Improved methods and compositions for synthesis of biomarkers

Abstract

This disclosure covers embodiments comprising nucleic acids containing genetic elements that can be used to detect diseased cells. In some aspects, this disclosure provides a method comprising: (a) administering a composition to a subject, wherein the composition induces in the subject the expression of a biomarker in diseased cells preferentially over the expression of the biomarker in non-diseased cells, such that the relative ratio of the biomarker expressed in the diseased cells to that in the non-diseased cells is greater than 1.0; (b) detecting the biomarker; and (c) using the biomarker detected in (b) to determine with at least 70% accuracy that the subject has the diseased cells.

Description

[0001] Cross-references

[0002] The rights of U.S. Provisional Application No. 62 / 830,279, filed April 5, 2019, entitled “IMPROVED METHODS AND COMPOSITIONS FOR SYNTHETIC BIOMARKERS,” and U.S. Provisional Application No. 62 / 955,925, filed December 31, 2019, entitled “IMPROVED METHODS AND COMPOSITIONS FOR SYNTHETIC BIOMARKERS,” are hereby claimed, each of which is incorporated herein by reference in its entirety. Background Technology

[0003] Cancer is a massive global health problem. The World Health Organization estimates that there were an estimated 18.1 million new cancer diagnoses and 9.6 million cancer-related deaths in 2018 alone. The timing of cancer detection—before initial diagnosis and during tumor recurrence—is one of the most significant factors influencing patient outcomes, as current treatments are often more effective if detected early. Regrettably, most cancers are detected relatively late, leading to high mortality rates. Unless more effective detection strategies and therapies are developed, these mortality rates are expected to double by 2030. To curb the enormous loss of life caused by this horrific disease, there is an urgent need for widely available tools capable of detecting cancer at its earliest stages.

[0004] Two current paradigms for improving cancer detection include the development of blood-based assays and molecular imaging assays. Blood-based assays detect endogenous cancer biomarkers (such as proteins, microRNAs, circulating tumor DNA, circulating tumor cells, etc.) that are shed or released into the bloodstream, while molecular imaging assays utilize imaging probes that target biomarkers to better visualize tumors that cannot be detected by conventional anatomical imaging.

[0005] Blood tests are of great interest because they facilitate affordable cancer screening procedures, but they often encounter sensitivity and specificity issues due to: (Nagrath et al., (2007) Nature 450: 1235-1239); low blood biomarker concentrations; rapid biomarker degradation in vivo and in vitro (Haun et al., (2011) Sci. Translational Med. 3: 71ra16); and highly variable background expression in non-malignant tissues (Diamandis E P (2010) J. National Cancer Inst. 102: 1462-1467). Using current clinical biomarker assays, tumors have been estimated to grow for 10-12 years and reach a spherical diameter greater than 2.5 cm before endogenous blood biomarker levels are sufficient to indicate disease (Hori & Gambhir (2011) Sci. Translational Med. 3: 109ra116). Of the thousands of potential blood biomarkers reported, less than 1% are used clinically (7), and the implementation of new blood biomarkers in clinical settings is limited due to their lack of validated specificity and diagnostic value (Haun et al., (2011) Sci. Translational Med. 3: 71ra16; Kern SE (2012) Cancer Res. 72:6097-6101). In summary, although numerous attempts have been made to develop tools for the detection of blood biomarkers for endogenous cancers, very few have been successful. Therefore, there is an urgent need for new strategies and tools that can sensitively and specifically detect cancers. Summary of the Invention

[0006] In some aspects, this disclosure provides a method comprising: (a) administering a composition to a subject, wherein the composition induces in the subject the expression of a biomarker in diseased cells preferentially over the expression of the biomarker in non-diseased cells, such that the relative ratio of the biomarker expressed in the diseased cells to that in the non-diseased cells is greater than 1.0; (b) detecting the biomarker; and (c) using the biomarker detected in (b) to determine with at least 70% accuracy that the subject has the diseased cells.

[0007] In some aspects, this disclosure provides a method of treating a subject who has or is suspected of having a disease, the method comprising administering a composition to the subject, the composition inducing in the subject disease-associated disease cells to express a therapeutic efficacy preferentially to non-disease cells, such that the relative concentration of the therapeutic efficacy expressed by the disease-associated cells compared to the non-disease cells is greater than 1.0, the therapeutic efficacy treating the subject with at least 10% therapeutic efficacy, as determined by a reduction in the cell population of the disease-associated cells.

[0008] In some aspects, this disclosure provides a composition comprising a first nucleic acid sequence encoding a first polypeptide or nucleic acid biomarker and a second nucleic acid sequence encoding a second polypeptide or second nucleic acid biomarker, wherein the composition is configured such that, when the composition is in a cell: the amount of the second polypeptide or nucleic acid biomarker expressed reflects the delivery of the first and second nucleic acids to the cell, and the first polypeptide or nucleic acid biomarker is differentially expressed in diseased cells relative to non-diseased cells.

[0009] In some aspects, this disclosure provides a method for detecting diseased cells in a subject, the method comprising administering a composition to the subject, wherein the composition comprises: a first nucleic acid sequence encoding a first polypeptide or nucleic acid biomarker, and a second nucleic acid sequence encoding a second polypeptide or a second nucleic acid biomarker, wherein the composition is configured such that when the composition is in cells: (i) the cells induce expression of the first nucleic acid sequence in diseased cells preferentially over expression of the first nucleic acid sequence in non-diseased cells, wherein the first polypeptide is a detectable biomarker or therapeutic agent; and (ii) the cells induce equivalent expression of the second nucleic acid sequence in both diseased and non-diseased cells and the second nucleic acid sequence produces the second polypeptide, which is not a detectable biomarker or therapeutic agent, such that the expression level of the second polypeptide provides a control for assessing the relative levels of the nucleic acid sequence in the cells.

[0010] In some aspects, this disclosure provides a composition comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, wherein the composition is configured such that 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 configured to combine to form a heterodimeric protein.

[0011] In some aspects, this disclosure provides a method for detecting or treating diseased cells, the method comprising administering the composition described above, the composition comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, wherein the first polypeptide and the second polypeptide are selectively transcribed or translated in the diseased cells.

[0012] In some aspects, this disclosure provides a composition comprising a non-naturally occurring recombinant gene construct comprising a sequence encoding a polypeptide or nucleic acid sequence, wherein the sequence comprises a first promoter that, when introduced into cells in vitro, selectively drives the expression of the polypeptide or nucleic acid biomarker sequence in a variety of different cell types isolated from a subject.

