the same cells. [Figure 37]Figures 37A, 37B, and 37C show, along with corresponding performance data, the design of a reporter-promoter construct designed to use a prefabricated lateral flow assay (e.g., a pregnancy hCG lateral flow immunoassay) to detect activation of the promoter-reporter construct in cancer cell lines. In this design, a cancer-specific promoter (Px, in this case represented by the survivin promoter) is used to drive expression of a secretion signal-modified luciferase that is also fused to a human chorionic gonadotropin (hCG) epitope. Figure 37B shows that, via transfection of various related constructs into H1299 cells, the hCG tag does not appreciably interfere with luciferase expression from the survivin promoter. Figure 37C shows that the supernatants from transfected cells can be loaded onto commercially available lateral flow immunoassay strips for hCG, and that the lateral flow immunoassay can detect hCG-tagged luciferase, demonstrating the utility of using existing epitope immunoassays to read the expression of promoter-reporter constructs in which the reporter is tagged with an epitope that has a reliable, off-the-shelf assay. [Figure 38] Figure 38 shows an example of a nanoplasmid-based promoter construct described herein, the sequence of which is shown in SEQ ID NO: 5, and which involves a mini-R6K origin, an RNA-out selectable marker, a survivin promoter, SEAP as a reporter, and a WPRE element. DETAILED DESCRIPTION OF THE INVENTION

[0033] Although various embodiments of the present invention have been shown and described herein, such embodiments It will be apparent to those skilled in the art that the following are provided by way of example only. Numerous variations, modifications, and substitutions may be made. However, it is possible for one skilled in the art to do so without departing from the present invention. Various alternatives to the embodiments of the invention described herein may be used.

[0034] Before describing the present disclosure in more detail, it should be understood that the present disclosure It is not intended to be limited to the particular embodiments illustrated, which may, of course, vary. It should be further understood that the scope of the present disclosure is limited only by the appended claims. Therefore, the terminology used herein is for the purpose of describing particular embodiments only. and is not intended to be limiting.

[0035] When a range of values ​​is provided, the lower limit is in tenths of a second unless the context clearly dictates otherwise. 1, each intermediate value between the upper and lower limits of the range, and any other value within the stated range. It is understood that any value or intermediate value of these smaller ranges is encompassed within the present disclosure. The upper and lower limits may independently be included in the smaller ranges, and in the stated ranges Subject to any limitations specifically excluded herein, any ranges not expressly set forth are still included in this disclosure. If the range includes one or both of the limits, the range excludes either or both of those included limits. are also included in the disclosure.

[0036] Unless otherwise defined, all technical and scientific terms used herein are within the meaning of the present disclosure. The terms "a," "b," and "c" have the same meaning as commonly understood by one of ordinary skill in the art. Any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of this disclosure. Although various methods and materials are possible, preferred methods and materials are described herein.

[0037] All publications and patents cited herein are to be construed as though each individual publication or patent were incorporated by reference. No. 6,017,713, filed Dec. 19, 2002, is incorporated herein by reference as if specifically and individually indicated to be incorporated. Reference is made to the method and / or material in which the publication is incorporated and in which the publication is cited. The publication citations are for their disclosure prior to the filing date. and acknowledges that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, any dates of publication provided should be independently confirmed. The actual release date may differ.

[0038] As will be apparent to those skilled in the art upon reading this disclosure, the individual components described and illustrated herein may be Each of the embodiments may be combined with any number of others without departing from the scope or spirit of the present disclosure. A separate component that can be easily separated or combined from any feature of any of the embodiments. elements and features in the order of events recited or in any other order which is logically possible. Either of the cited methods can be performed.

[0039] Embodiments of the present disclosure are directed to a wide variety of medical, organic, and other therapeutic applications within the skill of the art, unless otherwise specified. It uses techniques from chemistry, biochemistry, molecular biology, pharmacology, toxicology, etc. Such techniques are fully explained in the literature.

[0040] It should be noted that, as used in this specification and the appended claims, the singular "a," ​​"an," and "the" refer to plurals unless the context clearly indicates otherwise. Thus, for example, a reference to "a support" includes a plurality of supports. In the specification and claims that follow, the following meanings shall be used unless a contrary intention is apparent: This will refer to a number of terms that will be defined.

[0041] As used herein, the following terms have the meanings ascribed to them unless otherwise specified: In this disclosure, "comprises" and "comprises" "sing," "containing," and "having," etc. "includes" can have the meaning ascribed to them in U.S. patent law. "includes," "includes," "includes," etc. When applied to methods and compositions encompassed by "ing essentially of" or "consists essentially of" "(sentially)" and the like refer to compositions as disclosed herein, but with additional (or analogs or derivatives of the above), but However, such additional structural groups, composition components, or method steps, etc., are not intended to be limiting unless otherwise disclosed herein. The invention relates to basic and novel characteristics of a composition or process as compared with those of a corresponding composition or process. Virtually no impact.

[0042] Before describing the various embodiments, the following definitions are provided, and unless otherwise indicated, should be used.

[0043] definition The term "subject" includes human or non-human animals. The methods and compositions described herein are applicable to both human and animal diseases and models. Preferred subjects are "patients," i.e., living humans receiving medical treatment for a disease or condition. This includes people without apparent illness who are being investigated for signs of illness. It also includes people who have or are suspected to be at risk of a definite disease.

[0044] As used herein, the term "gene" refers to a single hereditary unit that has a genetic function. Refers to all regulatory and coding sequences that are immediately associated with a gene. Nodes, DNA conformation, chromatin conformation, extent and location of base methylation , and those that specify the binding sites of proteins that control all of these (but are not limited to these) It may contain non-coding sequences that regulate genetic function (not intended to be a protein). The genes that code for a protein are made up of "exons" (coding sequences), which are called "intrinsic" sequences. In some cases, multiple transcription factors may be present. A complex of proteins or nucleic acids or other molecules, or a complex of any two of the above, On the other hand, the genetic function of a gene may depend on RNA expression or It may require only protein production or the production of proteins and / or nucleic acids without associated expression. In some cases, adjacent genes may only require binding. Genes may share overlapping sequences with other genes. It may be recognized as a chromosome, a plasmid, any other type of vector, or a separate, isolated entity. It can be considered.

[0045] As used herein, the term "episomal replicating vector" or "episomal vector" refers to a vector that replicates an episomal replicating vector. The term refers to vectors that are not typically integrated into the genome of the host cell but exist in parallel. Episomal replicating vectors can be replicated during the cell cycle, and this replication During the process, vector copies are distributed across the resulting cells depending on the number of copies present before and after cell division. Replication can occur within the nucleus of the host cell, preferably In addition, episomal replicating vectors replicate during the S phase of the cell cycle. It can replicate at least once, i.e., one or more times, in the nucleus of the host cell.

[0046] The term "sample" refers to any sample that is tested in an analytical or experimental method described herein. The sample is typically taken from a subject as described herein. Samples may include blood or blood fractions, saliva, urine, stool, cerebrospinal fluid, semen, and vaginal secretions. , phlegm, sweat, milk, synovial fluid, mucus (mucosal secretions, etc.), tears, bile, gastric juice, interstitial fluid, naturally excreted A biopsy of tissue or epithelial cells (such as a scraping of cheek cells) that is taken or specifically collected from the body Examples include, but are not limited to, aqueous mucus, amniotic fluid, pleural fluid, or exhaled breath from the subject. In some embodiments, the sample is obtained via a non-invasive method (e.g., (e.g., non-invasive sampling). Exemplary non-invasive methods include passive collection of bodily fluids, or external sampling. These include atraumatic scraping of tissue accessible to the external environment (e.g., the epidermis or mouth). Exemplary non-invasive samples include, but are not limited to, saliva, sputum, mucus, sweat, urine, and stool. Examples include semen, cervicovaginal secretions, breast milk, mucosal secretions, tears, or buccal swab samples. In some embodiments, the sample is obtained via a minimally invasive method. Exemplary minimally invasive methods include capillary sampling, venipuncture, thoracentesis, amniocentesis, needle aspiration, Exemplary minimally invasive samples include, but are not limited to, blood, or blood fractions (e.g., plasma or PBMC specimens), interstitial fluid, bile, gastric juice, and amniotic fluid. In some embodiments, the sample is obtained by biopsy. Exemplary biopsy samples include skin biopsy samples (e.g., punch biopsies, shave biopsies, trough biopsies, etc.). obtained by scraping, wedge, incisional, or excisional biopsy), bone marrow samples (e.g., biopsy), lymph node or breast biopsy (e.g., fine needle aspiration, core needle biopsy) biopsy, vacuum-assisted biopsy, or image-guided biopsy), surgical biopsy samples (e.g., (e.g., visceral tissue obtained by resection or incision biopsy) or tissue from the mouth, digestive tract, lungs, bladder, or Examples include, but are not limited to, urinary tract biopsy samples (e.g., obtained by endoscopy). Not determined.

[0047] As used herein, the term "origin of replication" refers to a vector that initiates replication of a plasmid containing an origin of replication. It refers to the DNA sequence recognized by the replication initiator or DNA replicase that causes the replication. The expression "recognized by a replication initiator" means that the replication initiator recognizes the entire or entire sequence of the replication origin. physically interact with a portion of the DNA molecule, ultimately resulting in the replication origin being replicated. This means that it can induce or stimulate the molecular mechanisms that cause the replication of a gene or part of a gene. Therefore, replication origins typically contain functionally necessary elements. An example of such a functionally required element is the repeat family of the EBV origin of replication (OriP) ( FR (Frequency Reversal) or dyad symmetry (DS) elements. Further replication containing functionally required elements. Origins are well known in the art and are described, for example, in Bode et al., (2001) G Ther. Mol. Biol. 6:33-46. The parent nucleic acid plasmid vector preferably contains at least one origin of replication.

[0048] A "vector" is a molecule that transfers other operably linked heterologous or recombinant nucleic acid sequences to a target cell. In some instances, the vector may be a minicircle, a plasmid, Nanoplasmid, Yeast Artificial Chromosome (YAC), Bacterial Artificial Chromosome (BAC), Cosmid, phagemid, bacteriophage genome, or baculovirus genome. The vectors include bacteriophages or plant, invertebrate, or animal (including human) viruses. vectors derived from viruses, such as CELiD vectors, adeno-associated virus vectors (e.g. For example, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AA V9, or AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8, etc. pseudotype combinations thereof), retroviral vectors (e.g., MLV or its self-inhibiting activated or SIN versions, or their pseudoversions), herpes viruses (e.g., HSV- or EBV-based), lentiviral vectors (e.g., HIV, FIV , or EIAV-based, or pseudotyped versions thereof), or adenovirus vectors (e.g., Ad5 systems such as replication-deficient, replication-competent, or helper-dependent versions). In some embodiments, the vector is a replication-competent virus-derived vector. In some embodiments, the vector is a replication-incompetent virus-derived vector. In this case, the vector may contain one or more target regions, such as a scaffold / matrix attachment region (S / MAR). The S / MAR element may contain episomal maintenance elements to facilitate replication in target cell types. , which are particularly useful for facilitating replication in the context of "naked" nucleic acid vectors such as minicircles. Exemplary suitable S / MAR elements include EμMAR from the immunoglobulin heavy chain locus. , apoB MAR from the human apolipoprotein B locus, chicken lysozyme inheritance Ch-LysMAR from the IFNβ locus, and huIFNβ MAR from the human IFNβ locus The vector may be expressed in the target cell, but is not limited to these. Therefore, as used herein, a "vector construct" may contain a coding sequence capable of The terms "construct," "expression vector," and "gene transfer vector" refer to vectors that are used to express a gene of interest. Any nucleic acid construct capable of directing expression and useful for introducing a gene of interest into a target cell. The vectors described herein can further direct the expression of mRNA therefrom. It may further comprise one or more cis-acting elements for stabilization or improvement. Elements include, for example, Johansen et al. Gene Medicine. (5)12:1080-1089(doi:10. 1 002 / jgm.444) or Vlasova-St. Louis and Sagarsk y. Mammalian CIS-Acting RNA Sequence Element ments (doi:10. 5772 / intechopen. 72124) These include, but are not limited to, any of the elements listed above.

[0049] As used herein, the term "minicircle" refers to one form of vector. Small double-stranded circular fragments that provide sustained, high-level expression of sequences of interest present on the vector. It refers to a DNA molecule, the target sequence of which may be a polypeptide, shRNA, antisense RNA, The sequence of interest may encode a regulatory sequence present on the minicircle vector. The regulatory sequence is operably linked to the sequence, and the regulatory sequence controls its expression. Minicircle vectors are described, for example, in published applications, which are specifically incorporated herein by reference. The different forms of vectors are described in U.S. Patent Application No. 20040241329. Therefore, "nanoplasmids" are plasmids containing minimized bacterial ColE1 or R6K replication origins (bacterial (Providing nanoplasmids that are replicable in the host strain), bacterial RNA selectable It refers to a vector that can contain a marker and a eukaryotic gene region. The plasmid contains the mini-R6K origin of SEQ ID NO: 3 and / or the RNA-OUT selection of SEQ ID NO: 4 Such elements (nanoplasmid origin and RNA-U) can contain a gene-specific marker. Further examples of T selectable markers are described, for example, in the Nanoplasmid sequence elements. No. 9,737,620B2, which is incorporated herein by reference.

[0050] The length of the minicircle vector is sufficient to contain the desired elements described below. The vector is capable of infecting a target cell upon contact with the cell, e.g., via systemic administration to a host containing the cell. not so long as to prevent or substantially impair the ability to enter the facility to an unacceptable level. Therefore, minicircle vectors are generally at least about 0.3 kb in length, and in most cases The parent vector may be at least about 1.0 kb in length, but may be 6 kb, 10 kb, or It can be longer than that.

[0051] Minicircle vectors lack an origin of replication or lack a natural origin of replication (e.g., minimized (which may contain a synthetic bacterial origin of replication) and a selectable marker commonly found on bacterial plasmids. - e.g., p-lactamase, tetracycline resistance (tet), kanamycin resistance (k and other antibiotic selection markers. This results in smaller minicircle sizes and more efficient delivery. Minicircles are vector backbone fragments of the parent plasmid from which the minicircle vector is excised. Lack of transgene expression silencing effects associated with born nucleic acid sequences. The sequence is a hybrid of the recombinase product sequence and the sequence of interest, i.e., the transcriptional sequence required for expression. It may be substantially free of vector sequences other than transcription and regulatory sequences.

[0052] As used herein, the term "nanoplasmid" refers to a minimized bacterial ColE1 or R6K replication origin (providing such nanoplasmids capable of replicating in bacterial host strains). (providing a gene for a gene encoding ... Some embodiments of nanoplasmids are described in, for example, US201502752 In some embodiments, the nanoplasmid is a fusion bacterium-R In some embodiments, the fusion protein may comprise a NA selectable marker / minimized origin of replication. Synthetic bacterial RNA selectable marker / minimized replication origin for eukaryotic nanoplasmids It may be located within a synthetic intron located within the gene region.

[0053] RNA selectable markers regulate chromosomally expressed target genes to facilitate vector selection. A vector-mediated expressed non-translated RNA provides an alternative to the use of a recombinant vector. Crouzet J and Soubrier F 2005 US Nonsense-suppressible selectable chromosomes described in Patent No. 6,977,174 It can be a plasmid-derived nonsense suppressor tRNA that regulates the target. Smid-mediated antisense repressor RNA, an RNA that suppresses RNA-IN regulatory targets The OUT gene, a pMB1 plasmid-origin encoding RNAI that suppresses RNAII regulatory targets, IncB plasmid pMU720 origin encodes RNAI, H, which suppresses RNAII regulatory targets ParB locus of plasmid RI that suppresses ok regulatory targets, Sok, flmA regulatory targets The Flm locus FlmB of the F plasmid, which represses ltuvia S, Romby P. 2002. Adv Genet 46:36 1 and Franch T, and Gerdes K. 2000. Current Other natural antisense molecules, such as those described in Opin Microbiol 3:159 Repressor RNA, or Park et al. Nature Biotechno logy volume 31, pp.170-174 (2013) engineered repressors such as small synthetic small RNAs like SgrS, MicC, or MicF scaffolds It can also be RNA.

[0054] Many suitable methods are available for transfecting cells according to the present disclosure. "Transfected" refers to the transformation of a cell resulting from the uptake of foreign nucleic acid, usually DNA. The use of the term "transfection" specifies the introduction of exogenous nucleic acid. Therefore, suitable methods include those for detecting viruses. Infection / transduction, conjugation, nanoparticle delivery, electroporation, particle gun technology, phosphate calcium precipitation, direct microinjection, etc. The choice of method depends on the The type of cell being transfected and the circumstances under which the transfection is being performed (i.e., The method depends on the type of experiment (in vitro, ex vivo, or in vivo). For a comprehensive discussion, see Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley&Sons, 1995, which are incorporated herein by reference.

[0055] The term "transfection agent" refers to the delivery of DNA to a host cell, e.g., a liposome. or any compound that mediates RNA uptake. A suitable method for transfecting is described by Sambrook et al. (MOLECULAR TRANSFER). CULAR CLONING:A LABORATORY MANUAL. 2nd e d., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold S Pring Harbor, NY, 1989), Ausubel, et al. , Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995, and other laboratory manuals ( ), which are incorporated herein by reference. Suitable transfection agents Examples include linear or branched polyethyleneimine, nanoparticles, liposomes, lipophilic particles, solids Nanoparticles, amphiphilic peptides, micelles, dendrimers, polymer compositions, hydrogels, synthetic Synthetic or naturally occurring exosomes, virus-like particles, or any combination thereof Examples include, but are not limited to:

[0056] As used herein, the term "EXO motif" refers to the incorporation of miRNAs into exosomes. In some embodiments, an EXO motif refers to an RNA sequence that controls the loading of exons. Heterogeneous ribonucleoprotein A2, which has been described to regulate miRNA loading into the endothelium Such sequences may mediate miRNA binding to hnRNPA2B1. Examples include, but are not limited to, 5'-GGAG-3' and 5'-CCCU-3'.

[0057] As used herein, the terms "nucleic acid molecule" and "polynucleotide" refer to a nucleic acid molecule of any length. A polymeric form of nucleotides, either deoxyribonucleotides or ribonucleotides A polynucleotide may have any three-dimensional structure. Polynucleotides can be used to synthesize and store information and can perform any function, known or unknown. Non-limiting examples include genes, gene fragments, exons, introns, messenger RNAs, and the like. A (mRNA), transfer RNA, ribosomal RNA, ribozyme, cDNA, s hRNA, single-stranded short or long RNA, recombinant polynucleotides, branched polynucleotides , plasmids, vectors, isolated DNA of any sequence, control regions, isolated RNA of any sequence , nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0058] The term "promoter" refers to a DNA sequence that directs transcription of a polynucleotide. Typically, the promoter is located in the 5' region of the polynucleotide to be transcribed. It can be proximal to the transcription start site of such a polynucleotide. The promoter is located in the region upstream of the first exon; more typically, the first exon of multiple transcription start sites. The promoter is often defined as the region upstream of the promoter. Directing the transcription of genes located on each of the complementary DNA strands that are 3' to each other In other words, many promoters are bidirectional and can be expressed in either direction. (i.e., 5' to 3' or 3' to 5' to the coding region of the gene) In addition, promoters can also direct the transcription of downstream genes. Such elements may include at least one regulatory element, such as an upstream activator. A polynucleotide that influences transcription, such as a UAR, and optionally a synthetic upstream element. There are other DNA sequences that

[0059] "Coding sequence" and "encode" when used in reference to polypeptides herein The term refers to, for example, when a nucleic acid is present in a living cell (in vivo) and is free of appropriate regulatory sequences (or " When placed under the control of a specific regulatory element, transcription (in the case of DNA) and translation (in the case of a specific polypeptide) occur. The boundaries of a coding sequence are typically determined by the 5' (amino) end of the coding sequence. It is determined by a start codon at the end and a translation stop codon at the 3' (carboxy) end. Coding sequences can include cDNA from viral, prokaryotic, or eukaryotic mRNA, Genomic DNA sequences from genus, eukaryotic or prokaryotic DNA, and synthetic DNA sequences The transcription termination sequence may optionally include, but is not limited to, an enhancer. 3 relative to the coding sequence along with additional regulatory sequences such as introns and polyadenylation sites The promoter may be located 5' to the coding sequence. The DNA sequence encoding the peptide was selected using codons preferred by the selected cells. Representing a DNA copy of the desired polypeptide coding sequence optimized for expression in a cell. This can be done.

[0060] As used herein, the term "barcode" or "barcode molecule" generally refers to a Barcodes / barcode molecules convey or convey information about the molecule to which they are attached A barcode / barcode molecule is a unique label or identifier that can be used to identify a specific object. Barcodes / barcode molecules can come in a variety of different formats For example, barcodes / barcode molecules can be polynucleotide barcodes; random nucleic acid and / or amino acid sequences; and synthetic nucleic acid and / or amino acid sequences. The barcode / barcode molecule can be reversibly or irreversibly attached to the molecule. The code may be generated, for example, by deoxyribonucleic acid before, during, and / or after sequencing of the sample. Barcodes can be added to fragments of DNA or RNA samples. allows for the identification and / or quantification of individual sequencing reads.

[0061] As used herein, the term "operably linked" means that Refers to an arrangement of elements so that the elements are configured to perform their usual function. Thus, where it is operably linked to a coding sequence (e.g., a reporter expression cassette), A given promoter is capable of causing expression of a coding sequence when the proper enzymes are present. The promoter or other control elements function to direct the expression of the gene. It need not be contiguous with the coding sequence, so long as there is no intervening untranslated The transcribed sequence can be located between the promoter sequence and the coding sequence. A control sequence can still be considered to be "operably linked" to a coding sequence. Cut.

[0062] As used herein, the term "expression cassette" refers to a gene / coding sequence of interest, as well as and non-coding RNAs such as shRNA, microRNA, siRNA, and antisense RNA. This refers to any nucleic acid construct capable of directing the expression of any RNA transcript comprising this Such cassettes are referred to as "vectors," "vectors," and "transfectants" for transferring the expression cassettes into target cells. The vector may be constructed into an "antigen construct," "expression vector," or "gene transfer vector." Thus, the term includes cloning and expression vehicles, as well as viral vectors. Included.

[0063] As used herein, the term "target cell" refers to a cell in which genetic modification is desired. Target cells can be isolated (e.g., in culture) or can be derived from a multicellular organism (e.g., the blastocyst). The term may be used in the context of a mammalian cell, fetus, postnatal animal, etc.

[0064] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier in which the probes of the present disclosure are administered and approved by a federal or state regulatory agency for use in animals, and more particularly in humans. approved by the U.S. Pharmacopoeia or listed in other generally recognized pharmacopeias. Such pharmaceutical carriers refer to liquids, adjuvants, excipients, or vehicles. For example, water and oils, such as those of animal, vegetable or synthetic origin, e.g., peanut oil; Soybean oil, mineral oil, sesame oil, etc. Pharmaceutical carriers include saline, acacia gum, , gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. When administered to a patient, the probe and pharmaceutically acceptable carrier are sterile. If the probe is administered intravenously, water is a useful carrier. Aqueous dextrose and glycerol solutions also are useful as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers include glucose, lactose, sucrose, glycerol monostearate, and the like. Glycerol, sodium chloride, glycerol, propylene, glycol, water, ethanol The composition, if desired, may also contain minor amounts of wetting or emulsifying agents, or p The composition may also contain a H buffer. The composition is advantageously in the form of a solution, emulsion, sustained release formulation, or It may take any other form suitable for use.

[0065] The term "detectable" refers to the ability to detect a signal above background signal. A detectable signal is one that can be used to generate an acceptable image using equipment available for preclinical use. A detectable signal is defined as an amount sufficient to produce a detectable signal. The dosage will depend on the degree of sensitivity of the individual, the individual's age, Dosage may vary depending on factors such as sex and weight, individual idiosyncratic response, and dosimetry. , and also depends on equipment and digital processing-related factors.

[0066] As used herein, the term "in vivo imaging" refers to imaging of a subject in a life-ending, sacrificial A method or method capable of examining the structural, functional, or physiological state of an organism without the need for sacrifice refers to a process.

[0067] As used herein, the term "non-invasive in vivo imaging" refers to imaging of structures present in vivo. The physical, functional or physiological state of the body is determined by the external (skin) or internal (accessible orifice) surface It can be probed by remote physical probing without the need to destroy the physical integrity of the surface. This refers to a method or process that can

[0068] An "imaging moiety" is a moiety that is administered externally to a human or non-human animal subject's body or intravascularly. detectors designed for in vivo use, such as radiological or endoscopic photodetectors, or can be detected through the use of radiation detectors designed for intraoperative use. The binding moiety is preferably a reporter suitable for in vivo optical imaging, This is not limited to:

[0069] As used herein, the term "bioluminescence" refers to the emission of light by biological molecules, particularly proteins. The prerequisite for bioluminescence is that the enzyme binds or is released in the presence of oxygenase. The luciferase reacts with the substrate and luciferin reacts with the substrate in the presence of molecular oxygen. converts the substrate to an excited state, which, upon returning to a lower energy level, emits light. Releases energy.

[0070] As used herein, the term "luciferase" refers to an oxygenase that catalyzes a light-emitting reaction. For example, bacterial luciferase is a enzyme that binds flavin mononucleotide and aliphatic aldehydes. It catalyzes the oxidation of aldehydes, producing light in the reaction. One type of luciferase catalyzes the oxidation of Cypridina luciferin, while another type of luciferase Luciferase catalyzes the oxidation of beetle luciferin. Hence, "luciferase." refers to an enzyme or photoprotein that catalyzes bioluminescence reactions. Luciferases, such as luciferase, are catalytic enzymes that act in bioluminescence-producing reactions. Luciferin is non-covalently bound to aequorin and obelin luminescent proteins. Luciferase photoproteins, such as proteins, induce the release of luciferin during the bioluminescence reaction. Luciferases may be produced by any organism or any of its variants or mutants, e.g. For example, mutants that have one or more properties, such as thermal or pH stability, that differ from the native protein. Luciferase and mutants are naturally occurring proteins produced by transgenesis. and modified mutants or variants thereof are known. " refers to enzymes isolated from members of the Renilla genus, or other species such as other Anthozoans. It refers to the equivalent molecule obtained from a commercial source or produced synthetically.

[0071] A "bioluminescent protein" is a molecule that produces or emits bioluminescence by acting on a bioluminescence initiator molecule substrate. It refers to a protein that can be excreted.

[0072] The "bioluminescence initiator molecule" reacts with the bioluminescence donor protein to generate bioluminescence. Bioluminescence initiator molecules include coelenterazine, its analogs, and Coelenterazine derivatives include, but are not limited to, functional derivatives thereof. Coelenterazine 400a, Coelenterazine CP, Coelenterazine F, Coelenterazine FCP, coelenterazine h, coelenterazine hcp; coelenterazine ip, coelenterazine n, coelenterazine 0, coelenterazine c, coelenterazine c, coelenterazine i, se Coelenterazine ICP, Coelenterazine 2-methyl, Benzyl-coelenterazine bisdeoxy Coelenterazine and Deep Blue Coelenterazine (DBC) (U.S. Patent No. 6,022, 5,968,750 and 5,874,304. ) but are not limited to these.