[0013] In some aspects, this disclosure provides a method for detecting diseased or impaired cells in vitro, the method comprising delivering a non-naturally occurring recombinant gene construct to a cell population isolated from a subject in vitro, wherein the non-naturally occurring recombinant gene construct comprises: a sequence encoding a polypeptide or nucleic acid biomarker sequence, wherein the sequence comprises a first promoter that, when transduced into cells, selectively drives the expression of the polypeptide or nucleic acid biomarker sequence in a variety of different cell types isolated from the subject.

[0014] In some aspects, this disclosure provides a composition comprising a vector, wherein the vector comprises a plurality of different promoters operatively linked to a plurality of different nucleic acid sequences, wherein each of the promoters drives the expression of the plurality of nucleic acid sequences in a cell to produce a plurality of polypeptide or nucleic acid biomarker sequences, wherein the level of each polypeptide or nucleic acid biomarker sequence in the plurality of nucleic acid sequences indicates a stage of disease of the cell or the tissue from which the cell is derived.

[0015] In some aspects, this disclosure provides a method for detecting a stage of a disease, the method comprising administering to a subject a composition comprising a vector, wherein the vector comprises a plurality of different promoters operatively linked to a plurality of different nucleic acid sequences, wherein each of the promoters drives the expression of the plurality of nucleic acid sequences in cells to produce a plurality of polypeptide or nucleic acid biomarker sequences, wherein the level of each polypeptide of the plurality of nucleic acid sequences indicates a stage of disease in the cells or a tissue from which the cells are derived.

[0016] In some aspects, this disclosure provides a composition comprising an engineered nucleic acid encoding an expressible reporter gene that exhibits about 10% or less expression in normal cells relative to diseased cells when compared to a recombinant nucleic acid comprising a reporter gene containing the nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.

[0017] In some aspects, this disclosure provides a method comprising administering the composition to a subject, the composition comprising an engineered nucleic acid encoding the above-described expressible reporter gene.

[0018] In some aspects, this disclosure provides a composition that exhibits about 10% or less expression in normal cells compared to diseased cells and comprises a recombinant nucleic acid containing a nucleic acid sequence encoding a reporter gene, wherein the reporter gene contains one or more miRNA binding sequences in its 3′ untranslated region.

[0019] In some aspects, this disclosure provides a method for detecting diseased cells, the method comprising administering the above-described composition to a subject, the composition exhibiting about 10% or less expression in normal cells compared to diseased cells.

[0020] In some aspects, this disclosure provides a composition that exhibits significantly longer expression of a synthesized biomarker compared to plasmid DNA or microcircular DNA, the composition comprising a linear vector comprising a double-stranded nucleic acid containing a promoter operatively linked to a DNA sequence encoding the synthesized biomarker, wherein the forward and reverse strands of the double-stranded nucleic acid are covalently linked at their respective ends, wherein the promoter induces expression of the synthesized biomarker in diseased cells preferentially over expression of the synthesized biomarker in non-diseased cells, such that the relative concentration of the synthesized biomarker expressed in the diseased cells compared to the non-diseased cells is greater than 1.0.

[0021] In some aspects, this disclosure provides a method for identifying diseased cells, the method comprising administering to a subject a composition that exhibits significantly longer expression of a synthetic biomarker compared to plasmid DNA or microcircular DNA, and detecting the synthetic biomarker, wherein expression of the synthetic biomarker in diseased cells is preferred in the subject than expression of the synthetic biomarker in non-diseased cells, such that the relative concentration of the synthetic biomarker expressed in the diseased cells compared to the non-diseased cells is greater than 1.0.

[0022] In some aspects, this disclosure provides a composition that exhibits significantly longer expression of a synthetic biomarker compared to plasmid DNA or microcircular DNA, the composition comprising a linear vector comprising a double-stranded nucleic acid containing a promoter operatively linked to a DNA sequence encoding a therapeutic agent, wherein the forward and reverse strands of the double-stranded nucleic acid are covalently linked at their respective ends, wherein the promoter induces expression of the therapeutic agent in diseased cells preferentially over expression of the synthetic biomarker in non-diseased cells, such that the relative concentration of the therapeutic agent expressed in the diseased cells compared to the non-diseased cells is greater than 1.0.

[0023] In some aspects, this disclosure provides a method for treating diseased cells, the method comprising administering the above-described composition to a subject and detecting the synthetic biomarker, wherein the expression of the synthetic biomarker in the subject is preferential in diseased cells over the expression of the synthetic biomarker in non-diseased cells, such that the relative concentration of the synthetic biomarker expressed in the diseased cells compared to the non-diseased cells is greater than 1.0.

[0024] In some aspects, this disclosure provides a composition comprising a nonviral vector expressing a synthetic biomarker, wherein the synthetic biomarker exhibits expression of about 10% or less in normal organ cells compared to diseased cells.

[0025] In some aspects, this disclosure provides an engineered particle that mimics one or more functions of biological cells or macrophages, including inducing the expression of a biomarker in diseased cells preferentially over the expression of the biomarker in non-diseased cells, such that the relative concentration ratio of the biomarker expressed in the diseased cells to that expressed in the non-diseased cells is greater than 1.0.

[0026] In some aspects, this disclosure provides at least one vector comprising: a plurality of different promoters operatively linked to a plurality of different nucleic acid sequences, wherein the promoters drive the expression of the plurality of nucleic acid sequences in cells to produce a plurality of polypeptide or nucleic acid biomarker sequences, wherein in a subject, the promoters induce expression of the plurality of polypeptide or nucleic acid biomarker sequences in diseased cells preferentially over expression of the plurality of polypeptide or nucleic acid biomarker sequences in non-diseased cells, such that the relative ratio of the plurality of polypeptide or nucleic acid biomarker sequences expressed in the diseased cells to those expressed in the non-diseased cells is greater than 1.0.

[0027] In some aspects, this disclosure provides a method for detecting a disease in a subject, the method comprising: administering a composition to the subject, the composition comprising at least one vector comprising a variety of different promoters operatively linked to a variety of different nucleic acid sequences; detecting a variety of polypeptide or nucleic acid biomarker sequences to obtain an expression profile; and detecting an expression profile based on diseased cells to detect the disease.