[0073] In general, coelenterazines are used to synthesize a wide variety of bioluminescent proteins, specifically luciferases. It is known that it emits light when acted upon by ze. Razin, in U.S. patent application Ser. No. 10 / 053,482, filed Nov. 2, 2001 (the disclosure of which is incorporated herein by reference). The disclosure of which is incorporated herein by reference in its entirety. The products are manufactured by Promega Corporation, Madison, Wisconsin. ion and Molecular Probes, Eugene, Oregon. Coelenterazines are also available from, for example, Shimomura et al., (1989) Biochem. J. 261: 913-920 ; Inouye et al., (1997) Biochem. Biophys Res. Comm. 233: 349-353, 1997; and Ter. anishi et al., (1997) Anal. Biochem. 249 : 37-43.

[0074] As used herein, the term "survivin" refers to a baculovirus apoptotic receptor. This refers to a protein called BIRC5, also known as BIRC5. The protein encoded by the RCS gene (NCBI reference sequence: NG0290 69.1). Survivin is a member of the inhibitor of apoptosis (IAP) family. The survivin protein inhibits caspase activation, thereby preventing apoptosis or programming. This results in negative regulation of regulated cell death, leading to increased apoptosis and reduced tumor growth. This is revealed by disrupting the survivin-induced pathway that leads to the survivin protein is highly expressed in most human tumors and fetal tissues, but is completely absent in terminally differentiated cells. Survivin expression is also highly regulated by the cell cycle and is expressed only during the G2-M phase. Survivin is localized to the mitotic spindle by interacting with tubulin during mitosis. It is known that it may play a role in regulating mitosis. Regulation of bin seems to be related to the p53 protein. It also regulates the Wnt pathway. It is a direct target gene of and is upregulated by β-catenin.

[0075] However, the minicircles of the present disclosure may be used to express a reporter gene that is desired to be expressed in target cells. Any tumor-specific promoter operably linked to a promoter or other heterologous nucleic acid sequence can be used. For example, but not by way of limitation, it is contemplated that Suitable promoters known in the art include the CXCR4 promoter, which is tumor-specific in melanoma. ;Tumor-specific hexokinase type II promoter;TRPM4 (transient The receptor potential-melastatin 4 promoter is preferentially active in prostate cancer; The lomelysin 3 promoter is specific to breast cancer cells (Basset et al., (1990) Nature 348:699); a specific antibody for non-small cell lung cancer cells Factor protein A promoter (Smith et al., 1994, H um. Gene Ther. 5:29-35); SLPI-expressing carcinoma-specific secretion Type leukoprotease inhibitor (SLPI) promoter (Garver et al., (1994) Gene Ther. 1:46-50); melanoma cell-specific tyrosine kinase Synthase promoter (Vile et al., (1994) Gene Ther . 1:307); Stress-induced grp78 / specific to fibrosarcoma / tumorigenic cells BiP promoter (Gazit et al., (1995) Cancer Re s. 55: 1660); Interleukin-10 promoter specific for glioblastoma multiforme cells Nitta et al., (1994) Brain Res. 649: 122); aB-crystallin / heat shock protein 27 protease specific to brain tumor cells Motor (Aoyama et al., (1993) Int. J. Cancer r 55: 760); epithelial growth specific to squamous cell carcinoma, glioma, and breast tumor cells factor receptor promoter (Ishii et al., (1993) Proc. N atl. Acad. Sci. USA 90: 282); specific to breast cancer cells The mucin-like glycoprotein (DF3, MUC1) promoter (Abe et al., (1993) Proc. Natl. Acad. Sci. USA 90: 282); the metastatic tumor-specific mts1 promoter (Tulchinsky et al., (1992) Proc. Natl. Acad. Sci. US A. 89: 9146); NSE promoter specific to small cell lung cancer cells (Fors s-Petter et al., (1990) Neuron 5:187); Alveolar lung cancer cell-specific somatostatin receptor promoter (Bombardier) et al., (1995) Eur. J. Cancer 31A:184; oh et al., (1995) Int. J. Cancer 60:843) The c-erbB-3 and c-erbB-2 promoters are specific to breast cancer cells (Q uin et al., (1994) Histopathology 25:247 ); c-erbB4 promoter specific to breast and gastric cancer cells (Rajkumar et al., (1994) Breast Cancer Res. Trends 29:3); thyroid cancer cell-specific thyroglobulin promoter (Mario i et al., (1995) J. Clin. Endocrinol. Me th. 80:468); α-fetoprotein promoter specific to hepatocellular carcinoma cells (Zuibel et al., (1995) J. Cell. Phys. 16 2:36); gastric cancer cell-specific villin promoter (Osborn et al., (1988) Virchows Arch. A. Pathol. Anat. Histopathol. 413:303); and albumin specific to hepatocellular carcinoma cells promoter (Huber, (1991) Proc. Natl. Acad. Sci. USA 88:8099) (all of which are incorporated herein by reference). Other examples of promoters include the ATP-binding cassette subfamily C member 4 (ABCC4) promoter, forward gradient 2, protein disulfide isomerase AGR2 promoter, activation-induced cytidine deaminase ( AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetyl B3GNT3 promoter, cadherin 3 ( CDH3 promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, Sen Centrosome protein F (CENPF) promoter, Centrosome protein 55 (C EP55) promoter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter , collagen type I α1 chain (COL1A1) promoter, cystatin SN (CST1) promoter promoter, denticleless E3 ubiquitin protein Ligase homolog (DTL) promoter, sequence similarity 111 member B (FAM111 B) A family with a promoter, Forkhead box A1 (FOXA1) promoter Motor, kinesin family member 20A (KIF20A), laminin subunit γ2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter, Matrix metallopeptidase 1 (MMP1) promoter, matrix metallopeptidase MMP12 promoter, matrix metallopeptidase 13 (MM P13) promoter, mesothelin (MSLN) promoter, cell surface-associated mucin 1 ( MUC1 promoter, phospholipase A2 group IID (PLA2G2D) promoter G protein signaling 13 (RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα ( TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 C (UBE2C), USH1 protein network component harmonin ( USH1C), a V-set domain containing the T-cell activation inhibitor 1 (VTCN1) promoter Ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase CHEK1 promoter, epithelial cell transformation 2 promoter (ECT2), BC L2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) Promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide Flood synthetase 1 (FLAD1) promoter, protein phosphatase, Mg 2 + / Mn 2+ promoter dependent 1G (PPM1G), ubiquitin-conjugating enzyme E2S (UB E2S) promoter, aurora kinase A and ninein-interacting protein (AUNI) P) promoter, cell division cycle 6 (CDC6) promoter, centromere protein L(CENPL) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter tar, deoxythymidylate kinase (DTYMK) promoter, neurite outgrowth 1 (GP G protein-regulated inducer of the RIN1 promoter, a regulator of mitochondrial division MTFR2 promoter, RAD51-associated protein 1 (RAD51AP1) promoter promoter, small nuclear ribonucleoprotein polypeptide A' (SNRPA1) promoter -, ATPase family, AAA domain containing 2 (ATAD2) promoter, BU B1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, Calcyclin-binding protein (CACYBP) promoter, cell division cycle-associated 3 (C DCA3 promoter, centromere protein O (CENPO) promoter, flagella Forkhead box specific endonuclease 1 (FEN1) promoter FOXM1 (FOXM1) promoter, cell proliferation regulator protein phosphatase 2A (KI AA1524 promoter, kinesin family member 2C (KIF2C) promoter Tar, karyopherin subunit α2 (KPNA2) promoter, MYB proto-oncogene like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nuclear ribonucleoprotein polymerase Lipid B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore Transforming acidic coiled-coil-containing protein 3 complex component (SPC24) promoter (TACC3) promoter, TBC1 domain family member 31 (TBC1D3 1) Promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein Zinc nucleoside 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter tar, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame C17orf53 promoter, chromobox 3 (CBX3 0) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CC NE1 promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter promoter, cell division cycle 45 (CDC45) promoter, cell division cycle-associated 5 (CD CA5 promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter , cadherin EGF LAG 7-pass G-type receptor 3 (CELSR3) promoter, Sen Centrosome protein A (CENPA) promoter, centrosome protein 72 (C EP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2 ) promoter, collagen type X α1 chain (COL10A1) promoter, chromosome segregation 1 CSE1L-like promoter, DBF4 zinc finger promoter, GINS complex promoter G protein-coupled receptor 19 (GPR19) promoter, GIN subunit 1 (GINS1) promoter Promoter, kinesin family member 18A (KIF18A) promoter, kinesin Kinesin family member 4A (KIF4A) promoter, a kinesin family member C1 (KIFC1) promoter, minichromosome maintenance 10 replication initiator (MCM1 0) promoter, minichromosome maintenance complex component 2 (MCM2) promoter promoter, minichromosome maintenance complex component 7 (MCM7) promoter, M RG domain binding protein (MRGBP) promoter, methylenetetrahydrofolate dehydrogenase Hydrogenase (NADP+-dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit H (NCA PH) promoter, NDC80, kinetochore complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonucleic acid RNAse H2 subunit A (RNASEH2A) promoter, RuvB-like AAA ATP ase 1 (RUVBL1) promoter, serologically defined breast cancer antigen NY-BR -85 (SGOL1) promoter, SHC binding and spindle associated 1 (SHCBP1) promoter Motor, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter, Timeless circadian regulator promoter, thyroid hormone Mon receptor interactor 13 (TRIP13) promoter, trophinin-related protein (TROAP) promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) promoter , WD repeat and HMG box DNA binding protein 1 (WDHD1) promoter , a functional fragment thereof, or any combination thereof.

[0076] Further definitions are provided in the following contexts: Unless otherwise defined, as used herein, All technical and scientific terms used herein are those commonly understood by one of ordinary skill in the art of molecular biology. The same meaning as in the above. Methods and materials similar or equivalent to those described herein are also included in the present application. Suitable methods and materials can be used in the practice or testing of the disclosure, including those described herein. It is being done.

[0077] Abbreviation SEAP, secreted embryonic alkaline phosphatase; MRI, magnetic resonance imaging; SPECT, Single-photon emission computed tomography; MC, minicircle; PP, parental plasmid; WPRE , woodchuck hepatitis virus (WHP) posttranscriptional regulatory element (WPRE); Luc, Luciferase; BLI, bioluminescence imaging; ROI, region of interest; AUC, area under the curve RG, reporter gene; TS, tumor-specific; Fluc (FLUC), firefly lucid ase, ROC (Receiver Operating Characteristics).

[0078] Introduction Early detection of cancer can dramatically improve the effectiveness of available treatment strategies. However, despite decades of efforts in blood-based biomarker cancer detection, many Promising endogenous biomarkers include those with highly variable background expression from non-malignant tissues. Strategies for improving cancer diagnosis have traditionally been limited to the use of conventional diagnostic methods. Specifically, we identify over-expressed genes in cancer cells through either molecular imaging or blood-based assays. These strategies have relied on the measurement of expressed endogenous molecules. In this case, significant expression in non-cancerous tissues, thereby resulting in high background levels and Another strategy is to identify reporter genes that are precisely specific within the tumor. To promote the expression of the gene (RG), tumor-specific ( To achieve this strategy, we use the promoter of the TS protein. Safety, specificity, and sensitivity are of utmost importance for this method. Although it is safer than viral vectors, However, two drawbacks of non-viral vectors are low gene transfer efficiency and transient expression process. Minicircles (MCs) are plasmids lacking a bacterial backbone. , which is advantageous in overcoming the important problems mentioned above.

[0079] The present disclosure provides a secretable receptor that can be detected almost exclusively in the blood of subjects with tumors. utilizes the pan-tumor-specific survivin promoter to drive expression of the target gene, Implementation of an alternative advantageous detection strategy based on the systemic administration of safe and tumor-activatable minicircles Following systemic administration, the efficacy and safety of the drug can be assessed for up to two weeks by simply measuring blood reporter levels. A potent antibody that distinguishes mice bearing experimental human melanoma metastases from tumor-free controls. Cumulative changes in reporter levels also identified tumor-bearing subjects. Receiver operating characteristic curve analysis confirmed the results of this study, with an AUC of 0.918 ± 0.084. Lung tumor burden correlated with cumulative reporter levels (r2 = 0.714; p<0.05), indicating that the extent of the disease can be determined. The continued development of this system will allow for the detection of tumors through temporally controlled, high reporter expression in tumors. Potential for dramatically improved detectability, with near-zero background from healthy tissue There is a possibility that this will happen.

[0080] Secreted embryonic alkaline phosphatase (SEAP) or firefly luciferase (FL A tumor-specific nanoplasmid vector that drives the expression of either UC has been developed and is being administered to the blood. and / or their usefulness in detecting tumors after systemic administration using imaging-based assays. In the case of gene vectors used for cancer screening purposes, These include efficient tumor delivery, achieving strong expression for maximum sensitivity, and targeting the target gene to achieve tumor specificity. These include tight control of expression for tumor specificity and minimization of safety concerns. The IVectocal vector overcomes all of these challenges and is a tumor-specific minigene administered systemically. The vector was assayed in serum and via non-invasive imaging to assess tumor-bearing controls. It has been shown that it is possible to distinguish subjects from normal subjects. Notably, the survivin promoter drives expression across tumor cells in many different tumor types. Therefore, the tumor-specific minicircle vectors of the present disclosure are effective in many patient populations. The tumor-specific minicircle vectors of the present disclosure have advantageously broad applicability. tumor detection via genomic assays, tumor localization via molecular genetic imaging, and New approaches involve tumor therapy using tumor-specific minicircle vectors and theranostic tumor-specific minicircle vectors. Providing a new cancer management paradigm.

[0081] The present disclosure encompasses embodiments of nucleic acid nanoplasmid vectors that are most advantageous for detecting tumor cells. In particular, the minicircles of the present disclosure can be used to target tumor cells or tissues containing tumor cells. a tumor-specific nucleotide sequence operably linked to a nucleotide sequence desired to be selectively expressed. In some embodiments of the present disclosure, the minicircle vector incorporates a promoter. operably linked to a nucleotide sequence encoding a polypeptide useful as a reporter. The fusion protein contains a tumor-specific promoter linked to it, and is therefore expressed by the recipient tumor cells. When expressed, the reporter may be detectable, thereby providing a visual image of the tumor cells. and / or its location in the tissues of the human or non-human animal subject. do.

[0082] In some embodiments, the nanoplasmid vectors according to the present disclosure contain an expressible reporter gene. The reporter gene can be advantageously delivered to tumor cells. non-invasive imaging such as PET imaging, SPECT imaging, and luminescence imaging It is within the scope of this disclosure that the invention may be detectable by an invasive detection method. Although not intended to be a marker, the MRI reporter gene is creatine kinase; Encodes enzymes; transferrin receptor; ferritin; and Mag A. PET imaging The gene reporter gene was the herpes simplex virus type 1 thymidine kinase (HSV1- TK); Hypoxanthine phosphoribosyltransferase; L-amino acid decarboxylase enzymes; dopamine 2 receptors (D2R, mutant D2RA80, etc.); somatostatin Receptor; Estrogen receptor (hERL); Dopamine transporter; Sodium iodide cotransporter catecholamine transporters; 13-galactosidase, etc. PET / SPECT imaging reporter genes are not intended to Multiple optimized mutants, including HSV1-sr39tk and HSV1-thymidine kinase Dopamine type 2 receptor; Sodium iodide cotransporter; Somatostatin type 2 receptor; Human norepinephrine transporter; human estrogen receptor a; human deoxycytidine kinase and recombinant carcinoembryonic antigen. Reporter genes include firefly luciferase (fl); synthetic Renilla luciferase (Renilla luciferase); enhanced green fluorescent protein (egfp); red fluorescent protein (rf p); monomeric red fluorescent protein (mrfp1); The reporter genes suitable for incorporation into the minicircles of the present disclosure are not It is further possible to provide a multi-modality method of imaging. Although not intended as a reporter suitable for photoacoustic, MRI, and PET imaging, The gene is reported in Qin et al., (2013), Sci. Rpts. 3:Ar No. 1490, the entirety of which is incorporated herein by reference. It is a gene that encodes scinase.

[0083] Advantageous Uses of the Nanoplasmids of the Present Disclosure for Selective Detection of Recipient Tumor Cells In addition, the nucleotide sequence operably linked to the tumor-specific promoter is administered to the subject. A recipe for reducing or eliminating target tumor cells from a human or non-human animal. Encoding a polypeptide useful for regulating the growth or metabolic activity of tumor cells can be done.

[0084] For example, but not by way of limitation, targeting and therapeutically attacking tumor cells. Advantageous therapeutically effective polypeptides include HSVtk; cytosine deaminase; DT diaphorase; nitroreductase; guanine phosphoribosyltransferase; Purine nucleoside phosphorylase; Thymidine phosphorylase; Carboxylesterase hydroxybenzoylpolyglutamyl synthetase; carboxypeptidase A1; carboxypeptidase B1 cytochrome P-450 (CYP2B1), etc. The activity of these polypeptides to convert the polypeptides into effective therapeutic compositions is described, for example, in Harr et al. ington et al., (2002) Clinical Oncology 14:148-169, which is incorporated herein by reference in its entirety. There are.

[0085] In a further embodiment of the present disclosure, tumor-specifically expressed nucleosides from minicircles are The nucleotide sequence is not capable of being translated into a heterologous polypeptide, but rather is also at least one gene of the recipient tumor cells that modulates the proliferation or metabolic activity of the recipient tumor cells; Short interfering ribonucleotide sequences (siRNAs) capable of interacting with gene regulatory elements Alternatively, the nucleotide sequence may be expressed as a microRNA. sequences (miRNAs) or do not correspond to known endogenous sequences and are not identified by nucleic acid hybridization Drugs that are detectable by amplification-based or nucleic acid biomarkers It is contemplated that they may be expressed as synthetic RNA sequences that serve a single purpose.

[0086] Therefore, tumor cells cultured in vitro or, most advantageously, in vivo Selectively targeting to identify and / or locate a population of cancerous or tumor cells in a subject It is useful to obtain detectable signals that identify the target tumor cells, as well as to deliver therapeutic drugs (peptides) to target tumor cells. Nucleic acid minicircle vectors (and their derivatives) useful for delivering peptides, polypeptides, and nucleic acids It is contemplated that it is within the scope of this disclosure to provide embodiments in which the parent plasmid is used.

[0087] The present disclosure provides a method for detecting the presence of a targeted tumor cell or cells (including tumor tissue). and providing nucleic acid minicircle vectors useful for administration to human or non-human animal subjects. For example, the nucleotide sequence of SEQ ID NO: 1 shown in FIG. The minicircle construct MC-pSurv-SEAP-WPRE-SV40PolyA is , a detectable polypeptide operably linked to the tumor-specific promoter p-survivin It contains a nucleic acid fragment encoding secreted embryonic alkaline phosphatase (SEAP).

[0088] Cultured melanoma cells, subcutaneous melanoma xenografts, or tumor-bearing animals may be used for intravenous delivery. In this case, the minicircle vector of the present disclosure contains a serum-secreted alkaline phosphatase polypeptide and or as shown in Figure 4 (and as shown in Figure 14) a minisample in which SEAP was replaced by a luciferase reporter; The signal is detectable as a bioluminescent signal in the vector construct. Thus, the minicircle constructs of the present disclosure can be used to treat metastatic tumors in recipient animals or humans. It has been demonstrated that it can identify both tumor cells or localized tumors.

[0089] The present disclosure further provides a method for targeting tumor cells by delivering minicircle nucleic acids to said tumor cells. The physiology or proliferation of a cell is modulated and the target cell is operably linked to a tumor-specific promoter. The nucleic acid sequence is then allowed to express a nucleotide sequence, and the expressed product is then delivered to the target cell. and allowing the molecule to interact with the cell, thereby modifying the physiological state of the cell or cells. Provide a way to change.

[0090] In a first example, the present disclosure provides a method for treating a tumor-specific promoter comprising administering to a mammalian subject the present invention .... Nucleic acid minicircles, such as, but not limited to,

[0091] Therefore, to overcome the limitations of endogenous biomarker detection, the present disclosure provides a method for detecting tumor-driven Blood samples of exogenously delivered genetically encoded reporters producing type 2 biomarkers An embodiment of a strategy based on identifying individuals with tumors using fluid-based detection is provided. The main advantage of this strategy is that biomarker expression occurs exclusively in cells of a specific phenotype (i.e., tumor cells). expression can be adjusted to reduce the number of false positives due to protein production from non-malignant tissue Therefore, tumor-activatable vectors encoding secretable reporter genes are Systemic administration of thrombin can be used to identify subjects with tumors, but see Figure 1. As shown in Fig. 1, tumor-specific promoters (genes that express proteins that are only present in tumors) The transgene expression was transcriptionally targeted to cancer cells using a promoter from a gene encoding the α-terminal β-actin (α-ATR) gene. Safety, specificity, sensitivity, and broad applicability are important for clinical translation of this strategy. Each component of the disclosed system was selected to provide the greatest translation potential. Specifically, the present disclosure provides vectors, including but not limited to, survivin promoter (pSurv). Human secreted embryonic alkaline phosphatase (ESA) achieves tumor specificity through the use of tumor-specific promoters reporter genes, including but not limited to, phosphatase (SEAP) This paper provides a non-viral tumor-activatable minicircle (MC) that can induce tumorigenesis.

[0092] Although safer than viral vectors, it is not as effective as conventional non-viral vectors (i.e., plasmids). The two drawbacks of MC are its slow gene transfer rate and transient expression profile. Essentially, it is a plasmid lacking the prokaryotic backbone required only for bacterial growth. C is a plasmid due to its relatively small size and low promoter silencing. Showing improved expression profile (months in non-dividing cells, weeks in dividing cells) compared to counterparts It has been repeatedly shown that Gene Therapy 4: 1341-1349; Darquet et al ., (1999) Gene Therapy 6: 209-218; Chen et al., (2003) Mol. Therapy: J. Am. Soc. Gene Therapy 8: 495-500; Chen et al., ( 2004) Gene Therapy 11: 856-864). MC is an antibiotic. Regulatory "" known to be safer for administration to humans than constructs containing resistance genes The principle of "antimicrobial resistance gene-free plasmid" (pFAR) (Marie et al. l., (2010) J. Gene Med. 12: 323-332) Furthermore, the production of MC has traditionally been very labor-intensive and time-consuming. However, more recent advances in MC production schemes have made it relatively easy and inexpensive to produce large quantities in a short period of time. Production became possible (Kay et al., (2010) Nat. Biotec h. 28:1287-1289). Finally, integration is a common mechanism for many genetic (especially viral) vectors. Although this is a safety concern for the dermatologist, effective methods such as direct local injection and electroporation have been developed. Even with various in vivo delivery methods, the integration rate of non-viral vectors is limited by spontaneous gene inactivation. The rate of spontaneous mutation is approximately 1 to 3 orders of magnitude lower (Wang et al., (2004) Gene Therapy 11: 711-721; Nichols et al., (19 95) Annals New York Acad. Sci. 772: 30-3 9; Ledwith et al., (2000) Develop. Biolo Gicals 104: 33-43; Ledwith et al., (2000 )Intervirology 43: 258-272). Therefore, MC is a translation The most useful non-viral vector platform in terms of potential, efficacy, and safety It has become one of the

[0093] SEAP is a commonly used secretable reporter protein and has many ideal This is due to the human placental alkaline phosphatase, which is expressed only during embryonic development. It is an artificial C-terminally truncated secretory form of PLAP. It is a unique reporter that cannot be seen in other reporters, and background should be nearly zero. (Berger et al., (1988) Gene 66:1-10). Compared to LAP, SEAP is very thermostable. Therefore, the sample was heated to 65°C. SEAP can be specifically assayed by heating (Bronstein, n et al., (1994) BioTechniques 17:172-17 4, 76-177). Commercially available SEAP detection assays cover a concentration range of at least four logarithmic orders. It is highly sensitive in the range of 1000p and has a detection limit in the picogram / mL range. It is also a protein-based reporter that favors translation of the mouse have demonstrated effective longitudinal monitoring of non-viral gene transfer in small and large animals. (Brown et al., (2008) Methods Mol. Biol 423:215-224); 2) its human origin, as demonstrated by murine encephalopathy in immunocompetent mice Decreased or zero immunity in patients similar to that shown with mu-SEAP (mu-SEAP) It may have epidemiogenic potential (Wang et al., (2001) Gene 279 :99-108);3) SEAP has been shown to be clinically effective as an adjuvant vaccine against HPV16 / 18AS04. has been used to monitor antibody levels after administration of cutin (Kemp et al. , (2008)Vaccine 26:3608-3616).

[0094] The system of the present disclosure utilizes pSurv to drive expression of SEAP. The inhibitors of apoptosis (AAPs) are members of the apoptosis inhibitor family, which regulate mitotic progression and promote cell death. It helps prevent cancer and is overexpressed in many cancers, including melanoma, liver, lung, breast, colon, and ovarian cancers. However, it is not overexpressed in healthy adult tissues (Ito et al., (2000) H epatology 31: 1080-1085; Chen et al., (2 004) Cancer Gene Therapy 11: 740-747; Lu et al., (2005) Gene Therapy 12: 330-338 Therefore, pSurv is a potential cancer risk factor for lung cancer, melanoma, colon cancer, breast cancer, ovarian cancer, and liver cancer. This is advantageous for tumor transcriptional targeting, as demonstrated in a pancreatic cancer model (Lu et al. al., (2005) Gene Therapy 12: 330-338; et al., (2006) J. Gene Med. 8: 1232-124 2; van Houdt et al., (2006) J. Neurosurg ery 104: 583-592; Ahn et al., (2011) Gen e Therapy 18: 606-612; Ray et al., (2008 ) Mol. Therapy: J. Am. Soc. Gene Therapy 16: 1848-856). Therefore, the tumor-specific promoter-driven tumors of the present disclosure Activatable MCs are effective cancer screening agents across multiple tumor types and patient populations It offers broad applicability for

[0095] Therefore, diagnostic tumor-activatable MCs may be genetically encoded cancer biomarkers. By measuring blood levels of MC, tumor-bearing subjects were compared with healthy controls after systemic administration of MC. For delivery, MCs have been developed and tested for their ability to differentiate from subjects. It has no toxicological properties (Bonnet et al., (2008)Pharmac Eut. Res. 25:2972-2982), and has the ability to be administered repeatedly to animals. It efficiently transduced both primary and metastatic tumors in mice after systemic (tail vein) administration. (Yang et al., (2013) Proc. Nat. Acad. Sci. USA110:14717-14722; Bhang et al., (2011)) Nat. Med. 17:123-129) The results were compared with non-targeting transfection agents that have been shown to be tumor-activatable. The use of MCs is an advantageous and promising platform for safe and effective cancer screening. The system monitors patients at high risk of tumor recurrence. After screening, it can help screen high-risk populations before tumor diagnosis and This may be advantageous for screening populations.

[0096] Exogenously delivered gene-encoding cancer blood biomarker vector strategy according to the present disclosure The high background expression in healthy tissues and the significant changes in biomarker expression over time Cancer screening targeting endogenous cancer blood biomarkers, such as irregular changes in blood glucose levels, is The present disclosure provides a simple and relatively inexpensive blood-based A tumor that can be administered systemically to identify subjects with tumors using a tumor assay. An embodiment of a tumor-activatable MC system is provided. This assay provides reliable detection of demonstrated the ability and disease coverage to be evaluated as a highly robust and safe cancer screening system. This demonstrates the feasibility of tumor-activatable MCs.

[0097] Cancer gene therapy research is focused on avoiding unwanted effects in non-target or normal cells. To achieve this goal, researchers have sought ways to express therapeutic transgenes specifically within tumors. To achieve this, transcriptional targeting of tumors using tumor-specific promoters (Aim et al. al., (2011) Gene Therapy 18:606-612; et al., (2003) Biochem. Biophys. Res. C omms. 307:759-764; Iyer et al., (2005) T transgenic Res. 14:47-55), using endogenous miRNA regulation Transcriptional silencing or suppression in healthy tissues (Cawood et al., (20 09) PLoS Pathogens 5: e 1 000440; Ronald et al., (2013) Gene Therapy 20:1006-101 3) Increased tumor tropism of both viral (transcriptional targeting) and non-viral vectors Strong (Chisholm et al., (2009) Cancer Res. 69 : 2655-2662; Bachtarzi et al., (2008) Ex Pert Opinion Drug Delivery 5:1231-1240), or a combination of these strategies (Tsuruta et al., (2008) Cl in. CancerRes. 14:3582-3588; Sugio et al ., (2011) Clin. Cancer Res 17:2807-2818) Several strategies have been explored, including the use of secretable antibodies for cancer detection. This application of gene vectors provides a means for expressing a potential reporter gene. As a practical screening tool, vectors can be used even in the absence of clear, visible evidence of cancer. Being available for use in patients presents the additional challenge of overcoming increased safety concerns. Thus, all components of this type of system are the delivery vehicle (if required), D The NA vector itself, including the transgene (if expressed), must be safe.

[0098] Many delivery formulations are known in the art and can be used with the MC systems of the present disclosure. However, it is expected that the drug will have a favorable safety profile (i.e., no immune stimulation) (B onnet et al., (2008) Pharmaceut. Res. 25 :2972-2982), currently undergoing phase I / II clinical trials (Lisziewicz et al. , (2012) PLoS ONE 7:e35416) In addition, non-viral vectors are much more effective than viral vectors. Although it is relatively safe (i.e., low / near-zero integration rate, low immunogenicity potential), Concerns about immunostimulatory prokaryotic CpG motifs in the plasmid backbone There are still cases where these vectors lack a prokaryotic backbone or are in the bacterial domain. Due to their small size (less than 500 bp), MC and / or nanoplasmids do not address this concern. Since SEAP is of human origin, it should not induce an immunogenic response. (Wang et al., (2001) Gene 279:99-108), It has already shown promise in clinical settings (Kemp et al., (2008) SEAP was selected based on the results of the study (Vaccine 26:3608-3616).

[0099] Previously, viral infection was used to transfect the cells using the viral OriP promoter / origin of replication. Interferon in Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) MC-OriP-IFNγ driving the expression of IFNγ (Zuo et al., (201 1) Driving cancer-specific gene constructs such as PLoS ONE 6:e19407 In contrast, the disclosed MC system has been shown to be effective in targeting many different tumors beyond virus-infected cells. The non-viral MC vectors of the present disclosure may be widely applicable to blood-borne It was developed for use in cancer screening using a source assay.

[0100] Tumor-activatable reporter gene expression vectors have been developed for cancer detection. (Bhang et al., (2011) Nat. Med. 17: 123- 129; Chaudhuri et al., (2003) Technol. I n Cancer Res. & Treat. 2: 171-180; m et al., (2011) Mol. Imaging Biol. 13: 452-461; Warram et al., (2012) Cancer Ge ne Therapy 19: 545-552; Browne et al., ( 2011) PLoS ONE 6: e19530), however, in these cases The vector systems used (adenovirus, herpes simplex virus, and plasmids) ) have safety issues that hinder clinical translation. Viruses are highly immunogenic and Pre-existing viral immunity is a widespread problem (Browne et al., 2014). 2011) PLoS ONE 6: e19530; Sumida et al., (2005) J. Immunol. 174: 7179-7185; rmbeck et al., (2008) Mol. Therapy 16: 1 609-1616). Plasmids are the backbone of prokaryotes (necessary only for plasmid production). It can be immunogenic due to the unmethylated CpG sequences in the an et al., (1999) Human Gene Therapy 10: 2153-2161), typically encoding antibiotic resistance to endogenous bacterial flora Gene (Marie et al., (2010) J. Gene Med 12: 323-332). Therefore, the tumor-activatable MCs of the present disclosure can be used to inhibit these It has advantages over other vectors, mainly easier manufacturing practices (compared to viruses) and It offers translation possibilities for more desirable profiles.

[0101] The MC and / or nanoplasmid system of the present disclosure can be administered by two mechanisms: 1) S 1) the uniqueness of the biomarker in blood, since EAP cannot be detected at all; and 2) the specificity of the biomarker in tumors. Through its ability to reduce signaling in healthy, tumor-free subjects by tightly driving expression This can provide improved specificity. Tumor-free mice receiving MC The slight SEAP signal from pSurv may be due to leakage of pSurv. Therefore, the MC system of the present disclosure is not limited to this particular promoter and may be used with other promoters. Tumor-activatable promoters, such as the Idl or hTERT promoter (Warram et al., (2011) Mol. Imaging Biol. 13: 4 52-461; Zhang et al., (2008) Life science es 82: 1154-1161) are expected to be useful in the MC of the present disclosure. Furthermore, the sensitivity of endogenous biomarkers depends on the biomarkers produced by the tumor. Inherently limited by the amount of markers (Hori & Gambhir (2011 ) Sci. Translational Med. 3:109ra116). Control Consequently, the sensitivity of the MC system of the present disclosure can be varied.

[0102] One advantage of endogenous blood biomarkers is that they can be used to determine what type of cancer a person has. (For example, a high PSA level may indicate a prostate cancer.) However, the MC system provided by the present disclosure also It is advantageous to screen for all cancers rather than a specific tumor type. Furthermore, specific mutations, such as the prostate-specific antigen enhancer / promoter variants in prostate cancer, have been identified. Alternative promoters (Iyer et al.) are useful for screening patients at high risk for cancer. t al., (2005) Transgenic Res. 14:47-55; Iyer et al., (2004) Mol. Therapy 10:545- 552; Iyer et al., (2006) Human Gene Ther apy 17:125-132) or the mucin-1 promoter in breast cancer (Huyn et al., (2009) Clin. Cancer Res. 15:3126-3 134) and the like can be incorporated into the MC system of the present disclosure.

[0103] Another limitation of exogenous biomarkers (i.e., reporters) is the location of the biomarker. The problem is that it is not possible to determine the location of the site in the body where SEAP is expressed. Children (e.g., Yaghoubi SS and Gambhir SS (2006) N at Protoc. 1(6):3069-75. Herpes simplex virus thymidine kinase 1 (HSV-1) for PET imaging (PET) By expressing the tumor, the system of the present disclosure also allows visualization of the tumor location. Bhang et al. recently reported that suitable imaging reporter genes After systemic administration of a tumor-activatable plasmid expressing the gene, BLI and single-photon emission computer imaging were performed. described the ability to image tumors using both CT and SPECT. (Bhang et al., (2011) Nat. Med. 17:123-1 29) This strategy allows the use of these vectors for visualization of tumors using a fluorescent stereo microscope. Since the SEAP-expressing viral vectors co-expressed fluorescent proteins, (Chaudhuri et al., (2003) Techno l. In Cancer Res. & Treat. 2: 171-180; W arram et al., (2011) Mol. Imaging Biol. 13: 452-461; Warram et al., (2012) Cance r Gene Therapy 19: 545-552). In addition, two reporters Rather than one vector system expressing the same gene, two different vectors designed for specific applications are used. It is envisioned that it would be possible to deliver vectors containing secretable reporters. one for screening tumors expressing the imaging reporter, and the other for screening tumors expressing the imaging reporter. It is used for position determination.

[0104] Thus, one aspect of the present disclosure is a method for detecting a tumor-specific gene expression promoter, comprising: The present invention encompasses embodiments of recombinant nucleic acid minicircle vectors comprising nucleotide sequences that are Higher levels of expression by recipient tumor cells than by tumor cells .

[0105] In an embodiment of this aspect of the disclosure, the tumor-specific gene expression promoter is surubi BIRC5 promoter, CXCR4 promoter, ATP-binding cassette subframe Family C member 4 (ABCC4) promoter, forward gradient 2, protein disulfide Activation-induced cytidine deoxyribonucleotide isomerase family member (AGR2) promoter AICDA promoter, UDP-GlcNAc:βGal β-1,3- N-acetylglucosaminyltransferase 3 (B3GNT3) promoter, cad CDH3 promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter tar, centromere protein F (CENPF) promoter, centrosome protein CEP55 promoter, claudin 3 (CLDN3) promoter, claudin Collagen 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter promoter, collagen type I α1 chain (COL1A1) promoter, cystatin SN (C ST1 promoter, denticleless E3 ubiquitin Protein ligase homolog (DTL) promoter, sequence similarity 111 member B (F AM111B) promoter, Forkhead box A1 (FOX A1) Promoter, kinesin family member 20A (KIF20A), laminin Subunit γ2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter Motor, matrix metallopeptidase 1 (MMP1) promoter, matrix Metallopeptidase 12 (MMP12) promoter, matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface associated Mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2 D) Promoter, G protein signaling 13 (RGS13) promoter, secretory Topoglobin family 2A member 1 (SCGB2A1) promoter, topoisomer TOP2A promoter, ubiquitin D (UBD) promoter, ubiquitin UBE2C (ubiquitin-conjugating enzyme E2 C), USH1 protein network component V-cells containing the promoter of u-monin (USH1C), T-cell activation inhibitor 1 (VTCN1) hexokinase type II promoter, TRPM4 promoter, strome Lysin 3 promoter, surfactant protein A promoter, secretory leukoproter Proteinase inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / BiP-containing domain promoter, interleukin-10 promoter, α-B-clone Listerin / heat shock protein 27 promoter, epidermal growth factor receptor promoter , mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, So Tomatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein Protein promoter, villin promoter, albumin promoter, glycoprotein A 33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, Seq oxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2 (EGFR) promoter receptor 2, human telomerase reverse transcriptase promoter; kinase containing receptor promoter Domain insert; rad51 recombinase promoter; TTF-1, urokinase plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T( UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, Epithelial cell transformation 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter motor, centromere protein I (CENPI) promoter, E2F transcription factor 1 ( E2F1 promoter, flavin adenine dinucleotide synthetase 1 (FLAD1 ) promoter, protein phosphatase, Mg 2+ / Mn 2+ Dependency 1G(PPM1 G) Promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, check CHEK1 promoter, epithelial cell transformation 2 promoter (E CT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin a Denine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase Tase, Mg 2+ / Mn 2+ promoter-dependent 1G (PPM1G), ubiquitin-conjugating enzyme The uninhibited E2S (UBE2S) promoter, aurora kinase A, and ninein-interacting protein AUNIP promoter, cell division cycle 6 (CDC6) promoter, centro CENPL promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DS N1) promoter, deoxythymidylate kinase (DTYMK) promoter, neurite G protein-regulated inducer of elongation 1 (GPRIN1) promoter, mitochondrial compartment MTFR2 promoter, RAD51-related protein 1 (RAD 51AP1) promoter, small nuclear ribonucleoprotein polypeptide A′ (SNRPA 1) Promoter, ATPase family, AAA domain-containing 2 (ATAD2) promoter Motor, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin-binding protein (CACYBP) promoter, cell division Cycle-associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter Motor, flap structure-specific endonuclease 1 (FEN1) promoter, pho FOXM1 promoter, cell proliferation regulatory protein phosphatase Kinesin family member 2A (KIAA1524) promoter, kinesin family member 2C (KIF 2C) Promoter, karyopherin subunit α2 (KPNA2) promoter, M YB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) Promoter, RAN-binding protein 1 (RANBP1) promoter, micronuclear ribonuclease SPC24 / NDC, SPC24 / NDC promoter, SPC24 / NDC promoter 80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing TACC3 promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc filament Zinc fusion protein 695 (ZNF695) promoter, aurora kinase A (AUR KA) promoter, BLM RecQ-like helicase (BLM) promoter, chromosome 1 7 open reading frame 53 (C17orf53) promoter, chromoboc CBX30 promoter, cyclin B1 (CCNB1) promoter, cyclin B1 The promoter of cyclin E1 (CCNE1), cyclin F (CCNF), and the cell division cycle 20 ( CDC20 promoter, cell division cycle 45 (CDC45) promoter, cell division cycle Phase-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3 ) promoter, cadherin EGF LAG 7-pass G-type receptor 3 (CELSR3) promoter Motor, Centromere protein A (CENPA) promoter, Centrosome protein Protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit CKS2 promoter, collagen type X alpha 1 chain (COL10A1) promoter , chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinesin family member 18A (KIF18A) promoter Motor, kinesin family member 4A (KIF4A) promoter, kinesin phagocytosis Millimember C1 (KIFC1) promoter, minichromosome maintenance 10 replication initiation factor (MCM10) promoter, minichromosome maintenance complex component 2 (M CM2 promoter, minichromosome maintenance complex component 7 (MCM7) promoter promoter, MRG domain binding protein (MRGBP) promoter, methylenediaminetetraacetic acid Tetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohexyl MHFD2 promoter, non-SMC condensin I complex subunit NCAPH promoter, NDC80, kinetochore complex component (NDC80) Promoter, nudix hydrolase 1 (NUDT1) promoter , ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA ATPase 1 (RUVBL1) promoter, serologically defined breast cancer Antigen NY-BR-85 (SGOL1) promoter, SHC binding and spindle associated 1 (SH CBP1 promoter, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter promoter, timeless circadian regulator promoter -, thyroid hormone receptor interactor 13 (TRIP13) promoter, trophinin TROAP promoter, ubiquitin-conjugating enzyme E2 C (UBE2C ) promoter, WD repeats and HMG box DNA binding protein 1 (WDHD1 ) promoter, alpha-fetoprotein (AFP) promoter, fragments thereof, or The compound may be selected from the group consisting of any combination thereof.

[0106] In some embodiments of this aspect of the disclosure, the The resulting nucleotide sequence can be expressed as a polypeptide.

[0107] In some embodiments of this aspect of the disclosure, the The selected nucleotide sequence can encode a reporter polypeptide.

[0108] In some embodiments of this aspect of the disclosure, the reporter polypeptide is an MRI reporter. -, PET reporter; SPECT reporter, photoacoustic reporter, bioluminescence reporter , or any combination thereof.

[0109] In some embodiments of this aspect of the disclosure, the polypeptide is a polypeptide derived from secreted embryonic alkaline phosphatase. The enzyme may be SEAP.

[0110] In some embodiments of this aspect of the disclosure, the recombinant nucleic acid minicircle vector comprises SEQ ID NO: The nucleic acid sequence may be according to SEQ ID NO: 1.

[0111] In some embodiments of this aspect of the disclosure, the polypeptide is a bioluminescent reporter. It is possible.

[0112] In some embodiments of this aspect of the disclosure, the recombinant nucleic acid minicircle vector comprises SEQ ID NO: The nucleic acid sequence may be according to SEQ ID NO:2.

[0113] In some embodiments of this aspect of the disclosure, the The selected nucleotide sequence may be a small interfering RNA (siRNA) or a therapeutically active polypeptide. It can be expressed as

[0114] Another aspect of the present disclosure is a method for producing a non-tumor specific gene, comprising: Nucleotides that are expressible at higher levels by recipient tumor cells than by tumor cells. and a pharmaceutical composition comprising a recombinant nucleic acid minicircle vector containing a nucleotide sequence and a pharmaceutically acceptable carrier. A therapeutically acceptable composition, comprising: (i) a tumor-specific gene expression promoter selected from the group consisting of surviv CXCR4 promoter (BIRC5), CXCR4 promoter, ATP-binding cassette subfamily Milli C member 4 (ABCC4) promoter, anterior gradient 2, protein disulfide Isomerase family member (AGR2) promoter, activation-induced cytidine deaminase AICDA promoter, UDP-GlcNAc:βGal β-1,3-N -acetylglucosaminyltransferase 3 (B3GNT3) promoter, cadherin CDH3 promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter -, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter, claudin 3 (CLDN3) promoter, claudin Collagen XI α1 chain (COL11A1) promoter Motor, collagen type I α1 chain (COL1A1) promoter, cystatin SN (CS T1) promoter, denticleless E3 ubiquitination Protein ligase homolog (DTL) promoter, sequence similarity 111 member B (FA M111B) promoter-containing family, Forkhead box A1 (FOXA 1) Promoter, kinesin family member 20A (KIF20A), laminin subunit Unit γ2 (LAMC2) promoter, mitotic spindle positioning (MISP) promoter matrix metallopeptidase 1 (MMP1) promoter, matrix metalloproteinase ... Metallopeptidase 12 (MMP12) promoter, matrix metallopeptidase 1 3 (MMP13) promoter, mesothelin (MSLN) promoter, cell surface-associated Mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D ) promoter, G protein signaling 13 (RGS13) promoter, Secret Globin family 2A member 1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin Conjugation enzyme E2 C (UBE2C), a USH1 protein network component V-seq containing monin (USH1C), T-cell activation inhibitor 1 (VTCN1) promoter domain, hexokinase type II promoter, TRPM4 promoter, stromerase Synthase 3 promoter, surfactant protein A promoter, secretory leukoprotein A promoter ase inhibitor promoter, tyrosinase promoter, stress-inducible grp78 / B iP-containing domain promoter, interleukin-10 promoter, α-B-cre staphylococcus aureus / heat shock protein 27 promoter, epidermal growth factor receptor promoter, Mucin-like glycoprotein promoter, mts1 promoter, NSE promoter, soma Toxicantrone receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter Motor, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein tein promoter, villin promoter, albumin promoter, glycoprotein A3 3 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclo Oxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor Body 2, human telomerase reverse transcriptase promoter; kinase receptor containing promoter Main insert: rad51 recombinase promoter; TTF-1, urokinase Plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T(U BE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, top epithelial cell transformation 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter centromere protein I (CENPI) promoter, E2F transcription factor 1 (E 2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) Promoter, protein phosphatase, Mg 2+ / Mn 2+ Dependency 1G (PPM1G ) promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint CHEK1 promoter, epithelial cell transformation 2 promoter (EC T2), BCL2-like 12 (BCL2L12) promoter, centromere protein I ( CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide analogue (FAD) N-terminal dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase Ze, Mg 2+ / Mn 2+ promoter-dependent 1G (PPM1G) ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and ninein-interacting protein AUNIP promoter, cell division cycle 6 (CDC6) promoter, centromere CENPL promoter, DNA replicative helicase / nuclease 2 ( DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN 1) Promoter, deoxythymidylate kinase (DTYMK) promoter, neurite G protein-regulated inducer of elongation 1 (GPRIN1) promoter, mitochondrial fission MTFR2 promoter, RAD51-related protein 1 (RAD5 1AP1) promoter, small nuclear ribonucleoprotein polypeptide A′ (SNRPA1 ) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter promoter, calcyclin-binding protein (CACYBP) promoter, peri-mitotic Phase-associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter promoter, flap-specific endonuclease 1 (FEN1) promoter, fork Headbox M1 (FOXM1) promoter, cell proliferation regulating protein phosphatase Kinesin family member 2A (KIAA1524) promoter, kinesin family member 2C (KIF2 C) Promoter, karyopherin subunit α2 (KPNA2) promoter, MY B proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 (NEK2) promoter promoter, RAN-binding protein 1 (RANBP1) promoter, micronuclear ribonuclease Protein polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC8 Kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil-containing Protein 3 (TACC3) promoter, TBC1 domain family member 31 ( TBC1D31 promoter, thymidine kinase 1 (TK1) promoter, zinc phosphate Zinc nucleoside 695 (ZNF695) promoter, aurora kinase A (AURK A) Promoter, BLM RecQ-like helicase (BLM) promoter, chromosome 17 Open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin Cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (C DC20) promoter, cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) Promoter, Cadherin EGF LAG 7-pass G-type receptor 3 (CELSR3) promoter Centromere protein A (CENPA) promoter, centrosome protein Cytoplasmic protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen type X α1 chain (COL10A1) promoter, Chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, G INS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 ( GPR19 promoter, kinesin family member 18A (KIF18A) promoter motor, kinesin family member 4A (KIF4A) promoter, kinesin family Lee member C1 (KIFC1) promoter, minichromosome maintenance 10 replication initiator MCM10 promoter, minichromosome maintenance complex component 2 (MC M2) promoter, minichromosome maintenance complex component 7 (MCM7) promoter Motor, MRG domain binding protein (MRGBP) promoter, methylenetetramine Hydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclo Hydrolase (MTHFD2) promoter, non-SMC condensin I complex subunit NCAPH promoter, NDC80, kinetochore complex component (NDC80) promoter promoter, nudix hydrolase 1 (NUDT1) promoter, Ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like A AA ATPase 1 (RUVBL1) promoter, a serologically defined breast cancer anti- The original NY-BR-85 (SGOL1) promoter, SHC binding and spindle-associated 1 (SHC BP1 promoter, small nuclear ribonucleoprotein polypeptide G (SNRPG) promoter Motor, timeless circadian regulator promoter , thyroid hormone receptor interactor 13 (TRIP13) promoter, trophinin-related TROAP promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) Promoter, WD repeat and HMG box DNA binding protein 1 (WDHD1) promoter, alpha-fetoprotein (AFP) promoter, fragments thereof, or and (ii) a tumor-specific promoter. The nucleotide sequence operably linked to the motor is an MRI reporter, a PET reporter, or reporter, SPECT reporter, photoacoustic reporter, bioluminescence reporter, or any of these The present invention encompasses embodiments of compositions that can be expressed as polypeptides encoding any combination of do.

[0115] In some embodiments of this aspect of the disclosure, the recombinant nucleic acid minicircle vector comprises SEQ ID NO: The nucleic acid sequence may be according to SEQ ID NO: 1 or SEQ ID NO: 2.

[0116] Yet another aspect of the present disclosure is a method for detecting tumor cells in a human or non-human subject. (i) administering to a first human or non-human animal subject a tumor-specific gene operably linked to an expression promoter and expressed by recipient tumor cells in preference to non-tumor cells a recombinant vector comprising a nucleotide sequence capable of being expressed at higher levels by a recombinant vector containing a nucleotide sequence and a pharmaceutically acceptable carrier; A pharmaceutically acceptable composition comprising a nucleic acid minicircle vector, the composition comprising: a) a tumor-specific The gene expression promoters are survivin promoter (BIRC5), CXCR4 promoter promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, Anterior gradient 2, a protein disulfide isomerase family member (AGR2) pro Motor, activation-induced cytidine deaminase (AICDA) promoter, UDP-Gl cNAc:βGal β-1,3-N-acetylglucosaminyltransferase 3 ( B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion Centromere activator molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter Motor, centrosome protein 55 (CEP55) promoter, claudin-3 (CLDN3) promoter, claudin 4 (CLDN4) promoter, collagen Collagen type XI α1 chain (COL11A1) promoter, collagen type I α1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, denticleless (dent cleless E3 ubiquitin protein ligase homolog (DTL) promoter , family with sequence similarity 111 member B (FAM111B) promoter, Forkhead box A1 (FOXA1) promoter, kinesin family member 2 0A (KIF20A), laminin subunit γ2 (LAMC2) promoter, mitotic Spindle positioning disruption (MISP) promoter, matrix metallopeptidase 1 (MM P1) promoter, matrix metallopeptidase 12 (MMP12) promoter , matrix metallopeptidase 13 (MMP13) promoter, mesothelin (MS LN) promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (PLA2G2D) promoter, G protein signaling 13( RGS13) promoter, secretoglobin family 2A member 1 (SCGB2A 1) Promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 C (UBE2C), USH1 protein Protein network component harmonin (USH1C), inhibitor of T cell activation 1 (V V-set domain containing TCN1 promoter, hexokinase type II promoter, TRPM4 promoter, stromelysin 3 promoter, surfactant protein A promoter, secretory leukoprotease inhibitor promoter, tyrosinase promoter tar, stress-inducible grp78 / BiP-containing domain promoter, interleukin α-B-crystallin / heat shock protein 27 promoter , epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter Motor, NSE promoter, somatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglo Bulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter Min promoter, glycoprotein A33 promoter, B cell-specific Moloney leukemia virus Lus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor receptor 1 (FGF1) factor promoter; human epidermal growth receptor 2, human telomerase reverse transcriptase promoter; Kinase domain insert containing receptor promoter; rad51 recombinase promoter Motor: TTF-1, urokinase-type plasminogen activator receptor promoter -, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase CHEK1 promoter, epithelial cell transformation 2 promoter (ECT2), BC L2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) Promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide Flood synthetase 1 (FLAD1) promoter, protein phosphatase, Mg 2 + / Mn 2+ Promoter dependent 1G (PPM1G), ubiquitin-conjugating enzyme E2 T (U BE2T) promoter, checkpoint kinase 1 (CHEK1) promoter, top epithelial cell transformation 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter centromere protein I (CENPI) promoter, E2F transcription factor 1 (E 2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) Promoter, protein phosphatase, Mg 2+ / Mn 2+ Dependency 1G (PPM1G ) promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, Aurora A-unitase A and ninein-interacting protein (AUNIP) promoter, cell division cycle 6 ( CDC6) promoter, centromere protein L (CENPL) promoter, DN A replicative helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MI S12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase ( DTYMK) promoter, G protein in neurite outgrowth 1 (GPRIN1) promoter Quality control inducer, mitochondrial fission regulator 2 (MTFR2) promoter, R AD51-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein Protein polypeptide A' (SNRPA1) promoter, ATPase family, AAA BUB1 mitotic checkpoint serine, promoter, domain containing 2 (ATAD2) / Threonine kinase (BUB1) promoter, calcyclin-binding protein (CA CYBP promoter, cell division cycle associated 3 (CDCA3) promoter, centromere CENPO promoter-flap structure-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, Cell proliferation Reg protein phosphatase 2A (KIAA1524) promoter, kinesi Karyopherin family member 2C (KIF2C) promoter, karyopherin subunit α 2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, N IMA-associated kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP) 1) Promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) Promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter -, transforming acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC 1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter Tar, Aurora kinase A (AURKA) promoter, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame 53 (C17orf 53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 ( CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CD C45 promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent Dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG 7 pathway G-type receptor 3 (CELSR3) promoter, centromere protein A (CENPA) Promoter, centrosome protein 72 (CEP72) promoter, CDC28 Protein kinase regulatory subunit 2 (CKS2) promoter, collagen type X alpha 1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DB F4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter , G protein-coupled receptor 19 (GPR19) promoter, kinesin family members Kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF 4A) Promoter, kinesin family member C1 (KIFC1) promoter, Minichromosome maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance Nance complex component 2 (MCM2) promoter, minichromosome maintenance complex MCM7 promoter, MRG domain-binding protein (MRG BP) promoter, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-S MC condensin I complex subunit H (NCAPH) promoter, NDC80, Netcore complex component (NDC80) promoter, nudix hydrolase nuclease 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASE H2A) promoter, RuvB-like AAA ATPase 1 (RUVBL1) promoter tar, the serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein SNRPG promoter, timeless circulatory Diane regulator promoter, thyroid hormone receptor interactor 13 (TRIP 13) Promoter, trophin-related protein (TROAP) promoter, ubiquitin UBE2C promoter, WD repeats, and HMG box D NA-binding protein 1 (WDHD1) promoter, alpha-fetoprotein (AFP) promoter a promoter, a fragment thereof, or any combination thereof. (b) the nucleotide sequence operably linked to the tumor-specific promoter is RI reporter, PET reporter, SPECT reporter, photoacoustic reporter, bioluminescence or any combination thereof. (ii) delivering a composition to the first subject; and (ii) detecting the expression product in the first subject. wherein the expression product is a tumor-specific gene expression promoter of a minicircle vector. and detection of said expression product is performed by a first In one embodiment, the method includes a step of indicating the presence of tumor cells in a subject.