[0028] In some aspects, this disclosure provides a method for detecting a disease or the absence of a disease in a subject, the method comprising contacting one or more cells of the subject with a gene construct in vitro, wherein: the gene construct contains a disease-activated promoter operatively linked to a barcode molecule, and the disease-activated promoter drives the expression of the barcode molecule in cells suffering from the disease; quantifying the expression level of the barcode molecule; and detecting the disease or its absence based on the expression level.

[0029] By assigning unique markers to unique members within a larger group, barcoding provides the opportunity to identify and quantify a member (e.g., the expression of a reporter gene under the control of a specific cancer-specific promoter) within a larger and more complex mixture of many members (e.g., multiple promoter-reporter gene constructs expressed within the same cell), and also provides the opportunity to isolate individual members from complex mixtures. For example, in the case of nucleic acid-based barcoding, hybridization based on base pair complementarity can be used for capture and separation, or otherwise reduce the complexity of the mixture through said capture event. For peptide-based barcoding, unique characteristics (including immune capture or interaction of ligands and receptors) can be used for capture and separation, or otherwise reduce the complexity of the mixture through said capture event.

[0030] In some aspects, this disclosure provides a method for generating a disease spectrum of a subject, the method comprising contacting one or more cells of the subject with a plurality of gene constructs, wherein: the plurality of gene constructs include a plurality of disease-activated promoters operatively linked to a plurality of barcode molecules, and the disease-activated promoters drive the expression of the corresponding barcode molecules in cells suffering from the disease; and quantifying the expression levels of the plurality of barcode molecules to generate the spectrum.

[0031] Other aspects and advantages of this disclosure will become apparent to those skilled in the art from the following detailed description of illustrative embodiments shown and described therein only. It will be appreciated that this disclosure can have other and different embodiments, and that several details thereof can be modified in various obvious respects without departing from this disclosure. Therefore, the drawings and description are to be considered illustrative rather than limiting in nature.

[0032] Incorporation

[0033] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference. Where a publication, patent, or patent application incorporated by reference conflicts with the disclosure contained herein, this specification is intended to supersede and / or give precedence to any such contradictory material. Attached Figure Description

[0034] The novel features of the invention are set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description of illustrative embodiments in which the principles of the invention are utilized, along with the accompanying drawings (also referred to herein as “Figures”):

[0035] Figure 1 This paper exemplifies a blood-based tumor-activated microloop (MC) approach for cancer detection. (A) A tumor-activated MC, driven by a tumor-specific promoter and encoding a secreted reporter protein, is complexed with a non-targeted transfection agent (TA). These nanocomposites are delivered systemically (via the tail vein). (B) MCs are transfected into numerous tissues, but the production of the reporter protein occurs almost entirely within tumor cells, and the expressed reporter is secreted into the bloodstream (BS). Due to promoter leakage, minimal protein expression should occur in tumor-free subjects. (C) Collecting blood and detecting the secreted reporter in the plasma enables differentiation between tumor-positive (reporter-positive) and tumor-free (reporter-negative) subjects.

[0036] Figure 2A-2B This describes the design and construction of a tumor-activated carrier. Figure 2A This diagram illustrates the vector representations of the parental plasmid (PP; top) and MC (bottom) driven by the survival protein promoter (pSurv). These constructs encode embryonic alkaline phosphatase (SEAP), a reporter protein secreted by the embryo. PP and MC share the same transcriptional unit (pSurv-SEAP-WPRE-polyA), but MC lacks the prokaryotic backbone (light gray). WPRE (Marmot hepatitis virus posttranscriptional regulatory element (WPRE)). Figure 2BThis demonstrates the ability of agarose gel electrophoresis to produce both PP (7.9 kb) and MC (4.1 kb).

[0037] Figure 3 This is a schematic diagram of the microring carrier construct MC-pSurv-SEAP-WPRE-SV40 poly-A-pause.

[0038] Figure 4 This is a schematic diagram of the microring carrier construct MC-pSurv-Luc2-WPRE-SV40 poly-A-pause.

[0039] Figure 5 This is a diagram illustrating the comparison between tumor-specific plasmids (PP-SEAP) and microloops (MC-SEAP) in MeWo human melanoma cancer cells.

[0040] Figure 6 This is a diagram illustrating the comparison between tumor-specific plasmids (PP-SEAP) and microloops (MC-SEAP) in SK-MEL-28 human melanoma cancer cells.

[0041] Figure 7 This is a standard curve for SEAP assays based on blood tests for cancer detection following systemic administration of tumor-specific SEAP microcircles. Standard curve analysis of SEAP assays showed values ​​above approximately 10. 4 The RLU value is within the linear region of detectable SEAP levels in plasma.

[0042] Figure 8 This figure illustrates the effect of intratumoral administration of tumor-activated MC on detectable blood reporter activity. Nude mice with subcutaneous human melanoma xenografts were administered intratumorally (IT) with SEAP-expressing tumor-activated MC (n=4; MC IT) or 5% glucose (n=3; mock). Control mice also received intramuscular (IM) injections of MC (n=3; MCI.M.). Plasma SEAP measurements before and up to 2 weeks after MC administration showed elevated SEAP levels in MC IT mice from day 3 to day 14 (*p<0.05; **p<0.01; ***p<0.001). Data are presented as mean ± SD.

[0043] Figure 9 This study demonstrates the activity of SEAP in the blood of representative mice following systemic administration of tumor-specific SEAP microcircuits.

[0044] Figure 10This figure illustrates blood-based cancer detection following systemic administration of tumor-specific SEAP microcircles. Significantly higher SEAP activity was detected in blood samples from tumor-bearing mice from day 3 to day 14 after MC injection compared to control mice (p<0.05). No significant differences were observed between control mice receiving MC or 5% glucose. Error bars represent SD.

[0045] Figure 11 This is a series of digital images illustrating molecular genetic imaging cancer detection 3 days after systemic administration of tumor-specific FLUC microloops.

[0046] Figure 12 This is a diagram illustrating molecular genetic imaging for cancer detection following systemic administration of tumor-specific FLUC microloops.

[0047] Figure 13 Describe the nucleic acid sequence of the microcircular MC-pSurv-SEAP-WPRE-pA.

[0048] Figure 14 Describe the nucleic acid sequence of the microcircular MC-pSurv-Luc2-WPRE-pA.