[0117] In some embodiments of this aspect of the disclosure, the expression product can be a serum polypeptide. and step (ii) includes obtaining a serum sample from the first subject and This may include determining the serum level of the expression product produced from the vector.

[0118] In some embodiments of this aspect of the disclosure, the expression product detected is a secreted embryonic alkaline phosphatase. It can be a sulfatase (SEAP).

[0119] In some embodiments of this aspect of the disclosure, the minicircle vector is according to SEQ ID NO: 1. It can have a nucleic acid sequence.

[0120] In some embodiments of this aspect of the disclosure, the expression product is a bioluminescent polypeptide. and step (ii) is to generate a detectable signal derived from the expression product, measuring the level of detectable signal generated from the circle vector; and The level of signal from one subject was compared with that from a second subject who did not receive the minicircle vector. This may include comparing the levels obtained from the second subject with those obtained from the first subject. A high level of signal from the first subject compared to the first subject indicates that the first subject has tumor cells or tumors. It is shown to contain a population of cells.

[0121] In some embodiments of this aspect of the disclosure, step (ii) comprises non-invasively generating a detectable signal. converting said signal into an image; and imaging said image of a first subject. and positioning the detectable signal relative to the first subject. and determining the location of the tumor cell or cluster of tumor cells in the first subject. It can include.

[0122] In some embodiments of this aspect of the disclosure, the expression product can be luciferase. do.

[0123] In some embodiments of this aspect of the disclosure, the minicircle vector is according to SEQ ID NO:2. It can have a nucleic acid sequence.

[0124] Improved synthetic biomarkers for disease diagnosis, detection, and monitoring In some aspects, the present disclosure provides: (a) administering a composition to a subject, wherein the composition induces a non-lesional Induce the expression of synthetic biomarkers in diseased cells preferentially over the expression of biomarkers in cells. thereby determining the relative concentrations of biomarkers expressed in diseased cells compared to non-diseased cells. (b) detecting a synthetic biomarker; and (c) detecting a synthetic biomarker; c) Using the synthetic biomarkers detected in (b), the subject is identified as having diseased cells. In some embodiments, the detecting comprises detecting a protein comprising at least 90 It has an accuracy of %.

[0125] Optionally, the composition is administered to the subject intravenously, subcutaneously, intracerebroventricularly, intrathecally, or intravenously. administration, intracerebroventricular administration, transdermal administration, intramuscular administration, oral administration, inhalation administration, intranasal administration, rectal administration, tumor administration Intratumoral administration or tumor-proximal administration. areas predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes) Intratumoral or proximal approaches may include, for example, endoscopic ultrasound. Testing (e.g., Shirley et al., Gastroenterol Res P ract. 2013;2013:207129) or bronchoscope (e.g., Rojas -Solano et al. J Bronchology Interv Pulm onol. 2018 Jul;25(3):168-17) In some embodiments, the composition is applied to the neck, epitrochlear condyle, Supraclavicular, cervical, axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteal lymph nodes Optionally, lymph node-based administration is administered to at least one of the following tissue regions: It may serve as a method of intermediate local delivery.

[0126] In some cases, detection of diseased cells is at least about 50%, at least about 53%, or at least about 50%. At least about 55%, at least about 57%, at least about 60%, at least about 50%63% , at least about 65%, at least about 67%, at least about 70%, at least about 72% , at least about 75%, at least about 77%, at least about 78%, at least about 79% , at least about 80%, at least about 81%, at least about 82%, 83%, at least About 84%, 85%, at least about 86%, 87%, 88%, 89%, 90%, 91%, 9 2%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99. 2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 9 9.9%, or any range of accuracy between these values. Detection was up to approximately 53%, 55%, 57%, 60%, 63%, 65%, 67%, 70%, 72%, 75%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99 0.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or any of these It may have any range of precision between the values.

[0127] In some cases, detection of diseased cells is at least about 80%, 81%, 82%, 83%, 84%, %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 Sensitivity of %, 95%, 96%, 97%, 98%, 99%, or any range between these values In some cases, detection of diseased cells may be up to about 81%, 82%, 83%, 84%. ,85%,86%,87%,88%,89%,90%,91%,92%,93%,94% , 95%, 96%, 97%, 98%, 99%, or any range between these values. It can have.

[0128] In some cases, detection of diseased cells is at least about 80%, 81%, 82%, 83%, 84%, %, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, or any range between these values In some cases, detection of diseased cells can be up to about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or any range between these values It may have specificity.

[0129] In some cases, detection of diseased cells is at least about 80%, 81%, 82%, 83%, 84%, %, 85%, 86%, 87%88%, 89%, 90%, 91%, 92%, 93%, 94% ,95%,95.2%,95.5%,95.7%,96%,96.2%,96.5%,9 6.7%, 97%, 97.2%, 97.5%, 97.7%, 98%, 98.2%, 98. 5%, 98.7%, 99%, 99.2%, 99.5%, 99.7%, or 99.9%, or may have a negative predictive value (NPV) in any range between these values. Detection of cells was at least approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95.2%, 95 0.5%, 95.7%, 96%, 96.2%, 96.5%, 96.7%, 97%, 97.2 %, 97.5%, 97.7%, 98%, 98.2%, 98.5%, 98.7%, 99%, 99.2%, 99.5%, 99.7%, or 99.9%, or any range between these values The NPV may be in the range

[0130] In some cases, detection of diseased cells is at least about 30%, 31%, 32%, 33%, 34%, %, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44 %, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54 %, 55%, 56%, 57%, 60%, 63%, 65%, 67%, 70%, 72%, 75 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 8 6%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9 Positive predictive values ​​(P) of 6%, 97%, 98%, or 99%, or any range between these values In some cases, detection of diseased cells may be up to about 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 60%, 63%, 65%, 67%, 70%, 72% ,75%,77%,78%,79%,80%,81%,82%,83%,84%,85% ,86%,87%,88%,89%,90%,91%,92%,93%,94%,95% , 96%, 97%, 98%, 99%, or any range between these values. do.

[0131] In some embodiments, the composition comprises a vector encoding the synthetic biomarker. Suitable vectors include minicircles, plasmids, nanoplasmids, miniintro Nicked plasmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), cosmids, including, but not limited to, phagemids, bacteriophages, and baculoviruses. Suitable vectors include vectors suitable for administration to cells in vivo (not including the vector itself). The vectors include bacteriophages or plant, invertebrate, or CELiD vectors. Vectors derived from animal (including human) viruses, such as adeno-associated virus vectors ( For example, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, A AV9, or AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8, etc. These pseudotyped combinations), retroviral vectors (e.g., MLV or its autologous variants), Inactivated or SIN versions, or their pseudo-versions), herpes virus vectors (e.g., HSV- or EBV-based), lentiviral vectors (e.g., HIV, FI V, or EIAV-based, or pseudotyped versions thereof), or adenovirus vectors - (e.g., Ad5 systems such as replication-deficient, replication-competent, or helper-dependent versions). In some cases, the vector may contain a single region, such as a scaffold / matrix attachment region (S / MAR). It may contain episomal maintenance elements to facilitate replication in the above target cell types. R elements are particularly useful in facilitating replication in the context of "naked" nucleic acid vectors such as minicircles. Exemplary suitable S / MAR elements include E from the immunoglobulin heavy chain locus. μMAR, apoB MAR from the human apolipoprotein B locus; chicken lysozyme Ch-LysMAR from the IFNβ locus, and huIFNβ from the human IFNβ locus In some embodiments, vectors include, but are not limited to, vector MARs. The vector can be a non-viral vector.

[0132] Optionally, the composition comprises a synthetic biomarker operably linked to a promoter. Suitable promoters include naturally occurring pan-tumor specific promoters. promoters, natural tissue-specific promoters, natural disease-specific / disease-activated promoters Promoters include promoters, naturally occurring constitutive promoters, and any combinations thereof. The survivin promoter (BIRC5), CXCR4 promoter, and ATP-binding cassette ABCC4 promoter, anterior gradient 2, protein Disulfide isomerase family member (AGR2) promoter, activation-induced AICDA promoter, UDP-GlcNAc:βGal β -1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) promoter tar, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) ) promoter, centromere protein F (CENPF) promoter, Centrosau CEP55 promoter, claudin 3 promoter ter, claudin 4 (CLDN4) promoter, collagen type XI α1 chain (COL1 1A1) promoter, collagen type I α1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, denticleless E3 Ubiquitin protein ligase homolog (DTL) promoter, sequence similarity 111 members Family with BarB (FAM111B) promoter, forkhead box A 1 (FOXA1) promoter, kinesin family member 20A (KIF20A), Laminin subunit γ2 (LAMC2) promoter, mitotic spindle positioning (MI SP) promoter, matrix metallopeptidase 1 (MMP1) promoter, Matrix metallopeptidase 12 (MMP12) promoter, matrix metallopeptidase Mesothelin (MSLN) promoter, myocarditis Cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 group IID (P LA2G2D) promoter, G protein signaling 13 (RGS13) promoter -, secretoglobin family 2A member 1 (SCGB2A1) promoter, topo Isomerase IIα (TOP2A) promoter, ubiquitin D (UBD) promoter , ubiquitin-conjugating enzyme E2 C (UBE2C), USH1 protein network component Usentharmonic1C, T-cell activation inhibitor 1 (VTCN1) promoter V-set domain containing, hexokinase type II promoter, TRPM4 promoter , stromelysin 3 promoter, surfactant protein A promoter, secreted Leukoprotease inhibitor promoter, tyrosinase promoter, stress-induced g rp78 / BiP-containing domain promoter, interleukin-10 promoter, α-B-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter promoter, mucin-like glycoprotein promoter, mts1 promoter, NSE promoter somatostatin receptor promoter, c-erbB-3 promoter, c-er bB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α -Fetoprotein promoter, villin promoter, albumin promoter, glycoprotein promoter Protein A33 promoter, B cell-specific Moloney leukemia virus insertion site 1 promoter tar, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth receptor 2, human telomerase reverse transcriptase promoter; receptor promoter kinase domain insert; rad51 recombinase promoter; TTF-1, Urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (CHEK1) promoter Motor, epithelial cell transformation 2 promoter (ECT2), BCL2-like 12 (BCL2L 12) Promoter, centromere protein I (CENPI) promoter, E2F transcription factor transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 ( FLAD1) promoter, protein phosphatase, Mg 2+ / Mn 2+ Dependency 1G (PPM1G) promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter , checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 2 promoter ECT2, BCL2-like 12 (BCL2L12) promoter, centromere CENPI promoter, E2F transcription factor 1 (E2F1) promoter, Flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein Protein phosphatase, Mg 2+ / Mn 2+ promoter dependent 1G (PPM1G), ubiquitin Interaction of the ubiquinone-binding enzyme E2S (UBE2S) promoter, aurora kinase A, and ninein AUNIP promoter, cell division cycle 6 (CDC6) promoter , centromere protein L (CENPL) promoter, DNA replicative helicase / nuclease DNA2 promoter, DSN1 homolog, and MIS12 kinetochore complex component (DSN1) promoter, deoxythymidylate kinase (DTYMK) promoter -, G protein-regulated inducer of neurite outgrowth 1 (GPRIN1) promoter, mitochondrial androcyte division regulator 2 (MTFR2) promoter, a RAD51-related protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polypeptide A′ ( SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATA D2) promoter, BUB1 mitotic checkpoint serine / threonine kinase ( BUB1 promoter, calcyclin-binding protein (CACYBP) promoter , cell division cycle associated 3 (CDCA3) promoter, centromere protein O (CEN PO) promoter, flap structure-specific endonuclease 1 (FEN1) promoter -, Forkhead box M1 (FOXM1) promoter, cell proliferation Reg protein Phosphatase 2A (KIAA1524) promoter, kinesin family member 2 C (KIF2C) promoter, karyopherin subunit α2 (KPNA2) promoter motor, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related kinase 2 ( NEK2 promoter, RAN-binding protein 1 (RANBP1) promoter, micronucleus Ribonucleoprotein polypeptide B and B1 (SNRPB) promoter, SPC2 4 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coil TACC3 promoter, a member of the TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter -, zinc finger protein 695 (ZNF695) promoter, Aurora kinase A (AURKA) promoter, BLM RecQ-like helicase (BLM) promoter , chromosome 17 open reading frame 53 (C17orf53) promoter, CBX30 promoter, cyclin B1 promoter -, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell division Cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter, Cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 ( CDKN3 promoter, cadherin EGF LAG 7-pass G-type receptor 3 (CELS R3) promoter, centromere protein A (CENPA) promoter, centromere Cortical protein 72 (CEP72) promoter, CDC28 protein kinase regulated Subunit 2 (CKS2) promoter, collagen type X α1 chain (COL10A1) promoter promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter Motor, GINS complex subunit 1 (GINS1) promoter, G protein binding receptor 19 (GPR19) promoter, kinesin family member 18A (KIF1 8A) Promoter, kinesin family member 4A (KIF4A) promoter, Nesin family member C1 (KIFC1) promoter, minichromosome maintenance 1 Replication initiator (MCM10) promoter, minichromosome maintenance complex component Minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome maintenance complex component 7 (M CM7 promoter, MRG domain binding protein (MRGBP) promoter, Tetrylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, methenyltetrahydrofolate MHFD2 promoter, non-SMC condensin I complex NCAPH promoter, NDC80, kinetochore complex component (N DC80 promoter, nudix hydrolase 1 (NUDT1) promoter promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA ATPase 1 (RUVBL1) promoter, serologically defined Breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC binding and spindle association SHCBP1 promoter, small nuclear ribonucleoprotein polypeptide G (SN RPG promoter, timeless circadian regulator Promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, TROAP promoter, ubiquitin-conjugating enzyme E2 C ( UBE2C) promoter, WD repeats and HMG box DNA binding protein 1 ( WDHD1 promoter, alpha-fetoprotein (AFP) promoter, and their fragments pieces, or any combination thereof.

[0133] Optionally, the synthetic biomarker can be a polypeptide or nucleic acid biomarker. The polypeptide may include any of the reporter polypeptides described herein. Nucleic acids include natural or engineered miRNAs, RNA hairpins, and RNA aptamers or If the nucleic acid is an miRNA, the miRNA For example, degenerate primer-based annealing and ligation, poly(A) polymerization, Enzyme labeling followed by RT or ligation, or q-PCR, sequencing or Standard library analysis, such as sequential adapter ligation combined with electrophoretic detection If the biomarker is a polypeptide, it can be detected by a purification technique. The polypeptide may contain an N-terminal secretory signal sequence (e.g., the N-terminal secretory signal sequence from CD33 or CD8a). The nucleotide sequence may include a signal peptide (terminal signal peptide).

[0134] By attributing dedicated labels to unique members of a larger group, Codes can be larger and more complex mixtures of many members (e.g., expressed in the same cell). Identifying and quantifying its members within the context of multiple promoter-reporter constructs Given the opportunity (e.g., expression of a reporter under the control of a particular cancer-specific promoter), and offer the opportunity to isolate single members from complex mixtures. In the case of barcodes, hybridization of the barcode based on base pair complementarity is used. The capture event captures and isolates or otherwise reduces the complexity of the mixture. In the case of peptide-based barcodes, immunocapture or ligand-receptor binding can be used. The capture event captures and isolates the mixture using unique characteristics, including interactions. , otherwise the complexity can be reduced.

[0135] When the nucleic acid is an engineered miRNA, the nucleic acid may be prepared using the method described by Ronald et al. al., PLoS ON E 11(7):e0159369) It can be a Sec-miR or miR-neg construct. Such a construct can be a) A coding sequence that is not endogenously expressed and has no known vertebrate target (e.g., Sec- miR 5′-AAAUGUACUGCGCGUGGAGAC-3′); (b) the miRNAs that provide processing of pre-miRNAs into mature miRNAs flanked by miRNA sequences R backbone sequence (e.g., miR-155 or miR-130 backbone sequence); (c) Contains an EXO motif that enhances loading into exosomes (e.g., GGAG). Such miRNA constructs can be used, for example, to encode a gene encoding a reporter polypeptide. in the 3′-UTR of a protein or in an appropriately non-toxic protein (e.g., actin or tubulin). Any endogenous structural protein or highly expressed protein such as ubiquitin In some embodiments, the gene can be expressed from the 3'-UTR of the gene it manipulates. Multiple copies (e.g., at least two, at least four) of a miRNA are tandemly linked. It can be provided in

[0136] In some cases, the synthetic biomarker is detected by non-invasive imaging performed on the subject. The method may be a polypeptide biomarker detectable by a method comprising: This includes detecting synthetic biomarkers by non-invasive imaging. Invasive imaging methods include MRI imaging, PET imaging, and SPECT imaging. imaging, photoacoustic imaging, and bioluminescence imaging. Synthetic biomarkers detectable by ELISA include ferritin (or Pyrococcus furiosus). Pyrococcus furiousus ferritin mutants L55P and F57S , mutants thereof such as F123S), or lanthanide binding proteins (or Daughter ry et al., ChemBioChem 2012, 13, 2567-25 74) and engineered fusions thereof, such as the LBT-ubiquitin fusion described in Contrast agents include synthetic biomarkers that are detectable by PET or SPECT imaging. The human sodium iodide symporter (e.g., administration of PET-active iodine / iodide isotopes) In combination with, for example, Penheiter et al. Curr Gene Th er. 2012 Feb;12(1):33-47), HSV-tk or HSV -sr39tk or its variants (e.g., positron-labeled, e.g., [18F]FHBG) In combination with administration of acycloguanosine or pyrimidine analog PET reporters, ghoubi SS et al., Nat Protoc. 2006;1(6): 3069-75), and dopamine D2 receptors or D2R80A or D2R194A, etc. and variants thereof (e.g., 3-(2′-[18F]-fluoroethyl)-spiperone, etc. In combination with the administration of a positron-labeled D2 binder, Therefore, detectable synthetic biomarkers include β-galactosidase (e.g., X-gal). in combination with tyrosinase, chromogenic enzymes such as tyrosinase, autofluorescent proteins (e.g., G GFP, mCherry, or its derivatives), non-fluorescent GFP-like chromoproteins (e.g., a eCP597 and cjBlue and their derivatives), bacteriophytochrome-based Near-infrared fluorescent proteins (e.g., IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP713 / V256C, iRFP682, iRFP702, iRFP670, mIFP, iBlueberry, GAF-FP, BphP1-FP / C20S, or AphB variants), and reversible Photoswitchable proteins (Dronpa, Dronpa-M159T, BphP) 1 or a variant thereof). Biomarkers include Gaussia luciferase, Renilla luciferase, and Foci luciferase. Nussulus luciferase (e.g., Branchini et al., Anal. Bi 396(2010):290-297 Luciferases (e.g., coelenterases described herein, including E9 versions) In some embodiments, the synthetic biomarker is , a contrast agent, an enzyme that produces a detectable molecule, or a transactivator that drives the accumulation of a detectable molecule. The synthetic biomarkers can be used in situ in the subject's body. It can be measured.

[0137] Synthetic biomarkers are polypeptide biomarkers that can be detected by non-invasive imaging methods If the synthetic biomarker is a marker, a composition that induces expression of the synthetic biomarker in diseased cells is used as a target. (d) localizing diseased cells within the subject's body; The positioning may be performed at a specific resolution, for example, 10 mm to 10 cm, or at least The positioning can be related to a specific minimum detection limit of 10 mm or a maximum of 10 cm. Removable tumor size, e.g., 3 mm 3 ~5cm 3 This may be related to the size of the tumor. , the specific minimum range is 1cm 3 ~5cm 3 , or 900mm 3 ~1cm 3 , or 800m m 3 ~900mm 3 , or 700mm 3 ~800mm 3 , or 600mm 3 ~700mm 3 , or 500mm 3 ~600mm 3 , or 400mm 3 ~500mm 3 , or 300m m 3 ~400mm 3 , or 200mm 3 ~300mm 3, or 100mm 3 ~200mm 3 , or 50mm 3 ~100mm 3 , or 10 mm 3 ~50mm 3 , or 3 mm 3 ~10 mm 3 Optionally, non-invasive imaging scans (PET, MR Localization is performed using imaging techniques (e.g., MRI, SPECT, etc.). In some cases, in situ surgical intervention is required. Sometimes, for example, by using visual inspection (in the case of visible absorption reporters) or in combination with fluorescence excitation. Positioning is accomplished by the use of visual inspection.

[0138] Optionally, after the additional positioning step described above, the detected and / or positioned A surgical step may be performed to remove diseased cells. The surgical step may involve biomarker detection. administering a composition encoding the same or different The surgical step may be performed by any person. The surgical or non-surgical removal step can be radiosurgery (cancer Manaif, Reflexion, CyberKnife, and targeted ionizing radiation for disease control Minimally invasive killing techniques such as (but not limited to) Can be given.

[0139] Optionally, the synthetic biomarker is administered with a composition that induces expression of the synthetic biomarker. In some cases, the synthetic biomarkers can be detected in biological samples from subjects to which the biomarkers have been administered. The markers are detected in vivo and the location of diseased cells is determined.

[0140] Optionally, the composition administered to the subject may include a transfection agent. Suitable transfection agents include linear or branched polyethyleneimine, nanoparticles, lipophilic polymeric particles, peptides, micelles, dendrimers, hydrogels, synthetic or naturally occurring exosomes Examples of such compounds include cellulose, polymeric compositions, virus-like particles, and any combination thereof. , but not limited to these.

[0141] Optionally, the composition may further comprise a pharmaceutically acceptable carrier. Acceptable carriers include water, peanut oil, soybean oil, mineral oil, sesame oil, saline, and acacia gum. , gelatin, starch paste, talc, keratin, colloidal silica, urea, dextrose Sugar solution, glycerol solution, glucose, lactose, sucrose, glycerol monostearate acrylate, sodium chloride solution, propylene, glycol or ethanol, or Examples include, but are not limited to, any combination of:

[0142] A biological sample can be a sample obtained by non-invasive means from a subject. Exemplary non-invasive samples include saliva, sputum, sweat, urine, stool, semen, cervicovaginal secretions, breast milk, These include, but are not limited to, mucosal secretions, tears, and buccal swabs. The sample can be a sample collected from a subject by a minimally invasive method. Suitable minimally invasive samples include blood samples (e.g., obtained by venipuncture or capillary tubes) pleural fluid samples (e.g., obtained by thoracentesis); amniotic fluid samples (e.g., obtained by ovine samples (obtained by hydrocentesis), and gastric fluid samples (obtained by amniocentesis, for example). (e.g., obtained by gastric lavage). Skin biopsy samples (e.g., punch biopsy, shave biopsy, trough scrape biopsy, wedge biopsy, incision biopsy) biopsy, or excision biopsy), bone marrow sample (obtained by aspiration biopsy, for example), lymph node or breast biopsy (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, or imaging) biopsy samples (obtained by image-guided biopsy), surgical biopsy samples (e.g., obtained by excision or incisional biopsy), of internal organs obtained by smearing), or biopsy samples of the mouth, gastrointestinal tract, lung, bladder or urinary tract (e.g. It can be a sample obtained by biopsy (e.g., obtained by endoscopy). Cut.

[0143] Optionally, the biological sample is derived from administration of a composition that induces expression of a synthetic biomarker. After a period of time, a biological sample can be obtained to induce expression of the synthetic biomarker. At least about 15 minutes, at least about 30 minutes, at least about 1 hour, or at least about 1 hour after administration of the composition. at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 16 hours, At least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days , at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, At least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least The biological samples may be obtained over a period of about 5 months, or at least about 6 months. up to about 15 minutes, up to about 30 minutes, ...30 minutes, up to about 15 minutes, up to about 30 minutes, up to about 30 minutes 1 hour, max. 2 hours, max. 4 hours, max. 8 hours, max. 16 hours, max. Large: approx. 24 hours, max. approx. 36 hours, max. approx. 48 hours, max. approx. 3 days, max. approx. 4 days, max. approx. 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 1 1 day, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 1 months, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to In some embodiments, biological samples are obtained over a period of about six months. Any biomarker detection protocol can be used to induce expression of the synthetic biomarker. The method can be performed multiple times after administration of the composition (e.g., to assess synthetic biomarker levels). (To monitor the expression of synthetic biomarkers over time). at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 20, 22, 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 20 or 25 times the number of biological samples induced to express synthetic biomarkers. After administration of the composition, the dose can be given weekly or monthly.

[0144] In some cases, the diseased cells may be cancerous cells, cells indicative of an autoimmune disease (e.g., autologous activated T cells or lymphocytes, or normal cells damaged by autoimmunity), Cells exhibiting a degenerative disease (e.g., cells with or near toxic amyloid) the subject from whom the cells are obtained is suffering from or at risk of suffering from the disease. These cells may have altered gene expression profiles due to their A cell population containing cells with an altered gene expression profile can be identified by transcriptional regulation. In some cases, the diseased cells are cancerous cells and can be described as translocated or translocated cells (TACs). Exemplary cancers include, but are not limited to, carcinoma, sarcoma, lymphoma, leukemia, and adenoma. Carcinomas can arise from cells lining the inside and outside of the body, such as in the lung, breast, and colon. Sarcomas can occur in bone, cartilage, fat, connective tissue, muscle, and other supportive tissues. Lymphoma can arise from cells that are in the lymph nodes or immune system tissues. Leukemia can develop in the bone marrow and accumulate in the bloodstream. Adenomas can develop in the thyroid, pituitary, and Specific illustrative examples of cancer types include: , suitable for detection by the methods of the present disclosure, including acute lymphoblastic leukemia, acute bone marrow leukemia, Myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, adrenal cancer Intestinal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumors, e.g. cerebellar astrocytoma , cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, Visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt lymphoma, and cancer of unknown primary origin Cancer, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia disease, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplasia small round cell tumor, endometrial cancer, acanthoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, biliary Bladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, Head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma tumors, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver Lung cancer, including non-small cell and small cell lung cancer, lymphoma, leukemia, macroglobulinemia Malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastasis with occult primary Metastatic squamous neck cancer, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia , nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer , oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer , ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell cancer, paranasal sinus and nasal cancer, parathyroid cancer, Penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary Blastocytoma, plasma cell neoplasm, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell Cancer, renal pelvis and ureter transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin Cancer, skin cancer Merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T cell lymphoma Lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (during pregnancy), primary site Cancer of unknown location, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroscopic cancer These include globulinemia and Wilms' tumor.

[0145] In some cases, the diseased cells may be virus-infected cells. Exemplary viruses include HIV. , Hepatitis C virus, Hepatitis B virus, Hepatitis D virus, Herpes virus, EPS Tympanic virus, cytomegalovirus, and human T-lymphotropic virus type III These include, but are not limited to:

[0146] In some cases, the diseased cells may be indicative of an autoimmune disease. Exemplary autoimmune diseases include aca Alopecia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyotrophic lateral sclerosis Loidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune Angioedema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune Inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, auto Immune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN) , Baro's disease, Behcet's disease, benign mucosal emphysema, benign emphysema, Castleman's disease (CD), Achon disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal Osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), or eosinophilic granulomatosis (E GPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, herpes dermatitis, dermatomyositis, Big's disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagus Eosinophilic fasciitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia Arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, polycythemia Granulomatous ductulitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura (HSP), herpes gestationis or pemphigoid gestationis, suppurative sweat glands Acne hives (HS) (anticoagulant type), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis sexually transmitted diseases, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis ( IC), juvenile arthritis, juvenile diabetes (type I diabetes), juvenile myositis (JM), Kawasaki disease, Bennett-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis , linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis ( MPA), mixed connective tissue disease (MCTD), Mullen's ulcer, Mucha-Habermann disease, multifocal Motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, Narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic nerve rheumatism, palindromic rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD) ), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars plana inflammation (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, peripheral Encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome Type I, Type II, Type III, Polymyalgia rheumatica, Multifocal myositis, Post-myocardial infarction syndrome, Pericardium Post-incision syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, Psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, Radicular sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal Membrane fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, Scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), subcutaneous Acute bacterial endocarditis (SBE), Sasac syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis , temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome ( THS), transverse myelitis, type I diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCT) D), uveitis, vasculitis, vitritis, and Vogt-Koyanagi-Harada disease. Not limited to these.

[0147] In some cases, the diseased cells may be indicative of a neurodegenerative disease, such as multiple sclerosis. (MS), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis ( ALS), or Herpesviridae, Polyomaviridae, Bornaviridae, Orthoviridae Myxoviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Pill Neurodegeneration caused by infection with Conavirus or Retrovirus viruses However, it is not limited to these (Zhou et al., Virol J. 2013;10:172).

[0148] Gene / DNA-based therapeutics for diseased cells In some embodiments, the present disclosure provides a method for treating a subject having or suspected of having a disease. A method of treating a subject, comprising: By first inducing the expression of therapeutically effective drugs in disease-related diseased cells, The relative concentration of the therapeutically effective agent expressed by the diseased cells is greater than 1.0, This allows therapeutically effective drugs to be identified at least as determined by a reduction in the cell population of diseased cells. and administering to a subject a composition that treats the subject with at least 10% therapeutic efficacy. do.

[0149] Optionally, the composition is administered to the subject intravenously, subcutaneously, intracerebroventricularly, intrathecally, or intravenously. administration, intracerebroventricular administration, transdermal administration, intramuscular administration, oral administration, inhalation administration, intranasal administration, rectal administration, tumor administration Intratumoral administration or tumor-proximal administration. areas predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes) Intratumoral or proximal approaches may include, for example, endoscopic ultrasound. Testing (e.g., Shirley et al., Gastroenterol Res P ract. 2013;2013:207129) or bronchoscope (e.g., Rojas -Solano et al. J Bronchology Interv Pulm onol. 2018 Jul;25(3):168-175) In some embodiments, the composition is applied to the cervical, epitrochlear, and / or epicondylar areas. supraclavicular, cervical, axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteal lymph node sacs Optionally, lymph node-based administration is administered to at least one of the tissue regions. It may serve as a method of focused local delivery.

[0150] Optionally, administered to treat a subject having or suspected of having a disease. The composition to be prepared is operably linked to a nucleotide sequence encoding a therapeutically effective agent. The promoter may be a cancer-specific promoter. Suitable promoters include natural pan-tumor specific promoters, natural tissue specific promoters, , naturally occurring disease-specific / disease-activated promoters, naturally occurring constitutive promoters, and their The promoter may be any complex of the survivin promoter (BIRC5 ), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABC C4) Promoter, forward gradient 2, protein disulfide isomerase family member Bar (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyl Transferase 3 (B3GNT3) promoter, Cadherin 3 (CDH3) promoter tar, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter ter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter Motor, collagen type XI α1 chain (COL11A1) promoter, collagen type I α 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, dente denticleless E3 ubiquitin protein ligase homolog ( DTL) promoter, sequence similarity 111 member B (FAM111B) promoter Family with, Forkhead box A1 (FOXA1) promoter, kinesin Family member 20A (KIF20A), laminin subunit γ2 (LAMC2) Promoter, Mitotic Spindle Positioning (MISP) Promoter, Matrix Metallo peptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MM P12 promoter, matrix metallopeptidase 13 (MMP13) promoter -, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter ter, phospholipase A2 group IID (PLA2G2D) promoter, G protein Regulatory signaling 13 (RGS13) promoter, secretoglobin family 2A member Bar1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter activator, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 C (UBE2 C), USH1 protein network component harmonin (USH1C), T cell membrane Hexokinase, V-set domain containing cytoplasmic activation inhibitor 1 (VTCN1) promoter Type II promoter, TRPM4 promoter, stromelysin 3 promoter, Surf Actant protein A promoter, secretory leukoprotease inhibitor promoter, Tyrosinase promoter, a stress-inducible grp78 / BiP-containing domain promoter Tar, interleukin-10 promoter, α-B-crystallin / heat shock protein Protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter promoter, mts1 promoter, NSE promoter, somatostatin receptor promoter -, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter promoter, thyroglobulin promoter, α-fetoprotein promoter, villin Promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter tar, fibroblast growth factor promoter; human epidermal growth receptor 2, human telomerase reverse transcription transcriptase promoter; receptor promoter containing kinase domain insert; rad5 1 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, Checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 2 promoter -(ECT2), BCL2-like 12 (BCL2L12) promoter, centromeric protein CENPI promoter, E2F transcription factor 1 (E2F1) promoter, flagella Filamentous adenine dinucleotide synthetase 1 (FLAD1) promoter, protein Sphatase, Mg 2+ / Mn 2+ promoter, ubiquitin dependent 1G (PPM1G) UBE2T promoter, checkpoint kinase 1 (CHEK 1) promoter, epithelial cell transformation 2 promoter (ECT2), BCL2-like 12 (B CL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase FLAD1 promoter, protein phosphatase, Mg 2+ / Mn 2+ Depends PPM1G promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter promoter, aurora kinase A and ninein-interacting protein (AUNIP) promoter -, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL ) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, D SN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxy Thymidylate kinase (DTYMK) promoter, neurite outgrowth 1 (GPRIN1) promoter G protein-regulated inducer of motor, mitochondrial fission regulator 2 (MTFR) 2) Promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small Nuclear ribonucleoprotein polypeptide A′ (SNRPA1) promoter, ATPas e family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic thrombus Checkpoint serine / threonine kinase (BUB1) promoter, calcyclin Cell division cycle-associated 3 (CDCA3) promoter, cell cycle-binding protein (CACYBP) promoter Motor, centromere protein O (CENPO) promoter, flap structure specific Endonuclease 1 (FEN1) promoter, Forkhead box M1 (FOX M1) promoter, cell proliferation Reg protein phosphatase 2A (KIAA1524) Promoter, kinesin family member 2C (KIF2C) promoter, Karyof KPNA2 promoter, MYB proto-oncogene-like 2 (MYBL) 2) Promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein RANBP1 promoter, small nuclear ribonucleoprotein polypeptide B and and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (S PC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) Promoter, TBC1 domain family member 31 (TBC1D31) promoter -, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (Z NF695) promoter, aurora kinase A (AURKA) promoter, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame C17orf53 promoter, Chromobox 3 (CBX30) promoter -, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, Cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin E GF LAG 7-pass G-type receptor 3 (CELSR3) promoter, centromere protein Centrosome A (CENPA) promoter, Centrosome protein 72 (CEP72) promoter Motor, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter , collagen type X α1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L ) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 ( GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, Kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 (KIFC 1) Promoter, minichromosome maintenance 10 replication initiation factor (MCM10) promoter -, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome Chromoplast maintenance complex component 7 (MCM7) promoter, MRG domain binding Methylenetetrahydrofolate dehydrogenase (MRGBP) promoter (NADP+-dependent)2, methenyltetrahydrofolate cyclohydrolase (MTHFD2 ) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter tar, NDC80, kinetochore complex component (NDC80) promoter, Nudix ( nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit NitA (RNASEH2A) promoter, RuvB-like AAA ATPase 1 (R UVBL1) promoter, serologically defined breast cancer antigen NY-BR-85 (SGO L1) promoter, SHC binding and spindle-associated 1 (SHCBP1) promoter, micronucleus Ribonucleoprotein polypeptide G (SNRPG) promoter, timeless (ti meless) circadian regulator promoter, thyroid hormone receptor interaction Tricyclic anti-inflammatory protein 13 (TRIP13) promoter, trophinin-related protein (TROAP) Promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, WD repeat and HMG box DNA binding protein 1 (WDHD1) promoter, α-fetoprotein a promoter of the ribosomal protein (AFP), a fragment thereof, or any combination thereof. It is possible.

[0151] Optionally, operably linked to a nucleotide sequence encoding a therapeutically effective agent. The promoter can be present on a vector, which can be a component of a composition administered to a subject. Suitable vectors include minicircles, plasmids, nanoplasmids, miniintronics Plasmid, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), cosmid, fur including, but not limited to, zymides, bacteriophages, and baculoviruses. Suitable vectors include vectors suitable for administration to cells in vivo (e.g., vectors not specifically intended for use in human genomic DNA). are derived from bacteriophages or plant, invertebrate or animal (including human) viruses vectors, such as CELiD vectors, adeno-associated virus vectors (e.g., A AV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8 -type combinations), retroviral vectors (e.g., MLV or its self-inactivating or or SIN versions, or their pseudoversions), herpes viruses (e.g. , HSV- or EBV-based), lentiviral vectors (e.g., HIV, FIV, or E IAV systems, or pseudotyped versions thereof), or adenoviral vectors (e.g. There are also Ad5-based variants, such as replication-deficient, replication-competent, or helper-dependent versions. In some cases, the vector may contain one or more sequences, such as a scaffold / matrix attachment region (S / MAR). It may contain episomal maintenance elements to facilitate replication in the target cell type. S / MAR elements are particularly useful to facilitate replication in the context of "naked" nucleic acid vectors such as minicircles. Exemplary suitable S / MAR elements include EμMA from the immunoglobulin heavy chain locus. R, apoB MAR from the human apolipoprotein B locus, chicken lysozyme gene Ch-LysMAR from the gene locus and huIFNβ MA from the human IFNβ locus Examples include, but are not limited to, R. In some embodiments, the vector is a non- It can be a viral vector.

[0152] In some cases, the therapeutically effective agent may include a specific type of therapeutic agent. Exemplary classes of therapeutic agents suitable for use include therapeutically effective polypeptides (e.g., therapeutic Antibodies, fragments, or derivatives thereof; cytokines; growth factors; engineered or substituted protease inhibitors catabolic / catabolic enzymes, engineered short peptide agonists or antagonists, or prodrugs activating enzymes), small activating RNAs (saRNAs), microRNAs (miRNAs), small interfering RNA (siRNA), or any combination thereof. In some cases, the therapeutically active agent may be a prodrug-activating enzyme. Exemplary prodrug-activating enzymes include HSVtk, cytosine deaminase, DT dimerase, phosphodiesterase, nitroreductase, guanine phosphoribosyltransferase, purine Creoside phosphorylase, thymidine phosphorylase, carboxylesterase, phosphatase Polyglutamyl synthetase, carboxypeptidase A1, carboxypeptidase These include, but are not limited to, enzyme G2, and cytochrome P-450. When the agent is a prodrug-activating enzyme, the method can involve any of the routes described herein. If the therapeutically effective agent is a polypeptide, The polypeptide may comprise an N-terminal secretory signal sequence (e.g., the N-terminal secretory signal sequence from CD33 or CD8a). signal peptide).

[0153] Improved synthetic biomarker constructs and accurate transfection rates for individual subjects Methods for normalizing In some aspects, the present disclosure provides a method for detecting a first polypeptide or nucleic acid biomarker. a first nucleic acid sequence encoding a second polypeptide or a second nucleic acid biomarker; and a second nucleic acid sequence, wherein the composition, when the composition is in a cell, The second polypeptide or second nucleic acid biomarker is a polypeptide of at least one of the first and second nucleic acids. and the first polypeptide or nucleic acid biomarker is expressed in an amount reflective of delivery to the cell, ... The present invention provides a composition that is differentially expressed in diseased and non-diseased cells. Optionally, (i) the cells expression of the first nucleic acid sequence in diseased cells; expression of the first nucleic acid sequence in non-diseased cells; The first polypeptide is a detectable biomarker or a therapeutic agent. and (ii) the cells induce expression of the second nucleic acid sequence equally in diseased and non-diseased cells. and the second nucleic acid sequence is a second polypeptide that is not a detectable biomarker or a therapeutic agent. The expression level of the second polypeptide is determined by the relative level of the nucleic acid sequence in the cell. Optionally, a nucleic acid sequence encoding the first polypeptide provides a control for assessing the level of The first nucleic acid sequence and the second nucleic acid sequence encoding the second polypeptide are separate genetic constructs. Optionally, the composition may include a first polypeptide encoding the first polypeptide. and a second nucleic acid sequence encoding a second polypeptide are independent. Optionally, the vector can be on a genetic construct. (a) operably linked to a first nucleic acid sequence a linked first promoter, wherein the promoter is a first promoter in non-diseased cells; a first nucleic acid sequence that induces expression of the first nucleic acid sequence in diseased cells preferentially over expression of the second nucleic acid sequence; and (b) a promoter that induces expression equally in diseased and non-diseased cells and a second nucleic acid and a second promoter sequence operably linked to the

[0154] Optionally, the first polypeptide is both a detectable biomarker and a therapeutic agent. Optionally, the first polypeptide is a therapeutic antibody, a therapeutic antibody fragment or derivative, or a prodrug-activating enzyme. Exemplary prodrug-activating enzymes include HSVtk , cytosine deaminase, DT diaphorase, nitroreductase, guanine phosphoribosidase Silyltransferase, purine nucleoside phosphorylase, thymidine phosphorylase , carboxylesterase, folylpolyglutamyl synthetase, carboxypeptide These include carboxypeptidase A1, carboxypeptidase G2, and cytochrome P-450. The polypeptide may include, but is not limited to, an N-terminal secretory signal sequence (e.g., CD33 or an N-terminal signal peptide from CD8a).

[0155] Optionally, the first and / or second nucleic acid may be on a vector. Suitable vectors include , minicircle, plasmid, nanoplasmid, miniintronic plasmid, yeast YAC, bacterial artificial chromosome (BAC), cosmid, phagemid, bacterio In vivo expression vectors, such as, but not limited to, phage and baculovirus Suitable vectors include vectors suitable for administration to cells in vivo. or animal (including human) viruses such as plants, invertebrates, or CELiD vectors vectors derived from viruses, such as adeno-associated virus vectors (e.g., AAV1, AAV 2. AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV2 / 5 , AAV2 / 2, AAV-DJ, or AAV-DJ8, or their pseudotype combinations. retroviral vectors (e.g., MLV or its self-inactivating or SIN vectors) john, or pseudoversions thereof), herpes viruses (e.g., HSV- or EBV-based), lentiviral vectors (e.g., HIV, FIV, or EIAV-based, or or adenoviral vectors (e.g., replication-deficient, multiplying, or pseudotyped versions thereof) There are also Ad5-based versions (such as the Ad5-based version, which is a feature-rich or helper-dependent version). The target is a region that replicates in one or more target cell types, such as a scaffold / matrix-associated region (S / MAR). S / MAR elements may contain episomal maintenance elements to facilitate replication. It is particularly useful in the context of any "naked" nucleic acid vector to facilitate replication. The most common S / MAR elements include EμMAR from the immunoglobulin heavy chain locus, human apolipoprotein B1 apoB MAR from the protein B locus, Ch- from the chicken lysozyme locus LysMAR and huIFNβ MAR from the human IFNβ locus. In some embodiments, the vector is a non-viral vector. It can be.

[0156] Optionally, the cells to which the first and second nucleic acids are delivered may be diseased cells. Diseased cells include cancerous cells, cells exhibiting autoimmune disease (e.g., cells with autotropic activity), and T cells or lymphocytes, or normal cells damaged by autoimmunity), TACs, or Cells exhibiting degenerative disease (e.g., cells with toxic amyloid or cells expressing toxic amyloid) Such cells may be present in cancer, autoimmune diseases, and neurodegenerative diseases, including any of the cancers, autoimmune diseases, and neurodegenerative diseases described herein. In some cases, the diseased cells may be virally infected cells. Viruses include HIV, Hepatitis C, Hepatitis B, Hepatitis D, and herpes. Viruses, Epstein-Barr virus, cytomegalovirus, and human T lymphocyte-specific Examples include, but are not limited to, HIV type III.

[0157] Optionally, the first or second nucleic acid can be a detectable nucleic acid biomarker. Potentially detectable nucleic acids include natural or engineered miRNAs, RNA hairpins, and RNA These include, but are not limited to, aptamers or barcoded versions thereof. When the nucleic acid is a miRNA, the miRNA can be annealed using, for example, a degenerate primer. and ligation, poly(A) polymerase labeling followed by RT or ligation or sequential adapters in combination with q-PCR, sequencing or electrophoretic detection methods This can be detected by standard library generation techniques such as ligation. If the marker is a polypeptide, the polypeptide may contain an N-terminal secretory signal sequence (e.g. For example, the N-terminal signal peptide from CD33 or CD8a.

[0158] When the nucleic acid is an engineered miRNA, the nucleic acid may be prepared using the method described by Ronald et al. al., PLoS ON E 11(7):e0159369) It can be a Sec-miR or miR-neg construct. Such a construct can be a) A coding sequence that is not endogenously expressed and has no known vertebrate target (e.g., Sec- miR 5′-AAAUGUACUGCGCGUGGAGAC-3′); (b) the miRNAs that provide processing of pre-miRNAs into mature miRNAs flanked by miRNA sequences R backbone sequence (e.g., miR-155 or miR-130 backbone sequence); (c) Contains an EXO motif that enhances loading into exosomes (e.g., GGAG). Such miRNA constructs can be used, for example, to encode a gene encoding a reporter polypeptide. in the 3′-UTR of a protein or in an appropriately non-toxic protein (e.g., actin or tubulin). Any endogenous structural protein or highly expressed protein such as ubiquitin In some embodiments, the gene can be expressed from the 3'-UTR of the gene it manipulates. Multiple copies (e.g., at least two, at least four) of a miRNA are tandemly linked. It can be provided in

[0159] Optionally, the second polypeptide or the first polypeptide is administered to the subject. These non-invasive imaging methods can be used to detect the These include MRI imaging, PET imaging, SPECT imaging, and photoacoustic imaging. MRI imaging detectable compounds. Biomarkers include ferritin (or Pyrococcus furiosus) us furiousus) ferritin mutants such as L55P, F57S, and F123S or mutants thereof), or lanthanide-binding proteins (or Daughtry et al., LBT- described in ChemBioChem 2012, 13, 2567-2574 and polypeptide imaging agents such as ubiquitin fusions (and engineered fusions thereof, such as ubiquitin fusions). Synthetic biomarkers detectable by T or SPECT imaging include human sodium iodide in combination with administration of iodine / iodide isotopes in PET, e.g. Penheiter et al. Curr Gene Ther. 2012 F eb;12(1):33-47), HSV-tk or HSV-sr39tk its variants (e.g., positron-labeled acycloguanosine such as [18F]FHBG or in combination with the administration of a pyrimidine analogue PET reporter, Yaghoubi SS et al., Nat Protoc. 2006;1(6):3069-75), and and dopamine D2 receptors or variants thereof, such as D2R80A or D2R194A (e.g., For example, positron-labeled D2 such as 3-(2'-[18F]-fluoroethyl)-spiperone In combination with the administration of a binding agent, etc. Synthesis detectable by photoacoustic imaging Biomarkers include β-galactosidase (e.g., in combination with X-gal administration) and pigment-producing enzymes such as tyrosinase, autofluorescent proteins (e.g., GFP, mCherry) , or derivatives thereof), non-fluorescent GFP-like chromoproteins (e.g., aeCP597 and cj Blue and their derivatives), near-infrared fluorescent proteins based on bacteriophytochrome Quality (e.g., IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRF P713, iRFP720, iRFP713 / V256C, iRFP682, iR FP702, iRFP670, mIFP, iBlueberry, GAF-FP, Bph P1-FP / C20S, or AphB variant), and reversibly photoswitchable Protein (Dronpa, Dronpa-M159T, BphP1 or variants thereof) Synthetic biomarkers that can be detected by bioluminescence imaging include: Gaussia luciferase, Renilla luciferase, and Photinus luciferase ( See, for example, Branchini et al., Anal. Biochem. 396 (2010): 290-297, including the RE8 and RE9 versions of Ppy ), such as luciferases (e.g., in combination with the administration of coelenterazine as described herein) In some embodiments, the synthetic biomarkers may be used as imaging agents, detectable An enzyme that produces a molecule or a transporter that drives the accumulation of a detectable molecule Synthetic biomarkers can be measured in situ within the subject's body. .

[0160] Optionally, the present disclosure provides a method for treating diseased cells in a subject, comprising administering the composition to the subject. a method for detecting a cell, the method comprising: detecting a first polypeptide or nucleic acid biomarker; a first nucleic acid sequence encoding a second polypeptide or a second nucleic acid biomarker; a second nucleic acid sequence that encodes the second polypeptide, wherein the composition is present in a cell. or the second nucleic acid biomarker reflects delivery of at least the first and second nucleic acids to the cell. The composition is configured to express a first polypeptide or nucleic acid biomarker in an amount that satisfies the expression level of the first polypeptide or nucleic acid biomarker. The method further provides a method in which the marker is differentially expressed in diseased and non-diseased cells. (i) detecting expression of a first nucleic acid sequence in diseased cells and a first nucleic acid sequence in non-diseased cells; The first polypeptide preferentially induces expression of a detectable biomarker or is a therapeutic agent; and (ii) the cells express a second nucleic acid sequence equally in diseased and non-diseased cells. and the second nucleic acid sequence induces expression of a second nucleic acid sequence that is not a detectable biomarker or therapeutic agent. and the expression level of the second polypeptide is increased by the nucleic acid in the cell, thereby producing a polypeptide of Optionally, the first polypeptide is a control for assessing the relative levels of the sequence. The first nucleic acid sequence encoding the second polypeptide and the second nucleic acid sequence encoding the second polypeptide are independently Optionally, the first polypeptide may be present in the composition on a genetic construct. a first nucleic acid sequence encoding a second polypeptide and a second nucleic acid sequence encoding a second polypeptide; can be on separate genetic constructs. Optionally, the vector comprises (a) a first nucleic acid sequence a first promoter operably linked to a expression of the first nucleic acid sequence in the diseased cells preferentially over expression of the first nucleic acid sequence in the diseased cells. (b) a first promoter that induces expression equally in diseased and non-diseased cells; and , a second promoter sequence operably linked to the second nucleic acid. The method comprises: detecting a first polypeptide or nucleic acid biomarker and / or a second polypeptide or nucleic acid biomarkers. Optionally, such methods include detecting A non-invasive imaging method is performed on a subject. Imaging techniques include MRI imaging, PET imaging, SPECT imaging, and photoacoustic imaging. These include echo imaging, and bioluminescence imaging.

[0161] Synthetic biomarkers are polypeptide biomarkers that can be detected by non-invasive imaging methods If the marker, the method further comprises locating diseased cells in the subject. The positioning may be performed to a certain resolution, for example, 10 mm to 10 cm, at least 10 m. m, or up to 10 cm. Size, e.g. 3mm 3 ~10cm 3 This may be related to the size of the tumor during The minimum range for setting is 1cm 3 ~10cm 3 , or 900mm 3 ~1cm 3 , or 800mm 3 ~900mm 3 , or 700mm 3 ~800mm 3 , or 600mm 3 ~700mm 3 , or 500mm 3 ~600mm 3 , or 400mm 3 ~500mm 3 , or 300mm 3 ~400mm 3 , or 200mm 3 ~300mm 3 , or 100mm 3 ~200mm 3 , or 50mm 3 ~100mm 3 , or 10 mm 3 ~50mm 3 , or 3 mm 3 ~10m m 3 Optionally, non-invasive imaging scans (PET, MRI) , SPECT, etc.) and possibly during in situ surgical intervention. For example, use of visual inspection (for visual range absorption reporters) or in combination with fluorescence excitation. Positioning is accomplished by the use of enhanced visual inspection.

[0162] Optionally, after the additional positioning step described above, the detected and / or positioned A surgical step may be performed to remove diseased cells. The surgical step may involve biomarker detection. and / or administering a composition encoding the same or a part of the subject who is responsible for locating diseased cells. The surgical steps may be performed by different parties. The surgical or non-surgical removal step can include radiation therapy. Department (Gamma Knife, Reflexion, Cyberknife, and targeted ionizing radiation) Minimally invasive killing techniques such as, but not limited to, related techniques that kill diseased cells May be involved.