[0049] Figure 15 This is a graph illustrating a comparison of transfection of the constructs of this disclosure into cultured cancer cells. Transfection of equal masses of MC (n=3) and PP (n=3) into MeWo human melanoma cells using equal volumes of transfection reagent resulted in significantly higher SEAP concentrations in the medium containing MC from day 3 to day 8 (**p<0.01; ***p<0.001). Data are presented as mean ± SD.

[0050] Figures 16A-16D This demonstrates that systemic delivery of tumor-activated MCs allows for the identification of subjects with tumors. Figures 16A-16C This image illustrates the progression of human melanoma tumors in nude mice (n=7) monitored using bioluminescence imaging (BLI) following intravenous cellular administration (left image). Representative BLI images show tumor growth primarily within the lungs and a broad range of tumor burdens in each mouse within 3 days prior to MC administration. The BLI scale is shown in [image description missing]. Figure 16A and 16B The middle is the same, but Figure 16C The scale was one order of magnitude lower. Tumor-activated MC was administered systemically, and SEAP levels were measured before administration (day 0) and up to 14 days after administration (right figure). Variable SEAP concentrations were detected in mice with tumors during the 14-day period. Figure 16DThis study described healthy (tumor-free) mice receiving either MC (control + MC; n=7) or only 5% glucose load (control - MC; n=5). No statistically significant difference in plasma SEAP levels was detected between these two groups. Importantly, regardless of tumor burden, significantly higher plasma SEAP concentrations were detected in tumor-bearing mice receiving MC from day 3 to day 14 compared to both control groups (#*p<0.05; ##**p<0.01). Data are presented as mean ± SEM.

[0051] Figures 17A-17C This demonstrates that tumor-activated MCs can robustly identify subjects with tumors and measure tumor burden. Figure 17A Compared with healthy mice receiving MC (n=7) or 5% glucose (n=5), area under the curve (AUC) analysis of plasma SEAP measurements during the 2-week period showed a significant difference between tumor-bearing mice (n=7) receiving MC (*p<0.05; **p<0.01). Data are presented as mean SD. Figure 17B ROC (recipient operating characteristic curve) analysis showed that the tumor-activated MC system has a significant ability to distinguish between subjects with tumors and healthy subjects by measuring and calculating plasma SEAP AUC. Figure 17C Correlation analysis of SEAP AUC measurements and lung tumor burden (e.g., measured by BLI lung mean radiance). A significant positive correlation was observed between these two measurements in 6 mice, demonstrating the ability of our tool to assess tumor burden provided the tumor is located in a given area. One mouse was removed from the analysis (square symbol) because it had tumors in the lungs and multiple metastatic lesions outside the lungs (BLI measurements were performed only within the lungs, explaining the overall low BLI signal in this mouse). This mouse had a higher SEAP AUC level than might be expected based on its lung tumor burden.

[0052] Figures 18A-18D This study describes a comparison of promoter activity in vivo in healthy (tumor-free) mice. Mice were systemically administered a plasmid (30 µg; PGL4.2 backbone; complexed with PEI (N / P=6)) expressing a bioluminescence imaging (BLI) reporter gene codon-optimized firefly luciferase (Luc2) driven by pCMV (n=3), pSurv (n=5), or pPEG (n=3). Mice receiving a simulated injection received 5% glucose (n=3). Each mouse was also co-injected with a plasmid expressing a pCMV-driven BLI reporter gene humanized Renilla luciferase (hRluc) to assess transfection efficiency (3 µg; 10-fold lower than Luc2 plasmid mass). Figure 18AThis image illustrates representative BLI images 48 hours post-injection. The scale of the images used for pCMV mice is two orders of magnitude larger than that of all other mice. BLI signaling, primarily in the lungs, was observed in all mice that received the Luc2 plasmid. Figure 18B Analysis of the region of interest across the entire mouse population on BLI images revealed significantly higher (*p<0.05; approximately 100-fold) BLI signaling in mice receiving the pCMV-Luc2 plasmid compared to all other mice (*p<0.05). Significantly higher (*p<0.05) BLI signaling was also observed in pPEG mice compared to mice receiving the simulated injection. While qualitatively higher BLI signaling was observed in pSurv mice compared to mice receiving the simulated injection, quantitative measurements only showed a trend toward higher BLI signaling (p=0.16). Therefore, in this mouse strain, Luc2 expression was lowest in normal tissues under tumor-specific pSurv conditions. Figure 18C In vitro analysis of Luc2 activity in many tissues 48 h after plasmid injection showed significantly higher (*p<0.05) expression in the pCMV setting compared to all other groups. In the pPEG setting, significantly higher (*p<0.05) Luc2 activity was observed in the heart, lungs, and spleen compared to animals receiving the simulated injection. In the pSurv setting, Luc2 activity was significantly higher in the spleen (*p<0.05) and tended towards higher activity in the lungs (p=0.13). Figure 18D The only tissue exhibiting elevated hRluc activity above background levels was the lung (values ​​shown were normalized to the mean background value from mice receiving the simulated injection). Therefore, from imaging ( Figure 18B ) and in vitro tissue analysis ( Figure 18C The determined Luc2 values ​​were not normalized to hRluc values. No significant differences in hRluc values ​​were observed in the lungs across the three promoter mouse groups. Therefore, the differences in Luc2 measurements across the three groups are unlikely to be related to differences in transfection efficiency, but rather to differences in promoter activity. Data are presented as mean ± SD.

[0053] Figures 19A-19CThis study compares tumor-specific promoter activities 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 expressing Luc2 (1 µg) and co-transfected with promoter-free plasmids expressing hRluc (50 ng) to normalize transfection efficiency. No difference in Rluc transfection efficiency was observed among any of the three cell types. Compared with pSurv, pPEG-driven plasmids resulted in significantly higher Luc2 activity in fibroblasts (*p<0.05). pSurv-driven plasmids resulted in significantly higher Luc2 activity in MeWo cells (***p<0.001) and equivalent activity in MDA-MB-231 cells. Data are presented as mean ± SD.

[0054] Figure 20 This study compares tumor-activated PP and MC in cultured SK-MEL-28 melanoma cells. SK-MEL-28 human melanoma cells were transfected with equal masses of tumor-activated MC (n=3) and PP (n=3), and an equal volume of transfection agent PEI. Significantly higher SEAP activity was observed in cells transfected with MC from day 2 to day 7 (**p<0.01; ***p<0.001). Data are presented as mean ± SD.