[0163] Optionally, the non-invasive imaging method comprises administering to the subject a composition that induces expression of a synthetic biomarker. The non-invasive imaging method may be performed a period of time after the administration of the first and second At least about 15 minutes, at least about 30 minutes, at least about 1 minute, at least about 1 minute after administration of the composition comprising the nucleic acid hours, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 1 hour 6 hours, at least about 24 hours, at least about 36 hours, at least about 48 hours, or In all cases, the range is about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, or at least about 7 days. days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, At least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, At least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, It can be performed in at least about 5 months, at least about 6 months, or at least about 1 year. The non-invasive imaging method may be performed for up to 10 minutes after administration of a composition comprising the first and second nucleic acids. Approximately 15 minutes, up to approximately 30 minutes, up to approximately 1 hour, up to approximately 2 hours, up to approximately 4 hours, up to Approximately 8 hours, up to approximately 16 hours, up to approximately 24 hours, up to approximately 36 hours, up to approximately 48 hours , up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days days, up to about 14 days, up to about 15 days, up to about 1 month, up to about 2 months, up to about 3 months It can be carried out in about 1 month, about 4 months at the most, about 5 months at the most, about 6 months at the most, or about 1 year at the most. In some embodiments, the non-invasive imaging method comprises detecting the first and second nucleic acids. The method can be performed multiple times after administration of a composition comprising the compound (e.g., a synthetic biomarker level test). The non-invasive imaging method is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, The non-invasive imaging method may be performed 15, 20, or 25 times. The administration of the composition comprising the first nucleic acid and the second nucleic acid can be performed weekly or monthly.

[0164] Optionally, the first polypeptide or nucleic acid biomarker and / or the second polypeptide The nucleic acid biomarkers can be detected in a biological sample from a subject. A biological sample can be a sample obtained by non-invasive means from a subject. Exemplary non-invasive samples include saliva, sputum, sweat, urine, stool, semen, cervicovaginal secretions, breast milk, These include, but are not limited to, mucosal secretions, tears, and buccal swabs. The sample can be a sample collected from a subject by a minimally invasive method. Suitable minimally invasive samples include blood samples (e.g., obtained by venipuncture or capillary tubes) pleural fluid samples (e.g., obtained by thoracentesis); amniotic fluid samples (e.g., obtained by ovine samples (obtained by hydrocentesis), and gastric fluid samples (obtained by amniocentesis, for example). (e.g., obtained by gastric lavage). Skin biopsy samples (e.g., punch biopsy, shave biopsy, trough scrape biopsy, wedge biopsy, incision biopsy) biopsy, or excision biopsy), bone marrow sample (obtained by aspiration biopsy, for example), lymph node or breast biopsy (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy, or imaging) biopsy samples (obtained by image-guided biopsy), surgical biopsy samples (e.g., obtained by excision or incisional biopsy), of internal organs obtained by smearing), or biopsy samples of the mouth, gastrointestinal tract, lung, bladder or urinary tract (e.g. It can be a sample obtained by biopsy (e.g., obtained by endoscopy). Optionally, the biological sample may be analyzed by administering a composition that induces expression of a synthetic biomarker. The biological sample can be obtained after a period of time from a composition comprising the first and second nucleic acids. at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least about 1 hour after administration of about 2 hours, at least about 4 hours, at least about 8 hours, at least about 16 hours, at least at least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 1 month month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months The biological sample can be obtained within about six months, or at least about six months. at most about 15 minutes, at most about 30 minutes, at most about 1 hour, at most about 2 hours, after administration of a composition comprising Hours, Maximum approx. 4 hours, Maximum approx. 8 hours, Maximum approx. 16 hours, Maximum approx. 24 hours, Maximum approx. 36 hours, maximum approximately 48 hours, maximum approximately 3 days, maximum approximately 4 days, maximum approximately 5 days, maximum approximately 6 days, maximum approximately approximately 7 days, up to approximately 8 days, up to approximately 9 days, up to approximately 10 days, up to approximately 11 days, up to approximately 12 days, Up to about 13 days, up to about 14 days, up to about 15 days, up to about 1 month, up to about 2 months, It can be obtained in a maximum of approximately 3 months, 4 months, 5 months, or 6 months. In some embodiments, a biological sample can be obtained and any biomarkers can be analyzed. performing the car detection protocol multiple times after administration of the composition comprising the first and second nucleic acids; (e.g., to monitor synthetic biomarker levels over time). The sample may be collected at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 5, 6, 7, 8, 9, 10, 15, 20, or 25 times the biological sample can be obtained. The administration of the composition comprising the first and second nucleic acids can be given weekly or monthly.

[0165] Optionally, the method further comprises detecting the first or second nucleic acid biomarker by a particular nucleic acid detection method. The first or second nucleic acid biomarker may be detected by sequencing. Sequencing methods include next-generation sequencing, high-throughput sequencing, and pipetting. Sequencing, traditional Sanger sequencing, sequencing by ligation, sequencing by synthesis Sequencing, sequencing by hybridization, RNA-Seq (Illumina ), digital gene expression (Helicos), next-generation sequencing, single molecule sequencing by synthesis determination (SMSS) (Helicos), Ion Torrent sequencing machine (Life Technologies / Thermo-Fisher), massively parallel sequencing, Lone single molecule array (Solexa), shotgun sequencing, Maxim-Gilb These include ert sequencing, and primer walking.

[0166] Optionally, the first or second nucleic acid biomarker is detected by quantitative PCR (qPCR) or T It can be detected by "real-time amplification" methods, also known as aqman (e.g., G U.S. Patent No. 5,210,015 to Elfand and U.S. Patent No. 5,538,888 to Livak et al. No. 48, and No. 5,863,736 to Haaland, and Heid, CA et al., Genome Research, 6:986-994(1996 );Gibson, UEM et al., Genome Research 6:995-1001(1996);Holland, PM, et al., P roc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, KJ et al., PCR Methods and Applications 357-362(1995)). Formation of amplification products The basis of this method of monitoring is the use of dual-labeled fluorogenic oligonucleotide probes. The purpose of such an assay is to continuously measure PCR product accumulation using a Probes are typically short (approximately 20-25 bases) labeled with two different fluorescent dyes. The 5' end of the probe is typically conjugated to a reporter dye, and the 3' end is The ' end is bound to a quencher dye. The probe binds to the target mRNA or a nucleic acid derived from it. The sequence of the gene locus is designed to have at least substantial sequence complementarity with the region of the gene locus. Upstream and downstream PCR primers that bind to the region are also added to the reaction mixture. When intact, energy transfer between the two fluorophores occurs, and the quencher is released from the reporter. During the extension step of PCR, the probe is quenched by a nucleic acid polymerase such as Taq polymerase. The 5' nuclease activity of the polymerase cleaves the polynucleotide The reporter is released from the quencher, resulting in an increase in reporter luminescence intensity, which is suitable for detection. The recorded values ​​can then be used to calculate the normalized reporter -The increase in luminescence intensity can be continuously calculated, and the amount of mRNA amplified can be quantified. can.

[0167] In some embodiments, for qPCR or Taqman detection, an RT-PCR step is performed first. First, cDNA can be generated from cellular RNA by RT-PCR. Such amplification can be achieved using general (e.g., partially / fully degenerate) oligonucleotide primers. amplification by a gene) or targeted (e.g., directed against a specific gene to be analyzed at a later stage). amplification with directed oligonucleotide primers).

[0168] In some embodiments, qPCR or Taqman is performed on mRNA of isolated cells. This diversity can be used immediately after the reverse transcriptase reaction performed on the individual This is useful for quantifying individual mRNA levels.

[0169] In some embodiments, for qPCR or Taqman detection or RNA sequencing Alternatively, a "pre-amplification" step can be performed first on cDNA transcribed from cellular RNA. This allows for a signal to be detected under conditions where the natural level of RNA / cDNA is very low. Suitable preamplification methods include LM-PCR, random orientation PCR, and PCR using oligonucleotide primers (e.g., random hexamer PCR), PCR using A-specific primers, and any combination thereof. Preamplification can be general or targeted in the same manner as the reverse transcription reaction described above. It is possible.

[0170] RNA levels can also be measured using, for example, Panomics' QuantiGene® Using a branched nucleic acid probe such as a reagent system, hybridization to the probe Therefore, measurement can be performed without amplification.

[0171] Heterodimer-based synthetic biomarker design In some aspects, the disclosure provides a first nucleic acid sequence encoding a first polypeptide; A composition comprising a second nucleic acid sequence encoding a second polypeptide, the composition comprising: When the composition is in a cell, (i) the cell expresses the first nucleic acid sequence to produce the first polypeptide. (ii) the cell expresses the second nucleic acid sequence to produce the second polypeptide; and (iii) the first polypeptide and the second polypeptide expressed by the cell are linked and a composition configured to form a heterodimeric protein. Alternatively, the first polypeptide and the second polypeptide may be on separate genetic constructs. In some cases, the first polypeptide and the second polypeptide are on separate genetic constructs. obtain.

[0172] Optionally, the heterodimeric protein is a derivative of a naturally occurring heterodimer, or a complementary heterodimer of two The fragment may be a naturally occurring enzyme split into two halves of a typical polypeptide or an autofluorescent protein. Examples of such systems include the FRB / FKBP12 heterodimer, Split L. lucifera These include, but are not limited to, a ribosomal enzyme protein, or a split GFP protein. .

[0173] If the heterodimeric protein may be a derivative of a natural heterodimer (e.g. , FRB / FKBP12 pair), each half of the heterodimeric protein contains an enzyme or a detection pair is linked to the complementary half of the heterodimer, whereby dimerization of the heterodimer activates the enzyme, or Optionally, each half of the heterodimeric protein is capable of detecting its When activity is reconstituted by dimerization of the heterodimer, it can activate the expression of another element. A splitting agent such as Cre recombinase (e.g., a synthetic biomarker or therapeutic molecule) Possibly linked to a tricombinase. Optionally, a heterodimeric protein Each half of the protein is linked to one of two autofluorescent proteins that form a FRET pair. whereby FRET can be detected when heterodimers are formed. obtain.

[0174] Optionally, the first nucleic acid sequence and the second nucleic acid sequence are a first genetic element and a second genetic element. A first genetic element and a second genetic element can be operably linked to a child element. Both are selectively activated to inhibit expression of the first and second polypeptides in the same diseased cell type. The first or second genetic element may be a promoter, an enhancer, or an miRNA. Exemplary promoters include the survivin promoter (BIRC5 ), CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABC C4) Promoter, forward gradient 2, protein disulfide isomerase family member Bar (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter UDP-GlcNAc:βGal β-1,3-N-acetylglucosaminyl Transferase 3 (B3GNT3) promoter, Cadherin 3 (CDH3) promoter tar, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter ter, claudin 3 (CLDN3) promoter, claudin 4 (CLDN4) promoter Motor, collagen type XI α1 chain (COL11A1) promoter, collagen type I α 1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, dente denticleless E3 ubiquitin protein ligase homolog ( DTL) promoter, sequence similarity 111 member B (FAM111B) promoter Family with, Forkhead box A1 (FOXA1) promoter, kinesin Family member 20A (KIF20A), laminin subunit γ2 (LAMC2) Promoter, Mitotic Spindle Positioning (MISP) Promoter, Matrix Metallo peptidase 1 (MMP1) promoter, matrix metallopeptidase 12 (MM P12 promoter, matrix metallopeptidase 13 (MMP13) promoter -, mesothelin (MSLN) promoter, cell surface-associated mucin 1 (MUC1) promoter ter, phospholipase A2 group IID (PLA2G2D) promoter, G protein Regulatory signaling 13 (RGS13) promoter, secretoglobin family 2A member Bar1 (SCGB2A1) promoter, topoisomerase IIα (TOP2A) promoter activator, ubiquitin D (UBD) promoter, ubiquitin-conjugating enzyme E2 C (UBE2 C), USH1 protein network component harmonin (USH1C), T cell membrane Hexokinase, V-set domain containing cytoplasmic activation inhibitor 1 (VTCN1) promoter Type II promoter, TRPM4 promoter, stromelysin 3 promoter, Surf Actant protein A promoter, secretory leukoprotease inhibitor promoter, Tyrosinase promoter, a stress-inducible grp78 / BiP-containing domain promoter Tar, interleukin-10 promoter, α-B-crystallin / heat shock protein Protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter promoter, mts1 promoter, NSE promoter, somatostatin receptor promoter -, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter promoter, thyroglobulin promoter, α-fetoprotein promoter, villin Promoter, albumin promoter, glycoprotein A33 promoter, B cell specific Moloney leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter tar, fibroblast growth factor promoter; human epidermal growth receptor 2, human telomerase reverse transcription transcriptase promoter; receptor promoter containing kinase domain insert; rad5 1 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) promoter, Checkpoint kinase 1 (CHEK1) promoter, epithelial cell transformation 2 promoter -(ECT2), BCL2-like 12 (BCL2L12) promoter, centromeric protein CENPI promoter, E2F transcription factor 1 (E2F1) promoter, flagella Filamentous adenine dinucleotide synthetase 1 (FLAD1) promoter, protein Sphatase, Mg 2+ / Mn 2+ promoter, ubiquitin dependent 1G (PPM1G) UBE2T promoter, checkpoint kinase 1 (CHEK 1) promoter, epithelial cell transformation 2 promoter (ECT2), BCL2-like 12 (B CL2L12) promoter, centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase FLAD1 promoter, protein phosphatase, Mg 2+ / Mn 2+ Depends PPM1G promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter promoter, aurora kinase A and ninein-interacting protein (AUNIP) promoter -, cell division cycle 6 (CDC6) promoter, centromere protein L (CENPL ) promoter, DNA replicative helicase / nuclease 2 (DNA2) promoter, D SN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxy Thymidylate kinase (DTYMK) promoter, neurite outgrowth 1 (GPRIN1) promoter G protein-regulated inducer of motor, mitochondrial fission regulator 2 (MTFR) 2) Promoter, RAD51-associated protein 1 (RAD51AP1) promoter, small Nuclear ribonucleoprotein polypeptide A′ (SNRPA1) promoter, ATPas e family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic thrombus Checkpoint serine / threonine kinase (BUB1) promoter, calcyclin Cell division cycle-associated 3 (CDCA3) promoter, cell cycle-binding protein (CACYBP) promoter Motor, centromere protein O (CENPO) promoter, flap structure specific Endonuclease 1 (FEN1) promoter, Forkhead box M1 (FOX M1) promoter, cell proliferation Reg protein phosphatase 2A (KIAA1524) Promoter, kinesin family member 2C (KIF2C) promoter, Karyof KPNA2 promoter, MYB proto-oncogene-like 2 (MYBL) 2) Promoter, NIMA-related kinase 2 (NEK2) promoter, RAN-binding protein RANBP1 promoter, small nuclear ribonucleoprotein polypeptide B and and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (S PC24) promoter, transforming acidic coiled-coil-containing protein 3 (TACC3) Promoter, TBC1 domain family member 31 (TBC1D31) promoter -, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (Z NF695) promoter, aurora kinase A (AURKA) promoter, BLM RecQ-like helicase (BLM) promoter, chromosome 17 open reading frame C17orf53 promoter, Chromobox 3 (CBX30) promoter -, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter promoter, cyclin F (CCNF), cell division cycle 20 (CDC20) promoter, Cell division cycle 45 (CDC45) promoter, cell division cycle associated 5 (CDCA5) promoter cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin E GF LAG 7-pass G-type receptor 3 (CELSR3) promoter, centromere protein Centrosome A (CENPA) promoter, Centrosome protein 72 (CEP72) promoter Motor, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter , collagen type X α1 chain (COL10A1) promoter, chromosome segregation 1-like (CSE1L ) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 ( GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, Kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter, kinesin family member C1 (KIFC 1) Promoter, minichromosome maintenance 10 replication initiation factor (MCM10) promoter -, minichromosome maintenance complex component 2 (MCM2) promoter, minichromosome Chromoplast maintenance complex component 7 (MCM7) promoter, MRG domain binding Methylenetetrahydrofolate dehydrogenase (MRGBP) promoter (NADP+-dependent)2, methenyltetrahydrofolate cyclohydrolase (MTHFD2 ) promoter, non-SMC condensin I complex subunit H (NCAPH) promoter tar, NDC80, kinetochore complex component (NDC80) promoter, Nudix ( nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit NitA (RNASEH2A) promoter, RuvB-like AAA ATPase 1 (R UVBL1) promoter, serologically defined breast cancer antigen NY-BR-85 (SGO L1) promoter, SHC binding and spindle-associated 1 (SHCBP1) promoter, micronucleus Ribonucleoprotein polypeptide G (SNRPG) promoter, timeless (ti meless) circadian regulator promoter, thyroid hormone receptor interaction Tricyclic anti-inflammatory protein 13 (TRIP13) promoter, trophinin-related protein (TROAP) Promoter, ubiquitin-conjugating enzyme E2 C (UBE2C) promoter, WD repeat and HMG box DNA binding protein 1 (WDHD1) promoter, α-fetoprotein and the like, such as an AFP promoter, a fragment thereof, or any combination thereof. Exemplary miRNA binding sites include, but are not limited to, at least Another miR-15, miR-16, let-7, miR-122, or miR-34 binding Examples include, but are not limited to, fusion sequences.

[0175] Optionally, the genetic constructs encoding the first and second polypeptides are contained in a vector. Exemplary vectors include any of the vectors described herein.

[0176] In some aspects, the present disclosure provides a method for detecting or treating diseased cells comprising administering a composition. a first nucleic acid sequence encoding a first polypeptide; a second nucleic acid sequence encoding a second polypeptide, wherein the composition is When in the cell, (i) the cell expresses the first nucleic acid sequence to produce the first polypeptide. (ii) the cell expresses a second nucleic acid sequence to produce the second polypeptide; (iii) the first polypeptide and the second polypeptide expressed by the cell; and a method for preparing a heterodimeric protein comprising: Optionally, the composition is administered to the subject intravenously, subcutaneously, intracerebroventricularly, intrathecally, or intravenously. Intravenous administration, intracerebroventricular administration, transdermal administration, intramuscular administration, oral administration, inhalation, intranasal administration, rectal administration, Intratumoral administration or tumor-proximal administration. Tumor-proximal administration means administration to tissues within the vicinity of the tumor. or areas predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent Intratumoral or tumor-proximal approaches may include, for example, intra- and intra-ultrasound approaches. Endoscopy (e.g., Shirley et al., Gastroenterol Res Pract. 2013;2013:207129) or bronchoscope (e.g., Roj as-Solano et al. J Bronchology Interv Pu Ilmonol. 2018 Jul;25(3):168-175) The use of imaging techniques may be involved. In some embodiments, the composition is administered to the cervical, trochlear, Epicondylar, supraclavicular, cervical, axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteal lymph nodes Optionally, lymph node-based administration is administered to at least one of the tissue regions. This may serve as a method of focused local delivery to the The method may further comprise detecting the monodimeric protein.

[0177] In some cases, the detecting includes a non-invasive detection method performed on the subject. Invasive detection methods (e.g., in the case of autofluorescence or photoproteins) include SPECT imaging. These imaging techniques include, but are not limited to, fluoroscopy and bioluminescence imaging. The method comprises administering a composition encoding a heterodimeric protein to the subject for at least about 15 minutes, followed by administration of the composition encoding the heterodimeric protein. At least about 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, At least about 8 hours, at least about 16 hours, at least about 24 hours, at least about 36 hours at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, At least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least at least about 14 days, at least about 15 days, at least about 1 month, at least about 2 months, at least at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, or less This imaging method can be performed in at least about one year. up to about 15 minutes, up to about 30 minutes, up to about 1 hour, up to about 1 hour after administration of a composition encoding 2 hours, maximum 4 hours, maximum 8 hours, maximum 16 hours, maximum 24 hours, Maximum approx. 36 hours, Maximum approx. 48 hours, Maximum approx. 3 days, Maximum approx. 4 days, Maximum approx. 5 days, Maximum 6 days, maximum approx. 7 days, maximum approx. 8 days, maximum approx. 9 days, maximum approx. 10 days, maximum approx. 1 1 day, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 1 months, up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, up to about 6 months In some embodiments, the image can be generated in about 1 month, or up to about 1 year. The sizing method may be performed multiple times after administration of a composition encoding a heterodimeric protein. (e.g., to monitor synthetic biomarker levels over time). The imaging method comprises administering a composition encoding a heterodimeric protein to a subject, the subject being at least about Can be performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 times. The imaging method can be performed weekly after administration of the composition encoding the heterodimeric protein. It can be conducted once or once a month.

[0178] Optionally, detecting includes detecting the heterodimeric protein in a biological sample from the subject. The biological sample may be a sample obtained by a non-invasive method from the subject. Exemplary non-invasive samples include saliva, sputum, sweat, urine, feces, semen, and cervicovaginal secretions. These include, but are not limited to, breast milk, mucosal secretions, tears, and buccal swabs. The biological sample can be a sample collected from a subject by a minimally invasive method. Exemplary minimally invasive samples include blood samples (e.g., obtained by venipuncture or capillary tube). pleural fluid samples (e.g., obtained by thoracentesis), amniotic fluid samples (e.g., obtained by thoracentesis), samples of gastric juice (e.g., obtained by amniocentesis) and gastric juice samples (e.g., obtained by amniocentesis) Examples include, but are not limited to, gastric lavage. Samples may be skin biopsy samples (e.g., punch biopsy, shave biopsy, trough scrape biopsy, wedge biopsy) biopsy, incisional biopsy, or excisional biopsy), bone marrow samples (e.g., obtained by aspiration biopsy), obtained by fine needle aspiration, core needle biopsy, vacuum-assisted biopsy), lymph node or breast biopsy (e.g., fine needle aspiration, core needle biopsy, vacuum-assisted biopsy) or image-guided biopsy), surgical biopsy samples (e.g., excision or incision) Biopsy samples of internal organs (obtained by biopsy) or biopsy samples of the mouth, gastrointestinal tract, lungs, bladder or urinary tract A sample obtained by biopsy, such as a sample obtained by endoscopy It is possible.

[0179] Optionally, the biological sample is derived from administration of a composition that induces expression of a synthetic biomarker. The biological sample can be obtained after a period of time. At least about 15 minutes, at least about 30 minutes, at least about 1 hour, or at least about 1 hour after administration of the composition. at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 16 hours, At least about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days , at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, At least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least The biological sample can be obtained in about 5 months, or at least about 6 months. up to about 15 minutes, up to about 30 minutes, up to about 1 minute, Hours, up to about 2 hours, up to about 4 hours, up to about 8 hours, up to about 16 hours, up to about 24 hours, up to approximately 36 hours, up to approximately 48 hours, up to approximately 3 days, up to approximately 4 days, up to approximately 5 days, Approximately 6 days, maximum approximately 7 days, maximum approximately 8 days, maximum approximately 9 days, maximum approximately 10 days, maximum approximately 11 days, Up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 1 month, Up to about 2 months, up to about 3 months, up to about 4 months, up to about 5 months, or up to about 6 months In some embodiments, a biological sample can be obtained, Any biomarker detection protocol may be used to detect the presence of a composition encoding a heterodimeric protein. Multiple doses can be performed after administration. The biological sample contains the heterodimeric protein. at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 80, 95, 100, 150, 250, 350, 450, 5 ... The biological sample may be obtained 10, 20, or 25 times. After administration of the composition, the heterodimers in the biological sample can be obtained weekly or monthly. Monomeric proteins can be used in fluorescence assays, FRET assays, TR-FRET assays, or luminescence assays. It can be detected by an optical assay.

[0180] Alternatively or additionally, the heterodimeric protein may be used in a heterodimer-specific immunodetection assay. See, e.g., U.S. Patent Nos. 6,143,576; 6,113,855 No. 6,019,944; No. 5,985,579; No. 5,947,124; No. 5 , No. 939,272; No. 5,922,615; No. 5,885,527; No. 5,851 , No. 776; No. 5,824,799; No. 5,679,526; No. 5,525,524 and protein-containing immunoassays such as those described in US Pat. No. 5,480,792. Several methods and devices for measuring the level of quality are known. Various sands for generating signals related to the presence or amount of protein analytes in the sample. Any suitable immunoassay, e.g., switch, competitive, or non-competitive assay format. For example, lateral flow, enzyme-linked immunoassay (ELISA), radioimmunoassay (RI), A), competitive binding assays, or any combination thereof can be utilized.

[0181] Ex vivo construction of synthetic biomarkers for use in disease detection, monitoring, or diagnosis Buildings and methods In some aspects, the present disclosure provides non-human endothelial cells that contain sequences encoding polypeptides or nucleic acid sequences. A composition comprising a naturally occurring recombinant gene construct, wherein the sequence is transfected into cells ex vivo. When introduced, the polypeptides were detected in several different types of diseased cells isolated from the subject. a composition comprising a first promoter that selectively drives expression of a gene or nucleic acid biomarker sequence; Provide something.

[0182] Optionally, the composition may comprise cells transduced with a recombinant gene construct. Optionally, the plurality of different types of cells are diseased or damaged cells. blood cells, lymphocytes, leukocytes, epithelial cells, gastrointestinal cells, placental cells, amniotic cells, lung epithelial cells, Ureteral epithelial cells, or kidney cells.