[0055] Figure 21A and 21B This study compares transgene expression between MC and PP, driven by a strong constitutive promoter, in healthy (tumor-free) mice. Figure 21A Mice were shown to express hRluc driven by a strong constitutive EF1 promoter after systemic administration of MC (n=4) or PP (n=5) and complexation with PEI (40 µg; N / P=8). BLI imaging was performed using the substrate coelenterate on days 1, 2, 3, 5, and 7. Representative images show higher BLI signals in mice administered MC at all time points examined. For comparison, signals from mice receiving 5% glucose injection are shown (signals in the liver are derived from oxidized coelenterate). Figure 21B Analysis of regions of interest in the lung region showed significantly higher BLI signal in MC mice compared to PP mice on days 1, 2, and 5 (*p<0.05; **p<0.01). Data are presented as mean ± SD.

[0056] Figure 22AThis study describes the standard curve analysis for plasma SEAP assays. Three samples were measured in 25 µL of plasma with SEAP diluted 10-fold. SEAP activity was linear across five orders of magnitude and showed approximately 3 x 10⁻⁶ ppm in 25 µL of plasma. -7 The detection limit is µg (0.3 pg). Figure 22B This indicates that the SEAP measurements are reproducible across the entire linear range, with a coefficient of variation (% CV) of less than 4%.

[0057] Figure 23 This illustrates the tumor burden before and after MC administration. Bioluminescence (BLI) images (left) of two representative mice (top and bottom) before and two weeks after MC administration, and the corresponding in vitro image of the lungs at sacrifice (2 weeks after MC administration) (right). The value below each BLI image represents the mean radiance of the region of interest plotted on the lung. The image scales differ between the two mice. During the 2-week period after MC administration, both mice showed an approximately 4.5-fold increase in BLI signal, indicating continued tumor growth. At sacrifice, the tumors in the lungs were melanotic, and multiple tumor lesions were observed throughout the lungs in both mice (white arrows). Based on the changes in BLI signal, the total tumor burden at MC administration (two weeks before sacrifice) would be approximately 4.5-fold smaller than that seen in the in vitro images shown here.

[0058] Figure 24 The experimental results of Example 11 illustrate that when cells expressing FLuc are incorporated into normal PBMCs, the detection limit of this detection method is at least 3-10 diseased cells / 5 million normal PBMCs.

[0059] Figure 25 To illustrate the experimental results of Example 12, the cancer-activated DNA construct distinguished between mice with tumors and healthy mice: After intravenous administration of the viable-SEAP DNA nanoparticles, whole blood was collected via submandibular pleophoresis and processed into plasma. SEAP assays were performed on 20 µl aliquots. The cohort size was n=5.

[0060] Figures 26A-26FThis study describes the in vivo efficacy and in vitro cytotoxicity of the polymer / DNA complex. Figure 26A shows the experiments in which 40 µg of DNA encoding CMV-luciferase was formulated in the polymer formulation or complexed with JetPEI and then administered intravenously to Balb / C mice. Four days post-transfection, D-luciferin was injected into the animals, followed by animal sacrifice, and lungs were harvested for in vivo BLI analysis. For cytotoxicity assessment (Figures 26B, 26C, 26D, 26E, and 26F), a polyplex containing 250 ng of CMV-Luc DNA was added to each well of a 96-well plate on the day prior to transfection, seeding 10,000 cells / well. Each formulation was tested in triplicate. Forty-eight hours later, cell morphology was recorded by microscopy, and cell viability was measured during the MTT assay; Figure 26B shows blank cells; Figure 26C shows in vivo delivery of the JetPEI construct; Figure 26D shows delivery of DNA with high molecular weight amino-terminated poly(β-amino ester) C32-122; Figure 26E shows delivery of DNA with high molecular weight amino-terminated poly(β-amino ester) C32-145; and Figure 26F shows the cell viability results of the MTT assay.

[0061] Figure 27 The size of the protamine-condensed DNA polymeric complex was determined. 62.5 µg of DNA was condensed by completely mixing with 130 µg of protamine at a 1:1 v / v 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). The protamine:DNA LNP was assembled on a NanoAssemblr (Precision NanoSystems) at a total flow rate of 12 mL / min. The prepared particles were dialyzed against 1X PBS for at least 18 h, and their size was then determined using a Zetasizer.

[0062] Figure 28 Examples illustrating the use of luciferase to determine the biodistribution of a delivery formulation. A) In vivo bioluminescence imaging (BLI) of mice administered 40 mg of CMV luciferase carrier (pre-formulated in JetPEI) via tail vein injection. Four days after administration, mice were anesthetized, administered D-luciferin substrate, and immediately imaged on AMI-HT (Spectral Instruments Imaging). B) Following in vivo imaging, mice were sacrificed, and organs were harvested for in vitro BLI.

[0063] Figures 29A and 29B illustrate the sensitivity and specificity of Ad-survival protein-FLuc in in vitro assays in canine PBMCs and cells derived from canine tumors. Untransduced naïve cells from various subtypes of canine malignancies (including osteosarcoma, melanoma, and angiosarcoma) were infused into 5e⁵ canine PBMCs and then transduced with Ad-survival protein-FLuc at 0.3 MOI. A) Analysis shows single-cell detection of A17 osteosarcoma cells, or B) robustness of detection in cells derived from multiple tumor types.

[0064] Figures 30A-C illustrate the sensitivity and specificity of Ad-survival protein-FLuc in in vitro assays. A) H1299 cells engineered to constitutively express firefly luciferase protein were incorporated into 5e6 human normal PBMCs, and the whole sample was then processed, and luciferase expression was analyzed. B) Untransduced naïve H1299 cells were incorporated into human PBMCs and then transduced with a recombinant adenovirus containing an expression cassette (Ad-survival protein-FLuc) that drives the expression of firefly luciferase. After 48 hours of growth, the sample was processed, and luciferase expression was analyzed. C) Samples with only human PBMCs were transduced with Ad-survival protein-FLuc or Ad-CMV-FLuc, the latter controlled by a strongly constitutive promoter. After 2 days of incubation, luminescence assays were used to quantify FLuc expression.

[0065] Figure 31 This study demonstrated the ability of Ad-Survival Protein-Fluc to differentiate cancer cells from normal cells in an in vitro assay of human PBMCs. Commercially available samples of human PBMCs from healthy volunteers and cancer patients were counted, and then equivalent numbers of cells were transduced with Ad-Survival Protein-Fluc at a consistent MOI. Samples were then split into triplicate. After three days of incubation, cells were lysed, and luciferase activity was analyzed. Data were calculated as the mean and standard deviation of measurements from the triplicate samples. P-values ​​were calculated using a Student's t-test relative to normal PBMCs.

[0066] Figures 32A and 32B show the diagnostic performance of luciferase expression of the survival protein activation in distinguishing between healthy canine individuals and canine lymphoma cancer patients. (A) Comparison of fold changes in luciferase expression in healthy canine individuals (n=31) and canine lymphoma cancer patients (n=17). (B) Diagnostic predictive ability of luciferase activity of the survival protein activation in distinguishing between canine lymphoma cancer subjects and healthy canine subjects.

[0067] Figures 33A, 33B, 33C, 33D, 33E, and 33F illustrate the activation of various promoter-reporter constructs in specific cell lines from different tissue sources, where gene markers indicate the source of the promoters used in the constructs. Figure 33A shows various promoter-reporter constructs in liver-derived cell lines (e.g., HepG2 and Hep3B), indicating that CXCR4, TRIP13, MCM10, COL10A1, BIRC5, and BIRC5-501 are particularly activated in hepatocellular carcinoma. Figure 33B shows the activation of various promoter-reporter constructs in immortalized ovarian-derived cell lines (e.g., SKOV3 and OVCAR), indicating 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 pancreatic immortalized cell lines (e.g., ASPC1, BXPC3, and PANC1), indicating 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), indicating 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 lung-derived cell lines (e.g., A549, H460, and H1299), indicating 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 constructs as in 33E in normal lung cell lines, suggesting that genes other than MMP1 that are activated in 33E may be particularly useful for identifying lung cancer from normal tissue.

[0068] Figures 34A, 34B, and 34C illustrate the activation of the promoter-reporter construct group in melanoma, osteosarcoma, and angiosarcoma cancer cell lines, where gene markers indicate the promoters used in the constructs. Figure 34A shows the activation of members of this group (e.g., M2, M3, M4, M5, and CMGD) in melanoma-derived cell lines, indicating that BIRC5, BIRC5-501, CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are particularly activated in melanoma. Figure 34B shows the activation of members of this group (e.g., OS17, OS29, OS40, and OS484) in osteosarcoma-derived cell lines, indicating that BIRC5, BIRC5-501, CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are particularly activated in osteosarcoma. Figure 34C shows the activation of members of this group in angiosarcoma-derived cell lines, indicating that BIRC5, BIRC5-501, MMP13, CXCR4, UBE2C, TRIP13, CDKN3, MCM10, CDC20, TROAP, CEP55, KIF20A, and cBIRC5 are particularly activated in angiosarcoma.

[0069] Figures 35A, 35B, and 35C illustrate the design of a multiplex assay utilizing various cancer-specific promoters and linked barcodes. Figure 35A shows the design of multiplex constructs where various cancer-specific promoters (non-descriptively denoted as Px, Py, and Pz) are used to drive the expression of an orthogonal reporter generated by the fusion of a signal peptide with luciferase, wherein intercalated nucleic acid barcode sequences are unique for the promoters used to drive the constructs (barcodes A, B, and C are 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. This demonstrates that co-transfection of multiple reporter-promoter constructs into the same cell does not significantly alter the expression pattern of a given promoter in a given cell line, indicating that multiplex is an implementable form for generating a “spectrum” of promoter activation in a single cell type.

[0070] Figures 36A, 36B, and 36C illustrate designs for multiplex assays using various different peptide epitopes to detect reporter constructs driven by individual promoters. Figure 36A shows a multiplex construct design where different copies of the CMV promoter drive the expression of an orthogonal reporter generated by the fusion of a signal peptide with luciferase, where intercalation epitope peptides (e.g., FLAG, HA, V5, or HSV peptide epitopes) barcodes are unique to the promoter used to drive the construct (Figure 36A shows the CMV promoter used, but ultimately several different promoters are envisioned, such as Px, Py, Pz, etc. in Figure 35). Figure 36B shows how multiple epitope barcodes can be used in conjunction with epitope-specific capture antibodies (e.g., anti-FLAG, anti-HA, anti-V5, or anti-HSV) to isolate the secreted reporter construct for independent measurements of activity against each promoter. Figure 36C shows the co-transfection of luciferase constructs barcoded with FLAG / HA / V5 / HSV into cells, demonstrating that luciferase constructs tagged with each peptide epitope can be isolated and used to independently read out promoter activation in the same cells.

[0071] Figures 37A, 37B, and 37C show the design of a reporter-promoter construct designed to detect activation of the promoter-reporter construct in cancer cell lines using off-the-shelf lateral flow assays (e.g., pregnancy hCG lateral flow immunoassay), and corresponding performance data. In this design, a cancer-specific promoter (Px, represented in this case by the survival protein promoter) drives the expression of a secretory signal-modified luciferase (which is also fused to a human chorionic gonadotropin (hCG) epitope). Figure 37B shows that transfection of H1299 cells with various associated constructs did not significantly interrupt luciferase expression from the survival protein promoter. Figure 37C shows that the supernatant from transfected cells can be loaded onto a commercial lateral flow immunoassay strip for hCG, and the lateral flow immunoassay can detect hCG-tagged luciferase, demonstrating the utility of using existing epitope immunoassays to read out the expression of promoter-reporter constructs where the reporter is epitope-tagged with a high-confidence off-the-shelf assay.

[0072] Figure 38 This document shows an example of the nanoparticle-based promoter construct described herein. The sequence of this construct is summarized in SEQ ID NO: 5 and includes a small R6K origin, an RNA-out optional marker, a survival protein promoter, SEAP as a reporter, and a WPRE element. Detailed Implementation

[0073] While various embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

[0074] Before describing this disclosure in more detail, it should be understood that this disclosure is not limited to the specific embodiments described, and is therefore subject to change. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of this disclosure will be limited only by the appended claims.

[0075] Where a range of values ​​is provided, unless the context explicitly indicates otherwise, it should be understood that every intermediate value between the upper and lower limits of the range up to one-tenth of the lower limit, as well as any other stated value or intermediate value within the range, is included within this disclosure. The upper and lower limits of these smaller ranges may be independently included within that smaller range and also included within this disclosure, with any explicitly excluded limitations within the range. When the range includes one or both of the limits, the range excluding one or both of those included limits is also included in this disclosure.

[0076] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any methods and materials similar to or equivalent to those described herein may be used to implement or test this disclosure, preferred methods and materials are now described.