[0183] In some cases, the diseased or impaired cells are cancerous cells, cells indicative of an autoimmune disease (e.g., , T cells or lymphocytes with self-directed activity, or normal cells damaged by autoimmunity cells), TAC, or cells exhibiting a neurodegenerative disease (e.g., cells with toxic amyloid or cells in close proximity to toxic amyloid. Neurodegenerative diseases include any of the diseases described herein. The cells or damaged cells may be virally infected cells. Exemplary viral infections include HIV. , Hepatitis C virus, Hepatitis B virus, Hepatitis D virus, Herpes virus, EPS Tympanic virus, cytomegalovirus, and human T-lymphotropic virus type III These include, but are not limited to, those caused by:

[0184] Optionally, the first promoter is activated in the cell when the cell is in a diseased state. The first promoter may be a pan-tumor-specific promoter. Optionally, the first promoter is a cancer-specific promoter. Optionally, the final promoter is a The first promoters were the survivin promoter (BIRC5) and the CXCR4 promoter. , ATP-binding cassette subfamily C member 4 (ABCC4) promoter, forward gradient Sequence 2, protein disulfide isomerase family member (AGR2) promoter -, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNA c: βGal β-1,3-N-acetylglucosaminyltransferase 3 (B3G NT3 promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter -, centrosome protein 55 (CEP55) promoter, claudin-3 (CL DN3) promoter, claudin 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter, collagen type I α1 chain (COL1A1) promoter cystatin SN (CST1) promoter, denticleless eless) E3 ubiquitin protein ligase homolog (DTL) promoter, sequence Family with similarity 111 member B (FAM111B) promoter, fork Headbox A1 (FOXA1) promoter, kinesin family member 20A ( KIF20A), laminin subunit γ2 (LAMC2) promoter, mitotic spindle Body positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) Promoter, matrix metallopeptidase 12 (MMP12) promoter, matrix Metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) Promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 Loop IID (PLA2G2D) promoter, G protein signaling 13 (RGS 13) Promoter, Secretoglobin family 2A member 1 (SCGB2A1) promoter promoter, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UB D) Promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network network component harmonin (USH1C), T cell activation inhibitor 1 (VTCN 1) V-set domain containing promoter, hexokinase type II promoter, TRP M4 promoter, stromelysin 3 promoter, surfactant protein A promoter Motor, secretory leukoprotease inhibitor promoter, tyrosinase promoter, Stress-inducible grp78 / BiP-containing domain promoter, interleukin-1 0 promoter, α-B-crystallin / heat shock protein 27 promoter, epithelial Growth factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter -, NSE promoter, somatostatin receptor promoter, c-erbB-3 promoter promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter promoter, glycoprotein A33 promoter, B cell-specific Moloney leukemia virus insert Entry site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter human epidermal growth receptor 2, human telomerase reverse transcriptase promoter; receptor Kinase domain insert containing promoter; rad51 recombinase promoter TTF-1, urokinase-type plasminogen activator receptor promoter, Biquitin-conjugating enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 ( CHEK1 promoter, epithelial cell transformation 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, centromere protein I (CENPI) promoter E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter, protein phosphatase, Mg 2+ / M n 2+ promoter-dependent 1G (PPM1G), ubiquitin-conjugating enzyme E2 T (UBE2 T) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cells Transforming 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter , centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1 ) promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter ator, protein phosphatase, Mg 2+ / Mn 2+ Dependency 1G (PPM1G) Pro Motor, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, Aurora kinase A and ninein-interacting protein (AUNIP) promoter, cell division cycle 6 (CDC 6) Promoter, Centromere protein L (CENPL) promoter, DNA replication Helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 The promoter of the kinetochore complex component (DSN1), deoxythymidylate kinase (DTY G protein regulation of the MK promoter and neurite outgrowth 1 (GPRIN1) promoter Inducer, mitochondrial fission regulator 2 (MTFR2) promoter, RAD5 1-associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein polymerase SNP A' (SNRPA1) promoter, ATPase family, AAA domain ATAD2 promoter, BUB1 mitotic checkpoint serine / threonine Onion kinase (BUB1) promoter, calcyclin-binding protein (CACYB P) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere Protein O (CENPO) promoter, flap structure-specific endonuclease 1 (FE N1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation Growth factor receptor protein phosphatase 2A (KIAA1524) promoter, kinesin phosphatase Milli-member 2C (KIF2C) promoter, karyopherin subunit α2 (K PNA2 promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA Nek2-related kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter promoter, small nuclear ribonucleoprotein polypeptides B and B1 (SNRPB) promoter promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, Transformed acidic coiled-coil-containing protein 3 (TACC3) promoter, TBC1 domain TBC1D31 promoter, thymidine kinase 1 (T K1) promoter, zinc finger protein 695 (ZNF695) promoter, Aurora kinase A (AURKA) promoter, BLM RecQ-like helicase (BL M) Promoter, chromosome 17 open reading frame 53 (C17orf53) promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCN B1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CC NF), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45 ) promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase CDKN3 promoter, cadherin EGF LAG 7-pass G-type receptor Centromere receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter Centrosome protein 72 (CEP72) promoter, CDC28 protein Protein kinase regulatory subunit 2 (CKS2) promoter, collagen type X α1 chain (C OL10A1 promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 subunit lead finger promoter, GINS complex subunit 1 (GINS1) promoter; G protein-coupled receptor 19 (GPR19) promoter, a kinesin family member 18A (KIF18A) promoter, kinesin family member 4A (KIF4A) Promoter, Kinesin family member C1 (KIFC1) promoter, mini-staining MCM10 promoter, minichromosome maintenance Minichromosome maintenance complex component 2 (MCM2) promoter, Component 7 (MCM7) promoter, MRG domain-binding protein (MRGBP) Promoter, methylenetetrahydrofolate dehydrogenase (NADP+-dependent) 2, Thenyltetrahydrofolate cyclohydrolase (MTHFD2) promoter, non-SMC NDC80, Kinetoc. NDC80 promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A ) promoter, RuvB-like AAA ATPase 1 (RUVBL1) promoter, Serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC Binding and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein poly(A) Peptide G (SNRPG) promoter, timeless circadia Thyroid hormone receptor interactor 13 (TRIP13), a regulator promoter Promoter, trophinin-related protein (TROAP) promoter, ubiquitin-binding UBE2C promoter, WD repeats, and HMG box DNA binding site fusion protein 1 (WDHD1) promoter, alpha-fetoprotein (AFP) promoter The protein may be a nucleotide sequence, a fragment thereof, or any combination thereof.

[0185] Optionally, the recombinant gene construct for ex vivo detection is a retrovirus, May contain lentiviral or adenoviral packaging elements or long terminal repeats The recombinant gene construct may be a CELiD vector. Phage or vectors of plant, invertebrate, or animal (e.g., human) origin, e.g. Adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AA V6, AAV7, AAV8, AAV9, or AAV2 / 5, AAV2 / 2, AAV-DJ , or pseudotyped combinations thereof such as AAV-DJ8), retroviral vectors (e.g., MLV or its self-inactivating or SIN version, or their pseudotypes) versions), herpes viruses (e.g., HSV- or EBV-series), lentivirus vectors vectors (e.g., HIV, FIV, or EIAV systems, or pseudotyped versions thereof) ), or adenoviral vectors (e.g., replication-deficient, replication-competent, or helper-dependent vectors The recombinant gene constructs can be used in combination with these viral systems. It can also be a packaging vector compatible with either

[0186] The vector can be a non-viral vector. The non-viral vector is a minicircle vector. The minicircle may be a self-replicating minicircle. The vector may contain an S / MAR element. Non-viral vectors include nanoplasmids or miniplasmids. The MIP may be a transgenic plasmid (MIP). The MIP contains a bacterial origin of replication and a selectable marker. The car is placed in the transgene expression cassette as an intron. Furthermore, the MIP 3'; It is possible to maintain the juxtaposition of the ends of the transgene expression cassette as in the minicircle. (e.g., Lu et al., a mini-intronic plasmi d(MIP): a novel robust transgene expressi on vector in vivo and in vitro, mol. The r. 2013 May;21(5);954-963).

[0187] Optionally, a polypeptide or polypeptides that are selectively expressed when transduced ex vivo. The nucleic acid sequences are photoacoustic reporters, bioluminescent reporters, autofluorescent reporters, chemiluminescent reporters, reporters, luminescent reporters, colorimetric reporters, quantifiable nucleic acids, and any combination thereof Autofluorescent reporters include GFP, mC, and combinations thereof. herry, or their derivatives. Colorimetric reporters include β-galactosidase These include pigment-producing enzymes such as tyrosinase (in combination with the administration of X-gal) and tyrosinase. Bioluminescent, chemiluminescent, or luminescent reporters include Gaussia luciferase, Renilla luciferase, luciferase, and firefly luciferase (e.g., Branchini et al. Anal. Biochem. 396(2010):290-297 luciferases (e.g., those described herein) such as .py RE8 and RE9 versions In combination with the administration of coelenterazines described above, detection by photoacoustic imaging Possible reporters include β-galactosidase (in combination with administration of X-gal) and Chromogenic enzymes such as tyrosinase, autofluorescent proteins (GFP, mCherry, or their derivatives), non-fluorescent GFP-like chromoproteins (e.g., aeCP597 and c jBlue and its derivatives), bacteriophytochrome-based near-infrared fluorescent proteins (e.g., IFP1.4, Wi-Phy, IFP1.4rev, IFP2.0, iRFP 713, iRFP720, iRFP713 / V256C, iRFP682, iRFP70 2, iRFP670, mIFP, iBlueberry, GAF-FP, BphP1-F P / C20S, or AphB variants), and reversibly photoswitchable proteins (Dr onpa, Dronpa-M159T, and BphP1 or its variants) Quantifiable nucleic acids include ribozymes, self-splicing introns, and RNA hairpins. , microRNAs, or their barcoded versions, or other types of quantifiable RNAs The quantifiable nucleic acid can be a quantitative PCR or hybridization-based The polypeptide or nucleic acid may contain a unique sequence that is detectable by a specific technique. If the polypeptide is a polypeptide, the polypeptide may contain an N-terminal secretory signal sequence (e.g., CD33 or C The N-terminal signal peptide from D8a may be included.

[0188] In some cases, the composition may have a particular diagnostic efficacy, which diagnostic efficacy is determined by the subject. expression of said biomarker in diseased cells in the subject preferentially over expression in non-diseased cells; Thereby, the biomarkers expressed in the diseased cells are compared to the non-diseased cells. (b) detecting said biomarker; and (c) determining whether or not (b) is a relative ratio greater than about 1.0. Using the detected biomarker, the subject is detected to have the diseased cells. Determine with at least 90% accuracy that

[0189] In some cases, the composition administered to the cells ex vivo regulates the proliferation of diseased or disordered cells. The second polypeptide may comprise a second polypeptide or nucleic acid that inhibits the growth of diseased cells or lesions. a second promoter that selectively drives expression of a second polypeptide or nucleic acid in target cells. The second promoter may be under the control of a pan-cancer-specific promoter. The second promoter can be a cancer-specific promoter. The second polypeptide may be any of the specific promoters described in the subsection. The transforming agent may comprise a transforming agent or growth factor. The transforming agent may comprise telomerase or SV40 large T antigen. The growth factor may be, for example, EGF, PDGF, FGF, HGH, or IGF-1. It can be.

[0190] In some embodiments, the present disclosure provides methods for administering a non-naturally occurring recombinant genetic construct to a subject ex vivo. ex vivo transfection of diseased or damaged cells, including delivery to a population of cells isolated from the ex vivo transfection of diseased or damaged cells. a method for detecting in vivo the non-native recombinant gene construct containing a polypeptide or nucleic acid a sequence encoding a sequence that, when transduced into cells isolated from a subject, Selectively driving expression of a polypeptide or nucleic acid sequence in a plurality of different cell types. The method further comprises the step of:

[0191] In some embodiments, the present disclosure provides a method for the treatment of a subject, comprising ex vivo transfecting one or more cells of the subject with genes. a method for detecting disease or absence of disease in a subject, the method comprising contacting the subject with a construct; wherein the gene construct comprises a disease-activated promoter operably linked to a barcode molecule. wherein the disease-activated promoter is capable of activating the vector in cells affected by the disease. driving expression of said barcode molecule; quantifying the expression level of said barcode molecule; The present invention provides a method for detecting the disease or its absence based on the gene expression pattern.

[0192] In some cases, the method for detecting diseased or damaged cells ex vivo involves detecting the presence of normal cells. It may be possible to detect a certain number of diseased cells in the background. In one embodiment, the method comprises generating at least about 3, or a minimum of 10,000,000 normal cells per about 5 million normal cells. at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, 200, at least about 300, at least about 400, or at least about 500 diseased cells can be detected. In this embodiment, the normal cells are blood cells (eg, PBMCs).

[0193] Optionally, the method includes isolating a biological sample comprising the cells from the subject. Biological samples are samples obtained from subjects by non-invasive methods. Exemplary non-invasive samples include saliva, sputum, sweat, urine, stool, semen, mucus, and cervicovaginal secretions. of externally accessible tissues such as breast milk, mucosal secretions, tears, and buccal swabs. These include samples made from naturally released body material or non-destructive scrapings. Biological samples include, but are not limited to, samples collected from subjects by minimally invasive methods. Exemplary minimally invasive samples include blood samples or fractions thereof (e.g., e.g., obtained by venipuncture or capillary tube), pleural fluid samples (e.g., obtained by thoracentesis), amniotic fluid samples (obtained, for example, by amniocentesis), and gastric fluid samples. Examples include, but are not limited to, IVF (e.g., obtained by amniocentesis), and IVF (e.g., obtained by amniocentesis). , obtained by gastric lavage). Biological samples include skin biopsy samples (e.g., punch biopsies). , obtained by shave biopsy, trough scrape biopsy, wedge biopsy, incisional biopsy, or excisional biopsy), bone bone marrow samples (e.g., obtained by aspiration biopsy), lymph node or breast biopsies (e.g., obtained by needle aspiration, core needle biopsy, vacuum-assisted biopsy, or image-guided biopsy), surgical biopsy Biopsy samples (e.g., visceral samples obtained by resection or incision biopsy), or samples from the mouth, gastrointestinal tract biopsy samples (e.g., obtained by endoscopy) of the lung, bladder, or urinary tract The sample may be obtained by biopsy.

[0194] Optionally, the method comprises administering the recombinant gene construct to the cell for a specified period of time after delivery. The population of cells may include culturing the population of cells, wherein the gene construct is delivered to the cells. At least about 15 minutes, at least about 30 minutes, at least about 1 hour, or at least at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 16 hours, In both cases, the time is about 24 hours, at least about 36 hours, at least about 48 hours, at least about 3 days, or at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 1 2 days, at least about 13 days, at least about 14 days, at least about 15 days, or at least The population of cells can be cultured for about one month. The population of cells can be cultured for up to about one month after the gene construct is delivered to the cells. 15 minutes, up to approx. 30 minutes, up to approx. 1 hour, up to approx. 2 hours, up to approx. 4 hours, up to approx. 8 hours, Approximately 16 hours, maximum approximately 24 hours, maximum approximately 36 hours, maximum approximately 48 hours, maximum approximately 3 days, maximum approximately approximately 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, Maximum approximately 11 days, maximum approximately 12 days, maximum approximately 13 days, maximum approximately 14 days, maximum approximately 15 days, or maximum approximately It can be cultivated in one month.

[0195] Optionally, the method may include detecting the polypeptide or nucleic acid sequence. Detection can be performed before or after culturing the cell population. Detection can be performed using photoacoustic, bioluminescent, fluorescent, Detection may also include reporter, chemiluminescent, luminescent, colorimetric, or nucleic acid assays. Immunoassays may include immunoassays, such as those described in U.S. Patent No. 6,143,576 No. 6,113,855; No. 6,019,944; No. 5,985,579; No. 5 , No. 947,124; No. 5,939,272; No. 5,922,615; No. 5,885 , No. 527; No. 5,851,776; No. 5,824,799; No. 5,679,526 No. 5,525,524; and No. 5,480,792. Immunoassays involve generating a signal related to the presence or amount of a protein analyte of interest. There are a variety of sandwich, competitive, or non-competitive assay formats that can be generated. Suitable immunoassays, such as lateral flow, enzyme-linked immunoassay (ELISA), radioimmunoassay, Immunoassays (RIA), competitive binding assays, and the like can be used.

[0196] The detection method may include sequencing. Sequencing methods include next generation sequencing, high-throughput sequencing, and the like. put sequencing, pyrosequencing, traditional Sanger sequencing methods, and ligation sequencing by synthesis, sequencing by hybridization, RNA-Seq eq (Illumina), digital gene expression (Helicos), next-generation sequencing; Single Molecule Sequencing by Synthesis (SMSS) (Helicos), Ion Torrent sequencing machine (Life Technologies / Thermo-Fisher); Massively parallel sequencing, clonal single molecule array (Solexa), shotgun sequencing , Maxim-Gilbert sequencing, and primer walking.

[0197] Detection is by "real-time" PCR, also known as quantitative PCR (qPCR) or Taqman. These methods include "amplification" methods (see, e.g., U.S. Patent No. 5,210,014 to Gelfand). No. 5,538,848 to Livak et al., and No. 5,863 to Haaland. , No. 736, and Heid, CA et al., Genome Research rch, 6:986-994(1996);Gibson, UEM et al ., Genome Research 6:995-1001(1996);Holl and, PM, et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, K.J. et al., PCR Methods and Applications 357 -362(1995)). The basis of this method for monitoring the formation of amplification products is double-labeling. Continuous measurement of PCR product accumulation using a unique fluorogenic oligonucleotide probe. The probes used in such assays typically have two different fluorescent colors. It is a short (approximately 20-25 base) polynucleotide labeled with 5'-dimer. The first end is typically conjugated to a reporter dye, and the 3' end is conjugated to a quencher dye. having at least substantial sequence complementarity to a site on a target mRNA or nucleic acid derived therefrom. Upstream and downstream PCR primers that bind to the flanking regions of the locus are designed to When the probe is intact, the energy between the two fluorophores is During the extension step of PCR, the quencher quenches the reporter's light emission. The probe is synthesized by the 5' nuclease activity of nucleic acid polymerases such as Taq polymerase. The reporter is cleaved, thereby releasing the reporter from the polynucleotide quencher. The resulting luminescence intensity can be measured by a suitable detector. Using the calculated values, the increase in normalized reporter emission intensity was calculated successively, and finally The amount of mRNA that is amplified can be quantified.

[0198] In some embodiments, for qPCR or Taqman detection, an RT-PCR step is performed first. First, cDNA can be generated from cellular RNA by RT-PCR. Such amplification can be achieved using general (e.g., partially / fully degenerate) oligonucleotide primers. amplification by a gene) or targeted (e.g., directed against a specific gene to be analyzed at a later stage). amplification with directed oligonucleotide primers).

[0199] In some embodiments, qPCR or Taqman is performed on mRNA of isolated cells. This diversity can be used immediately after the reverse transcriptase reaction performed on the individual This is useful for quantifying individual mRNA levels.

[0200] In some embodiments, for qPCR or Taqman detection or RNA sequencing Alternatively, a "pre-amplification" step can be performed first on cDNA transcribed from cellular RNA. This allows for a signal to be detected under conditions where the natural level of RNA / cDNA is very low. Suitable preamplification methods include LM-PCR, random orientation PCR, and PCR using oligonucleotide primers (e.g., random hexamer PCR), PCR using A-specific primers, and any combination thereof. Preamplification can be general or targeted in the same manner as the reverse transcription reaction described above. It is possible.

[0201] Improved biomarkers, construct designs, and methods for disease staging In some aspects, the present disclosure provides a composition comprising a vector, wherein the vector comprises multiple The method comprises: providing a plurality of different promoters operably linked to a number of different nucleic acid sequences; a control vector driving expression of the plurality of nucleic acid sequences in a cell to produce a plurality of polypeptides or generating nucleic acid biomarker sequences and identifying individual polypeptide or nucleic acid biomarkers from the plurality of nucleic acid sequences; The present invention provides compositions in which the level of biomarkers is indicative of the stage of cellular disease. The disease state of the cell may be diseased, non-diseased, or in an intermediate state. In some cases, the different promoters may be administered simultaneously or separately. In some embodiments, the gene may be administered separately. Multiple independent gene constructs may be spaced 8, 16, 24, 36, 48, 60, or 72 hours apart from each other. In some embodiments, the disease stage progresses via metastasis to distant tissues. This can be assessed by the dissemination of cancer cells away from their tissue of origin. The different promoters are at least as important as the primary tissue site (e.g., where breast cancer is staged). promoter with high cancer specificity in the breast (when present) and different from the first site Highly specific and active promoters in common metastatic sites (e.g., lung, spleen, liver) Optionally, the plurality of different promoters may comprise at least one promoter from the initial tissue site (e.g., promoters with high cancer specificity (e.g., breast when breast cancer is being staged) and multiple promoters active with high specificity at multiple different metastatic sites (e.g., lung, spleen, liver). Such a system may therefore facilitate the migration of cancer from its original site. When translocated to the translocation site, the more distinct promoters (downstream of their operably linked This provides information about the activation of the immune system (which can be read by biomarkers) and how widespread the tumor is. In some embodiments, a general assessment of metastasis can be provided. One promoter that is highly specific and active in the lung is MMP-2, which is It provides high expression at all stages of cancer but is not overexpressed in breast cancer.

[0202] In some cases, the disease is cancer, an autoimmune disease (e.g., T-cell or T-cell mediated immune responses with autologous activity), or lymphocytes, or normal cells damaged by autoimmunity), or neurodegenerative diseases (e.g. , cells bearing toxic amyloid or cells in proximity to toxic amyloid). Exemplary cancers include, but are not limited to, carcinoma, sarcoma, lymphoma, leukemia, and adenoma. Carcinomas begin in cells lining the inside and outside of the body, such as the lung, breast, and colon. Sarcomas grow in cells found in bone, cartilage, fat, connective tissue, muscle, and other supportive tissues. Lymphoma can develop in lymph nodes or immune system tissues. Leukemias begin in the bone marrow and can accumulate in the bloodstream. Adenomas can occur in the thyroid, pituitary, and adrenal glands. Specific illustrative examples of cancer types include those described in this publication. Suitable for detection by diagnostic methods, including acute lymphoblastic leukemia, acute myeloid leukemia, Hematologic malignancies, adrenocortical cancer, AIDS-related cancer, AIDS-related lymphoma, anal cancer, adnexal cancer , astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone tumors, such as cerebellar astrocytoma, cerebral astrocytoma neurocyte tumor / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic nerve tumor tract and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt lymphoma, cancer of unknown primary origin, Central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic Myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, desmoplastic angioplasty Polycystic tumor, endometrial cancer, acanthoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer , gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer Cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic Islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, non- Lung cancer, including small cell and extracellular lung cancer, lymphoma, leukemia, macroglobulinemia, bone / Osteosarcoma, malignant fibrous histiocytoma, medulloblastoma, melanoma, mesothelioma, metastatic carcinoma with occult primary Neck cancer, oral cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer Cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian Germ cell tumors, pancreatic cancer, pancreatic islet cell cancer, sinus and nasal cancer, parathyroid cancer, penile cancer Pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma tumors, plasma cell neoplasms, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, Renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell skin cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma , throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (during pregnancy), primary site unknown Cancer of the urethra, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia These include phosphatemia, and Wilms' tumor.

[0203] In some cases, the disease may be a virally infected cell. Exemplary viral infections include HIV , Hepatitis C virus, Hepatitis B virus, Hepatitis D virus, Herpes virus, EPS Tyne-Barr virus, cytomegalovirus, and human T-lymphotropic virus II These include, but are not limited to, those caused by type I.

[0204] In some cases, when the disease is cancer, multiple different promoters are present in the early stages of the cancer. Optionally, multiple different promoters may be present. In some cases, the promoter may include a second promoter that is activated during intermediate stages of cancer. The promoters include a third promoter that is activated in later stages of cancer.

[0205] In some cases, the disease may be an autoimmune disease. Exemplary autoimmune diseases include achalasia Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis dosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune vascular Edema, autoimmune autonomic neuropathy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear Disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune Autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN), Behcet's disease, benign mucosal emphysema, benign emphysema, Castleman's disease (CD), celiac disease Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Relapsing Multiple Myelopathy (CRMP), Chagas' Disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Relapsing Multiple Myelopathy (CRMP) granulomatosis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGP) A), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Sackey's myocarditis, CREST syndrome, Crohn's disease, herpes dermatitis, dermatomyositis, David Neuromyelitis optica, discoid lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis ( EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia arterial Temporal arteritis, giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, polyangiitis Granulomatous disease with thyroiditis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Hach-Schönlein purpura (HSP), herpes gestationis or pemphigoid of gestationis, hidradenitis suppurativa (HS) (hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease) disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), and interstitial cystitis (IC). ), juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambe disease Connold-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, streptococcus aureus Lymphocytic inflammatory disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MP) A), Mixed connective tissue disease (MCTD), Mullen's ulcer, Mucha-Habermann disease, multifocal movement Neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, nal Colepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis , Palindromic rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis ( Peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, peripheral cerebral Myelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I , Type II, Type III, Polymyalgia rheumatica, Multifocal myositis, Post-myocardial infarction syndrome, Pericardotomy Post-inflammatory syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriasis Arthritis, Pure Red Cell Aplasia (PRCA), Pyoderma Gangrenosum, Raynaud's phenomenon, Reactive Arthritis, Reflex Sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal gland Fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), subacute Bacterial endocarditis (SBE), Sasac syndrome, sympathetic ophthalmia (SO), Takayasu arteritis, Cephalic arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (TH S), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD) , uveitis, vasculitis, vitritis, and Vogt-Koyanagi-Harada disease. Not limited to.

[0206] In some cases, the disease may be a neurodegenerative disease. Neurodegenerative diseases include multiple sclerosis (MS). S), Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis ( ALS), or Herpesviridae, Polyomaviridae, Bornaviridae, Orthoviridae Myxoviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Pill Neurodegeneration caused by infection with Conavirus or Retrovirus viruses However, it is not limited to these (Zhou et al., Virol J. 2013;10:172).

[0207] In some cases, the nucleic acid biomarker is, for example, a naturally occurring or engineered miRNA, RNA It can be a hairpin, an RNA aptamer, or a barcode version thereof.

[0208] Optionally, the vector provided in the composition for detecting the stage of disease is a vector as described herein. It can be any of the vectors described.

[0209] Optionally, at least one of the plurality of polypeptides is suitable for non-invasive imaging. Such non-invasive imaging methods may include polypeptides that can be detected by M RI imaging, PET imaging, SPECT imaging, photoacoustic imaging and bioluminescence imaging. Synthetic biomarkers detectable by MRI imaging. The target is ferritin (or Pyrococcus furiosus). uriousus) ferritin mutants L55P, F57S, F123S, and other mutations thereof ions), or lanthanide-binding proteins (or Daughtry et al., Che LBT-ubiquitin as described in mBioChem 2012, 13, 2567-2574 These include, but are not limited to, polypeptide contrast agents such as phosphodiesterase fusions and engineered fusions thereof. Synthetic biomarkers detectable by PET or SPECT imaging are not defined. Carriers include the human sodium iodide symporter (e.g., PET-activated iodine / iodide isotope In combination with the administration of, for example, Penheiter et al. Curr Gene Ther. 2012 Feb;12(1):33-47), HSV-tk or its variants, such as HSV-sr39tk (e.g., positron emission tomography (e.g., [18F]FHBG) in combination with administration of a fluorine-labeled acycloguanosine or pyrimidine analog PET reporter , Yaghoubi SS et al., Nat Protoc. 2006;1( 6):3069-75), and dopamine D2 receptor or D2R80A or D2R194 A variant thereof (e.g., 3-(2′-[18F]-fluoroethyl)-spiro[pi] In combination with the administration of positron-labeled D2 binders such as benzophenone. Synthetic biomarkers detectable by ELISA include β-galactosidase (e.g., X-galactosidase). in combination with the administration of tyrosinase, chromogenic enzymes such as tyrosinase, autofluorescent proteins ( GFP, mCherry, or their derivatives), non-fluorescent GFP-like chromoproteins (e.g., (e.g., aeCP597 and cjBlue and their derivatives), bacteriophytochrome Near-infrared fluorescent proteins based on IFP1.4 (e.g., IFP1.4, Wi-Phy, IFP1.4r) ev, IFP2.0, iRFP713, iRFP720, iRFP713 / V25 6C, iRFP682, iRFP702, iRFP670, mIFP, iBlueber ry, GAF-FP, BphP1-FP / C20S, or AphB variants), and and reversibly photoswitchable proteins (Dronpa, Dronpa-M159T, B phP1 or its variants) can be detected by bioluminescence imaging. Suitable synthetic biomarkers include Gaussia luciferase, Renilla luciferase, and Photinus luciferase (e.g., Branchini et al., Anal. Biochem. 396(2010):290-297 and RE9 versions) (e.g., selenium as described herein) In some embodiments, the synthetic biomarkers The carrier may be an imaging agent, an enzyme that produces a detectable molecule, or a molecule that drives the accumulation of a detectable molecule. The synthetic biomarker can be an in vivo transporter. It can be measured in meters.

[0210] Optionally, the barcode molecule is a detectable polypeptide in a biological sample from the subject. If the barcode molecule is a polypeptide, the polypeptide The peptide may contain an N-terminal secretory signal sequence (e.g., the N-terminal signal sequence from CD33 or CD8a). Exemplary polypeptide biomarkers include photoacoustic reporters. , bioluminescence reporter, autofluorescence reporter, chemiluminescence reporter, luminescence reporter, ratio color reporters, and any combination thereof. Autofluorescent reporters include GFP, mCherry, or their derivatives. Colorimetric reporters include β-galactosidase (in combination with X-gal administration) and and chromogenic enzymes such as tyrosinase. Bioluminescent, chemiluminescent or luminescent reporters Gaussia luciferase, Renilla luciferase, and firefly luciferase See, e.g., Branchini et al. Anal. Biochem. 39 6(2010):290-297, such as the Ppy RE8 and RE9 versions ), and the like (e.g., in combination with the administration of coelenterazines described herein). Reporters that can be detected by photoacoustic imaging include β-galactosidase. pigment-producing enzymes such as tyrosinase (in combination with the administration of X-gal), autoantibodies against Fluorescent proteins (e.g., GFP, mCherry, or their derivatives), non-fluorescent GFP-like Chromoproteins (e.g., aeCP597 and cjBlue and their derivatives), Near-infrared fluorescent proteins of the teriophytochrome family (e.g., IFP1.4, Wi-Phy , IFP1.4rev, IFP2.0, iRFP713, iRFP720, iRFP71 3 / V256C, iRFP682, iRFP702, iRFP670, mIFP, iBl ueberry, GAF-FP, BphP1-FP / C20S, or AphB variant ), and reversible photoswitchable proteins (Dronpa, Dronpa-M159T, and BphP1 or its variants). In this case, the detectable nucleic acid sequence may be a ribozyme, a self-splicing intron, an RNA hair pins, microRNAs, or their barcoded versions, or other types of quantifiable R Quantifiable nucleic acid sequences can include, but are not limited to, Unique sequences detectable by quantitative PCR or hybridization-based techniques It may include.

[0211] By attributing dedicated labels to unique members of a larger group, Codes can be larger and more complex mixtures of many members (e.g., expressed in the same cell). Identifying and quantifying its members within the context of multiple promoter-reporter constructs Given the opportunity (e.g., expression of a reporter under the control of a particular cancer-specific promoter), and offer the opportunity to isolate single members from complex mixtures. In the case of barcodes, hybridization of the barcode based on base pair complementarity is used. The capture event captures and isolates or otherwise reduces the complexity of the mixture. In the case of peptide-based barcodes, immunocapture or ligand-receptor binding can be used. The capture event captures and isolates the mixture using unique characteristics, including interactions. , otherwise the complexity can be reduced.

[0212] In some aspects, the disclosure includes administering to a subject a composition comprising the vector. A method for detecting a stage of a disease, wherein the vector is operably linked to a plurality of different nucleic acids. a plurality of different promoters, each promoter controlling the expression of a plurality of nucleic acid sequences in a cell; and generating a plurality of polypeptides or synthetic nucleic acid sequences, and The level of the polypeptide provides a method for indicating the stage of disease in the cell. The stage of the disease can be diseased, non-diseased, or an intermediate state. and a method for detecting different types of cancer, comprising administering to a subject a composition comprising the vector. The method of claim 1, wherein the vector is a multiple vector operably linked to multiple different nucleic acid sequences in a cell. By including a number of different promoters, multiple polypeptides or synthetic nucleic acid sequences can be produced; A method in which the levels of individual polypeptides of multiple nucleic acid sequences indicate different types of cancer in the body. In some cases, the cancer detected in the body is cancer of the breast, liver, colon, brain, lung, kidney, pancreas, organs, testes, ovaries, blood, or blood, bone, stomach, eyes, endocrine or neuroendocrine tissue, head and neck tissues of gastrointestinal, musculoskeletal, cutaneous, respiratory, nervous or genitourinary components, including but not limited to: The cancer may originate from the lining of the body (not the lining of the body) or from elsewhere in the body.

[0213] Optionally, the composition is administered to the subject intravenously, subcutaneously, intracerebroventricularly, intrathecally, or intravenously. administration, intracerebroventricular administration, transdermal administration, intramuscular administration, oral administration, inhalation administration, intranasal administration, rectal administration, tumor administration Intratumoral administration or tumor-proximal administration. areas predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes) Intratumoral or proximal approaches may include, for example, endoscopic ultrasound. Testing (e.g., Shirley et al., Gastroenterol Res P ract. 2013;2013:207129) or bronchoscope (e.g., Rojas -Solano et al. J Bronchology Interv Pulm onol. 2018 Jul;25(3):168-175) In some embodiments, the composition is applied to the cervical, epitrochlear, and / or epicondylar areas. supraclavicular, cervical, axillary, mediastinal, supratrochlear, mesenteric, inguinal, femoral, or popliteal lymph node sacs Optionally, lymph node-based administration is administered to at least one of the tissue regions. It may serve as a method of focused local delivery.

[0214] In some cases, when the disease is cancer, multiple different promoters are present in the early stages of the cancer. Optionally, multiple different promoters may be present. In some cases, the promoter may include a second promoter that is activated during intermediate stages of cancer. The promoters include a third promoter that is activated in later stages of cancer. Thus, the method comprises determining whether a mass of tissue or lesion in a subject is precancerous, benign, dysplastic, or otherwise in nature. can be identified as metastatic.

[0215] Optionally, the method can include isolating a biological sample from the subject. The sample may be a sample obtained from a subject by a non-invasive method. Invasive samples include saliva, sputum, sweat, urine, stool, semen, mucus, cervicovaginal secretions, milk, and mucosal secretions. naturally released from externally accessible tissues, such as tears, and buccal epithelial swab scrapings Examples include, but are not limited to, samples of body material or non-destructive scrapings. The biological sample may be a sample obtained from a subject by a minimally invasive method. Exemplary minimally invasive samples include blood samples or fractions thereof (e.g., venipuncture or hair tubule), pleural fluid samples (e.g., obtained by thoracentesis), amniotic fluid samples samples (e.g., obtained by amniocentesis), and gastric fluid samples (e.g., obtained by amniocentesis) Examples include, but are not limited to, gastric lavage. Biological samples include skin biopsy samples (e.g., punch biopsy, shave biopsy, trough scrape biopsy). biopsy, wedge biopsy, incisional biopsy, or excisional biopsy), bone marrow samples (e.g., aspirated lymph node or breast biopsy (e.g., fine needle aspiration, core needle biopsy, or cytomegalovirus); biopsy samples (obtained by air-assisted biopsy or image-guided biopsy), surgical biopsy samples (e.g., surgical of internal organs obtained by excision or incision biopsy), or the mouth, digestive tract, lungs, bladder or urinary tract Biopsy samples obtained by biopsy, such as biopsy samples from a patient (e.g., obtained by endoscopy) Optionally, over a period of time after administering the composition to the subject. , a biological sample can be taken.

[0216] The population of cells is incubated for at least about 15 minutes, at least about 20 minutes, after delivery of the gene construct to the cells. 30 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 16 hours, at least about 24 hours, at least about 36 hours, or less In both cases, at least about 48 hours, at least about 3 days, at least about 4 days, at least about 5 days, or at least About 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days , at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days The population of cells may be cultured for at least about 15 days, or at least about 1 month. After delivery of the construct to the cells, the 2 hours, up to approximately 4 hours, up to approximately 8 hours, up to approximately 16 hours, up to approximately 24 hours, up to 36 hours, up to 48 hours, up to 3 days, up to 4 days, up to 5 days, up to Approximately 6 days, up to approximately 7 days, up to approximately 8 days, up to approximately 9 days, up to approximately 10 days, up to approximately 11 days , up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, or up to about 1 It can be cultivated for months.

[0217] Optionally, the method may include detecting the polypeptide or nucleic acid sequence. Detection can be performed before or after culturing the cell population. Detection can be performed using photoacoustic, bioluminescent, fluorescent, Detection may also include reporter, chemiluminescent, luminescent, colorimetric, or nucleic acid assays. Immunoassays may include immunoassays, such as those described in U.S. Patent No. 6,143,576 No. 6,113,855; No. 6,019,944; No. 5,985,579; No. 5 , No. 947,124; No. 5,939,272; No. 5,922,615; No. 5,885 , No. 527; No. 5,851,776; No. 5,824,799; No. 5,679,526 No. 5,525,524; and No. 5,480,792. Immunoassays involve generating a signal related to the presence or amount of a protein analyte of interest. There are a variety of sandwich, competitive, or non-competitive assay formats that can be generated. Suitable immunoassays, such as lateral flow, enzyme-linked immunoassay (ELISA), radioimmunoassay, Immunoassays (RIA), competitive binding assays, and the like can be used.

[0218] The detection method may include sequencing. Sequencing methods include next generation sequencing, high-throughput sequencing, and the like. put sequencing, pyrosequencing, traditional Sanger sequencing methods, and ligation sequencing by synthesis, sequencing by hybridization, RNA-Seq eq (Illumina), digital gene expression (Helicos), next-generation sequencing; Single Molecule Sequencing by Synthesis (SMSS) (Helicos), Ion Torrent sequencing machine (Life Technologies / Thermo-Fisher); Massively parallel sequencing, clonal single molecule array (Solexa), shotgun sequencing , Maxim-Gilbert sequencing, and primer walking.

[0219] Detection is by "real-time" PCR, also known as quantitative PCR (qPCR) or Taqman. These methods include "amplification" methods (see, e.g., U.S. Patent No. 5,210,014 to Gelfand). No. 5,538,848 to Livak et al., and No. 5,863 to Haaland. , No. 736, and Heid, CA et al., Genome Research rch, 6:986-994(1996);Gibson, UEM et al ., Genome Research 6:995-1001(1996);Holl and, PM, et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, K.J. et al., PCR Methods and Applications 357 -362(1995)). The basis of this method for monitoring the formation of amplification products is double-labeling. Continuous measurement of PCR product accumulation using a unique fluorogenic oligonucleotide probe. The probes used in such assays typically have two different fluorescent colors. It is a short (approximately 20-25 base) polynucleotide labeled with 5'-dimer. The first end is typically conjugated to a reporter dye, and the 3' end is conjugated to a quencher dye. having at least substantial sequence complementarity to a site on a target mRNA or nucleic acid derived therefrom. Upstream and downstream PCR primers that bind to the flanking regions of the locus are designed to When the probe is intact, the energy between the two fluorophores is During the extension step of PCR, the quencher quenches the reporter's light emission. The probe is synthesized by the 5' nuclease activity of nucleic acid polymerases such as Taq polymerase. The reporter is cleaved, thereby releasing the reporter from the polynucleotide quencher. The resulting luminescence intensity can be measured by a suitable detector. Using the calculated values, the increase in normalized reporter emission intensity was calculated successively, and finally The amount of mRNA that is amplified can be quantified.

[0220] In some embodiments, for qPCR or Taqman detection, an RT-PCR step is performed first. First, cDNA can be generated from cellular RNA by RT-PCR. Such amplification can be achieved using general (e.g., partially / fully degenerate) oligonucleotide primers. amplification by a gene) or targeted (e.g., directed against a specific gene to be analyzed at a later stage). amplification with directed oligonucleotide primers).

[0221] In some embodiments, qPCR or Taqman is performed on mRNA of isolated cells. This diversity can be used immediately after the reverse transcriptase reaction performed on the individual This is useful for quantifying individual mRNA levels.

[0222] In some embodiments, for qPCR or Taqman detection or RNA sequencing Alternatively, a "pre-amplification" step can be performed first on cDNA transcribed from cellular RNA. This allows for a signal to be detected under conditions where the natural level of RNA / cDNA is very low. Suitable preamplification methods include LM-PCR, random orientation PCR, and PCR using oligonucleotide primers (e.g., r...

Claims

1. (a) administering a composition to a subject, wherein the composition induces a vasoconstriction in non-diseased cells in the subject; Induce expression of said biomarker in diseased cells preferentially over expression of said biomarker. and determining the relative expression of the biomarker in the diseased cells relative to the non-diseased cells. The ratio is greater than 1.0; (b) detecting the biomarker; and (c) using the biomarkers detected in (b), the subject is % accuracy in determining that the subject has diseased cells.

2. The method of claim 1 , wherein the relative ratio is a concentration ratio.

3. The method of claim 1 , wherein the biomarker is detected in a biological sample from the subject. Law.

4. The method of claim 3 , wherein the biological sample is a bodily fluid from the subject.

5. 10. The method of claim 1, wherein the biological sample is a blood or blood-based sample from the subject.

4. The method according to claim 4.

6. The method of claim 3 , wherein the biological sample is a gaseous sample from the subject.

7. 7. The method of claim 6, wherein the gaseous sample is exhaled breath from the subject.

8. The method of claim 1 , wherein the subject is a mammal.

9. The method of claim 8, wherein the subject is a human.

10. The method of claim 8, wherein the subject is an animal.

11. 4. The method according to claim 3, wherein the biological sample is measured in situ in a human or animal body. How to post.

12. The method of claim 1 , wherein the composition comprises a nucleic acid vector.

13. The vector may be a nanoplasmid, a plasmid, a minicircle, or a recombinant viral vector.

13. The method of claim 12, wherein the antibody is selected from the group consisting of:

14. the composition comprises a minicircle, the minicircle being a self-replicating minicircle; The method of claim 13.

15. The method of claim 14, wherein the self-replicating minicircle comprises an S / MAR element.

16. The composition is operably linked to a nucleotide sequence encoding the biomarker. The method of claim 12, comprising a promoter.

17. The promoter controls expression in multiple different types of diseased cells in the subject. The method of claim 16, further comprising driving the

18. The promoter inhibits expression of the biomarker in the non-diseased cells in the subject. and driving expression of said biomarkers in said plurality of different types of diseased cells preferentially over expression.

20. The method of claim 17, wherein the

19. The promoter is a survivin promoter (BIRC5), a CXCR4 promoter, ATP-binding cassette subfamily C member 4 (ABCC4) promoter, forward gradient 2. Protein disulfide isomerase family member (AGR2) promoter , activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc : βGal β-1,3-N-acetylglucosaminyltransferase 3 (B3GN T3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 ( CEACAM5 promoter, centromere protein F (CENPF) promoter , centrosome protein 55 (CEP55) promoter, claudin-3 (CLD N3) promoter, claudin 4 (CLDN4) promoter, collagen type XI alpha Collagen type I α1 chain (COL1A1) promoter, collagen type I α1 chain (COL1A1) promoter tar, cystatin SN (CST1) promoter, denticle-less (less) E3 ubiquitin protein ligase homolog (DTL) promoter, sequences Family with similar promoter, member B (FAM111B), forkhead FoxA1 promoter, kinesin family member 20A (K IF20A), laminin subunit γ2 (LAMC2) promoter, mitotic spindle Positioning (MISP) promoter, matrix metallopeptidase 1 (MMP1) promoter promoter, matrix metallopeptidase 12 (MMP12) promoter, matri Metallopeptidase 13 (MMP13) promoter, mesothelin (MSLN) promoter promoter, cell surface-associated mucin 1 (MUC1) promoter, phospholipase A2 PLA2G2D promoter, G protein signaling 13 (RGS1 3) Promoter, secretoglobin family 2A member 1 (SCGB2A1) promoter Motor, topoisomerase IIα (TOP2A) promoter, ubiquitin D (UBD ) promoter, ubiquitin-conjugating enzyme E2C (UBE2C), USH1 protein network network component harmonin (USH1C), T-cell activation inhibitor 1 (VTCN1 ) V-set domain containing promoter, hexokinase type II promoter, TRPM 4 promoter, stromelysin 3 promoter, surfactant protein A promoter promoter, secretory leukoprotease inhibitor promoter, tyrosinase promoter, Stress-inducible grp78 / BiP-containing domain promoter, interleukin-10 promoter, α-B-crystallin / heat shock protein 27 promoter, epithelial growth factor receptor 27 (EGFR) Long factor receptor promoter, mucin-like glycoprotein promoter, mts1 promoter , NSE promoter, somatostatin receptor promoter, c-erbB-3 promoter ter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, villin promoter, albumin promoter Motor, glycoprotein A33 promoter, B cell-specific Moloney leukemia virus insert Site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter Motor; human epidermal growth receptor 2, human telomerase reverse transcriptase promoter; receptor promoter kinase domain insert containing the promoter; rad51 recombinase promoter TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin Chitin-binding enzyme E2 T (UBE2T) promoter, checkpoint kinase 1 (C HEK1 promoter, epithelial cell transformation 2 promoter (ECT2), BCL2-like 1 2 (BCL2L12) promoter, centromere protein I (CENPI) promoter E2F transcription factor 1 (E2F1) promoter, flavin adenine dinucleotide sequence FLAD1 promoter, protein phosphatase, Mg ２＋ / Mn ２＋ PPM1G promoter, ubiquitin-conjugating enzyme E2 T (UBE2T) ) promoter, checkpoint kinase 1 (CHEK1) promoter, epithelial cell type Transformation 2 promoter (ECT2), BCL2-like 12 (BCL2L12) promoter, Centromere protein I (CENPI) promoter, E2F transcription factor 1 (E2F1) Promoter, flavin adenine dinucleotide synthetase 1 (FLAD1) promoter tar, protein phosphatase, Mg ２＋ / Mn ２＋ Dependent 1G (PPM1G) Promo promoter, ubiquitin-conjugating enzyme E2S (UBE2S) promoter, aurora kinase A and and ninein-interacting protein (AUNIP) promoter, cell division cycle 6 (CDC6 ) promoter, centromere protein L (CENPL) promoter, DNA replication DNA2 promoter, DSN1 homolog, MIS12 Netcore complex component (DSN1) promoter, deoxythymidylate kinase (DTYM K) Promoter, G protein-regulated induction of neurite outgrowth 1 (GPRIN1) promoter Inducer, mitochondrial division regulator 2 (MTFR2) promoter, RAD51 associated protein 1 (RAD51AP1) promoter, small nuclear ribonucleoprotein poly Peptide A' (SNRPA1) promoter, ATPase family, AAA domain containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine BUB1 promoter, calcyclin-binding protein (CACYBP) ) promoter, cell division cycle associated 3 (CDCA3) promoter, centromere protein CENPO promoter, flap structure-specific endonuclease 1 (FEN 1) Promoter, Forkhead box M1 (FOXM1) promoter, cell proliferation Reg protein phosphatase 2A (KIAA1524) promoter, kinesin family KIF2C promoter, karyopherin subunit α2 (KP NA2 promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-related Neonate kinase 2 (NEK2) promoter, Ran-binding protein 1 (RANBP1) promoter Motor, small nuclear ribonucleoprotein polypeptide B and B1 (SNRPB) promoter tar, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, trait Transgenic acidic coiled-coil containing protein 3 (TACC3) promoter, TBC1 domain TBC1D31 promoter, thymidine kinase 1 (TK 1) Promoter, zinc finger protein 695 (ZNF695) promoter, - Aurora kinase A (AURKA) promoter, BLM RecQ-like helicase (BLM ) promoter, chromosome 17 open reading frame 53 (C17orf53) promoter promoter, chromobox 3 (CBX30) promoter, cyclin B1 (CCNB 1) Promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCN F), cell division cycle 20 (CDC20) promoter, cell division cycle 45 (CDC45) promoter Promoter, cell division cycle associated 5 (CDCA5) promoter, cyclin-dependent kinase CDKN3 promoter, cadherin EGF LAG 7-pass G-type receptor Centromere 3 (CELSR3) promoter, centromere protein A (CENPA) promoter tar, centrosome protein 72 (CEP72) promoter, CDC28 protein Protein kinase regulatory subunit 2 (CKS2) promoter, collagen type X α1 chain (CO L10A1) promoter, chromosome segregation 1-like (CSE1L) promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G Protein-coupled receptor 19 (GPR19) promoter, kinesin family member 1 8A (KIF18A) promoter, kinesin family member 4A (KIF4A) promoter promoter, kinesin family member C1 (KIFC1) promoter, minichromosome Maintenance 10 replication initiator (MCM10) promoter, minichromosome maintenance Coalescence component 2 (MCM2) promoter, minichromosome maintenance complex component component 7 (MCM7) promoter, MRG domain binding protein (MRGBP) promoter promoter, methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, metha MTHFD2 promoter, non-SMC conjugate Densin I complex subunit H (NCAPH) promoter, NDC80, kinetochore Complex component (NDC80) promoter, nudix hydrolase 1 ( NUDT1 promoter, ribonuclease H2 subunit A (RNASEH2A) Promoter, RuvB-like AAA ATPase 1 (RUVBL1) promoter, blood Serologically defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC binding Synaptic and spindle-associated 1 (SHCBP1) promoter, small nuclear ribonucleoprotein polypeptide Petide G (SNRPG) promoter, timeless circadian Regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter promoter, trophinin-related protein (TROAP) promoter, ubiquitin binding Enzyme E2 C (UBE2C) promoter, WD repeat and HMG box DNA binding protein 1 (WDHD1) promoter, alpha-fetoprotein (AFP) promoter 17. The method of claim 16, wherein the amino acid sequence is selected from the group consisting of a nucleotide sequence of the amino acid ... The method described below.

20. The biomarker is an MRI reporter, a PET reporter, a SPECT reporter, or , photoacoustic reporter, bioluminescent reporter, fluorescent reporter, chemiluminescent reporter, luminescence reporters, colorimetric reporters, quantifiable nucleic acid biomarkers, and any combination thereof 17. The method of claim 16, wherein the compound is selected from the group consisting of:

21. 21. The method of claim 20, wherein the quantifiable nucleic acid biomarker is an engineered miRNA. How to do it.

22. 21. The method of claim 20, wherein detection of the biomarker determines the location of the diseased cells. Law.

23. The biomarkers are detected by non-invasive imaging or a combination thereof. The method of claim 1, which is detectable in a body sample of a subject.

24. 24. The method of claim 23, wherein the biomarker is detected using a blood-based assay. How to do it.

25. The method of claim 1 , wherein the composition further comprises a transfection agent.

26. The transfection agent may be a linear or branched polyethyleneimine, a nanoparticle, a parent Oily particles, solid nanoparticles, peptides, micelles, dendrimers, polymer compositions, hydrogen exosomes, synthetic or naturally derived exosomes, virus-like particles, or any combination thereof.

26. The method of claim 25, wherein the

27. The method of claim 1 , wherein the composition further comprises a pharmaceutically acceptable carrier.

28. The pharmaceutically acceptable carrier may be water, peanut oil, soybean oil, mineral oil, sesame oil, saline, Acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urine Dextrose, dextrose solution, glycerol solution, glucose, lactose, sucrose, monostearate Glycerol phosphate, sodium chloride solution, propylene glycol, cocoa butter, or 28. The method of claim 27, wherein is selected from the group consisting of ethanol.

29. 1. A method of treating a subject having or suspected of having a disease, comprising administering to the subject and (b) expressing the therapeutically effective agent in a manner that is more effective in treating a disease associated with the disease than in treating a disease associated with the disease, rather than in treating a disease associated with the disease. administering to the subject a composition that induces expression of a therapeutically effective agent by the transformed cells, the relative concentration of the therapeutically effective agent expressed by said diseased cells relative to said non-diseased cells; and wherein the therapeutically effective agent is a cell population of the diseased cells. The method of treating said subject with at least 10% therapeutic efficacy as determined by a decrease in

30. 30. The method of claim 29, wherein the composition comprises a vector.

31. The composition is operably linked to a nucleotide sequence encoding a therapeutically effective agent.

31. The method of claim 30, comprising a promoter.

32. The therapeutically effective agent may be a therapeutically effective polypeptide, a small activating RNA (saRN), or a A), microRNA (miRNA), small interfering RNA (siRNA), or polypeptide The method of claim 31, wherein the nucleic acid is selected from the group consisting of a combination of a nucleotide and a nucleic acid.

33. The therapeutically effective agent is selected from the group consisting of HSVtk, cytosine deaminase, DT diaphorase, and nitric oxide. thrombin reductase, guanine phosphoribosyltransferase, purine nucleoside phosphate Phosphorylase, thymidine phosphorylase, carboxylesterase, folylpolyglucose Tamil synthetase, carboxypeptidase A1, carboxypeptidase G2, cytosine 33. The method of claim 32, wherein the chromium P-450 is selected from the group consisting of: chromium P-450;

34. A first nucleic acid sequence encoding a first polypeptide or nucleic acid biomarker and a second polypeptide or nucleic acid biomarker. and a second nucleic acid sequence encoding a polypeptide or a second nucleic acid biomarker. wherein the composition, when the composition is in a cell, comprises the second polypeptide or the nucleic acid biomarkers are present in amounts that reflect delivery of the first and second nucleic acids to the cell. and the first polypeptide or nucleic acid biomarker is expressed in diseased and non-diseased cells. Differentially expressed compositions.

35. (i) the cells express the first nucleic acid sequence preferentially in diseased cells over non-diseased cells; Inducing expression of said first nucleic acid sequence of said first polypeptide or nucleic acid, are detectable biomarkers or therapeutic agents; (ii) the cells express the second nucleic acid sequence equally in diseased and non-diseased cells; wherein the second nucleic acid sequence is not the detectable biomarker or the therapeutic agent. the second polypeptide or nucleic acid biomarker, thereby The level of expression of the peptide or nucleic acid biomarker is determined by the relative level of the nucleic acid sequence in the cell.

35. The composition of claim 34, which provides a control for evaluating the function.

36. In the composition, the first nucleic acid sequence encoding the first polypeptide and The sequence comprising the second nucleic acid sequence encoding the second polypeptide is a separate gene.

36. The composition of claim 35, which is on a construct.

37. the first polypeptide is the detectable biomarker and the therapeutic agent; Item 36. The composition according to item 35.

38. 36. The composition of claim 35, wherein the cell is a diseased cell.

39. 36. The composition of claim 35, wherein the composition further comprises a vector comprising the first and second nucleic acids. The composition described in

40. The vector is (a) a first promoter operably linked to the first nucleic acid sequence, The promoter directs expression of the first nucleic acid sequence in diseased cells preferentially over expression in non-diseased cells. a first promoter that drives expression of the first nucleic acid sequence in (b) induces expression equally in diseased and non-diseased cells and is operable with said second nucleic acid; a second promoter sequence linked to the 40. The composition of claim 39, comprising:

41. 40. The composition of claim 39, wherein the vector is a non-viral vector.

42. 42. The composition of claim 41, wherein the non-viral vector is a minicircle vector.

43. 40. The composition of claim 39, wherein the vector is a nanoplasmid vector.

44. The first and second polypeptide or nucleic acid biomarkers are used for non-invasive imaging or and the combination thereof is detectable in a body sample of said subject.

41. The composition of claim 40.

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