[0077] All publications and patents referenced in this specification are incorporated herein by reference, as specifically and individually indicated by reference for each individual publication and patent, and are incorporated herein by reference to disclose and describe methods and / or materials relating to the referenced publication. References to any publication are for its publication prior to the date of this application and should not be construed as an admission that this disclosure is not prior to such publication in light of existing disclosures. Furthermore, the dates of the publications provided may differ from (and may require independent verification of) the actual publication date.

[0078] Upon reading this disclosure, it will become apparent to those skilled in the art that each individual embodiment described and illustrated herein has discrete components and features that can be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of this disclosure. Any described method may be performed in the order of the events described or in any other logically possible order.

[0079] Unless otherwise stated, embodiments of this disclosure will employ techniques from medicine, organic chemistry, biochemistry, molecular biology, pharmacology, toxicology, etc., which are within the scope of the art. Such techniques are well explained in the literature.

[0080] It must be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “a support” includes multiple supports. In this specification and the following claims, several terms will be referenced and defined as having the following meanings, unless it is obvious to the contrary.

[0081] As used herein, unless otherwise specified, the following terms have their respective meanings. In this disclosure, "comprising," "including," "containing," and "having," etc., may have their meanings under U.S. patent law and may mean "comprising," "including," etc.; "consisting substantially of," or "consisting substantially of," when applied to the methods and compositions covered by this disclosure, refers to a composition, such as those disclosed herein, but which may contain additional structural groups, compositional components, or method steps (or their analogues or derivatives as described above). However, such additional structural groups, compositional components, or method steps, etc., do not substantially affect the essential and novel features of the composition or method compared to those of the corresponding compositions or methods disclosed herein.

[0082] Before describing the various implementation schemes, the following definitions are provided and shall be used unless otherwise indicated.

[0083] definition

[0084] The term "subject" can include humans or non-human animals. Therefore, the methods and compositions described herein are applicable to human and veterinary diseases and animal models. A preferred subject is a "patient," i.e., a living human receiving medical care for a disease or condition. This includes individuals without a defined disease, for whom pathological signs are being studied. It also includes individuals suspected of having or at risk of a defined disease.

[0085] As used herein, the term "gene" refers to all regulatory and coding sequences associated with a single genetic unit that has a hereditary function. Genes may include non-coding sequences that regulate genetic function, including, but not limited to, those that specify polyadenylation, transcriptional regulation, DNA conformation, chromatin conformation, the degree and location of base methylation, and the binding sites of proteins that control all of these. Protein-coding genes include "exons" (coding sequences), which may be interrupted by "introns" (non-coding sequences). In some cases, a complex of multiple proteins or nucleic acids or other molecules, or a complex of any two of the foregoing, may be required for gene function. On the other hand, a gene's genetic function may require only RNA expression or protein production, or it may require only the binding of proteins and / or nucleic acids without related expression. In some cases, adjacent genes may share sequences such that one gene will overlap with another. Genes can be found within the genome of an organism, in artificial chromosomes, in plasmids, in any other kind of vector, or as separate, isolated entities.

[0086] As used herein, the term "epistone replication vector" or "epistone vector" refers to a vector that is typically not integrated into the host cell's genome but exists in parallel with it. Episone replication vectors can replicate during the cell cycle, and during this replication, vector copies are statistically distributed throughout the resulting cells according to the number of copies present before and after cell division. Replication can occur in the host cell's nucleus and preferably during the S phase of the cell cycle. Furthermore, episone replication vectors can replicate at least once, i.e., once or multiple times, in the host cell's nucleus during the S phase of the cell cycle.

[0087] The term "sample" is defined as any material to be tested in the analytical or experimental methods described herein. Samples are typically obtained from the subject described herein. Samples include, but are not limited to, blood or blood fractions, saliva, urine, feces, cerebrospinal fluid, semen, vaginal secretions, sputum, sweat, breast milk, synovial fluid, mucus (including inflammatory secretions), tears, bile, gastric juice, interstitial fluid, tissue biopsies or epithelial cells (which are naturally shed or intentionally collected from the body (e.g., cheek cell scraping), aqueous humor, amniotic fluid, pleural fluid, or the subject's breath. In some embodiments, the sample is obtained via a non-invasive method (e.g., a non-invasive sample). Exemplary non-invasive methods include, but are not limited to, passively collecting bodily fluids or harmlessly scraping tissue accessible to the external environment (e.g., epidermis or mouth). Exemplary non-invasive samples include, but are not limited to, saliva, sputum, mucus, sweat, urine, feces, semen, cervical and vaginal secretions, breast milk, inflammatory secretions, tears, or cheek epithelial swabs. In some embodiments, the sample... This sample is obtained via a minimally invasive method. Exemplary minimally invasive methods include, but are not limited to, capillary collection, venipuncture, thoracentesis, amniocentesis, needle aspiration, or gastric lavage. Exemplary minimally invasive samples include, but are not limited to, blood or blood fractions (e.g., plasma or PBMC products), interstitial fluid, bile, gastric juice, and amniotic fluid. In some embodiments, the sample is obtained via biopsy. Exemplary biopsy samples include, but are not limited to, skin biopsy samples (e.g., obtained through perforation, scraping, discoid surgery, wedge insertion, incision, or excision biopsy), bone marrow samples (e.g., obtained through aspiration biopsy), lymph node or breast biopsy (e.g., obtained through fine-needle aspiration, core needle biopsy, vacuum-assisted biopsy, or image-guided biopsy), surgical biopsy samples (e.g., obtained through excision or incision biopsy of internal organs), or mouth, gastrointestinal tract, lung, bladder, or urethra biopsy samples (e.g., obtained through endoscopy).

[0088] As used herein, the term "origin of replication" refers to a DNA sequence of a plasmid containing that origin of replication that is recognized by a replication initiation factor or DNA replicase and leads to replication. The expression "recognized by a replication initiation factor" is intended to mean that the replication initiation factor can physically interact with all or part of the origin of replication sequence, thereby causing or stimulating a molecular mechanism that ultimately leads to the replication of all or part of the DNA molecule containing that origin of replication. Therefore, an origin of replication typically contains a functionally required element. An example of such a functionally required element is a repeat family (FR) element or a double symmetry (DS) element of the EBV origin of replication (OriP). Other origins of replication containing functionally required elements are well known in the art and are described, for example, in Bode et al., (2001) Gene Ther. Mol. Biol. 6: 33-46. The parental nucleic acid plasmid vectors of this disclosure preferably contain at least one origin of replication.

[0089] A "vector" is a nucleic acid sequence capable of transferring other operatively linked heterologous or recombinant nucleic acid sequences into target cells. In some embodiments, the vector is a microcircle, plasmid, nanoplasmid, yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), granule, phage particle, bacteriophage genome, or baculovirus genome. Suitable vectors also include vectors derived from bacteriophages or plants, invertebrates or animals (including humans), and viruses, such as CELiD vectors, adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or combinations thereof such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g., MLV, or its self-inactivating or SIN variants, or their pseudotype variants), herpesvirus vectors (based on, for example, HSV- or EBV), lentiviral vectors (e.g., based on HIV, FIV, or EIAV, or their pseudotype variants), or adenoviral vectors (e.g., based on Ad5, including its replication-deficient, replicable, or helper-dependent variants). In some embodiments, the vector is derived from a replicable virus. In some embodiments, the vector is derived from a non-replicable virus. In some cases, vectors may contain attachment maintenance elements to facilitate replication in one or more target cell types, such as scaffold / matrix attachment regions (S / MARs). S / MAR elements are particularly useful for facilitating replication in the context of “naked” nucleic acid vectors, such as microloops. Exemplary suitable S / MAR elements include, but are not limited to, EμMAR from the immunoglobulin heavy chain locus, apoB MAR from the human apolipoprotein B locus, Ch-LysMAR from the chicken lysozyme locus, and huIFNβ MAR from the human IFNβ-locus. Vectors may contain coding sequences capable of being expressed in target cells. Therefore, the terms “vector construct,” “expression vector,” and “gene transfer vector” as used herein can refer to any nucleic acid construct capable of directing the expression of a target gene and having the means for transferring that target gene into target cells. Vectors described herein may also contain one or more cis-acting elements to stabilize or improve the expression of mRNA derived therefrom. Such cis-acting elements include, but are not limited to, any elements described, for example, Johansen et al., The Journal of Gene Medicine. (5) 12:1080-1089 (doi: 10.1002 / jgm.444), or Vlasova-St. Louis and Sagarsky. Mammalian Cis-Acting RNA Sequence Elements (doi: 10.5772 / intechopen.72124).

[0090] As one form of vector, the term "microloop" as used herein refers to a small double-stranded circular DNA molecule that provides sustained high-level expression of a target sequence present on the vector, which may encode a polypeptide, shRNA, antisense RNA, siRNA, and the like. The target sequence is operatively linked to a regulatory sequence present on the microloop vector, which controls its expression. Such microloop vectors are described, for example, in published U.S. Patent Application US20040214329, which is expressly incorporated herein by reference. As a different form of vector, "nanoplasmid" refers to a vector that may contain a minimized bacterial ColE1 or R6K origin of replication (which allows such nanoplasmids to replicate in a bacterial host strain), a bacterial RNA-selective marker, and a eukaryotic gene region. Such nanoplasmids may contain a small R6K origin of SEQ ID NO: 3 and / or an RNA-OUT selective marker of SEQ ID NO: 4. Other examples of such elements (nanoplasmid origin and RNA-out optional markers) are described, for example, in US9737620B2, which is incorporated herein by reference for the purpose of describing nanoplasmid sequence elements.

[0091] The total length of the microcircular vector is sufficient to include the desired elements as described below, but not so long as to prevent or substantially inhibit the vector's ability to enter the target cell upon contact with the cell (e.g., by systemic administration to a host containing the cell). Therefore, microcircular vectors can typically be at least about 0.3 kb long, often at least about 1.0 kb long, while parental vectors can be as long as 6 kb, 10 kb, or longer.

[0092] Microcircular vectors differ from bacterial plasmid vectors in that they lack an origin of replication or a natural origin of replication (e.g., they may contain a minimized synthetic bacterial origin of replication) and lack selection markers commonly found in bacterial plasmids, such as β-lactamase, tetracycline resistance (TET), kanamycin resistance (KAN), or other antibiotic selection markers. Therefore, microcircles are smaller in size, allowing for more efficient delivery. Microcircles lack the transgene expression silencing effect associated with the vector backbone nucleic acid sequence of the parent plasmid, from which the microcircular vector is excised. Microcircles may be substantially devoid of vector sequences other than the recombinase hybridization product sequence and the target sequence (i.e., the sequence for transcription and the regulatory sequence required for expression).

[0093] As used herein, the term "nanoplasmid" refers to a vector that may contain a minimized bacterial ColE1 or R6K origin of replication (which allows the nanoplasmid to replicate in a bacterial host strain), a bacterial RNA-selective marker, and a eukaryotic gene region. Some embodiments of nanoplasmids are described, for example, in US20150275221A1. In some embodiments, the nanoplasmid may contain a fusion bacterial RNA-selective marker / minimized origin of replication. In some embodiments, the fusion bacterial RNA-selective marker / minimized origin of replication may be located within a synthetic intron situated within the eukaryotic gene region of the nanoplasmid.

[0094] RNA selectivity is a vector-carried, expressed, untranslated RNA that regulates the expression of target genes on chromosomes to provide vector selectivity. This could be a plasmid-carried nonsense repressor tRNA that regulates nonsense repressible selectivity chromosomal targets, as described in U.S. Patent 6,977,174 to Crouzet J and Soubrier F, 2005, which is incorporated herein by reference. This could also be antisense repressor RNA carried by plasmids, RNA-OUT genes targeting RNA-IN regulation, RNAI encoding the origin of pMB1 plasmid targeting RNA-II regulation, RNAI encoding the origin of IncB plasmid pMU720 targeting RNA-II regulation, ParB locus Sok targeting Hok regulation in plasmid RI, FlmB locus FlmA targeting flmA regulation in plasmid F, another natural antisense repressor RNA, such as those described in Wagner EGH, Altuvia S, Romby P. 2002. Adv Genet 46:361 and Franch T and Gerdes K. 2000. Current Opin Microbiol 3: 159, or engineered repressor RNA, such as synthetic small RNAs like SgrS, MicC, or MicF scaffolds, as described in Park et al., Nature Biotechnology Vol. 31, pp. 170–174 (2013).

[0095] According to this disclosure, a variety of suitable methods for transfecting cells are available. “Transfection” means a cellular alteration resulting from the uptake of exogenous nucleic acids (usually DNA). The use of the term “transfection” is not intended to limit the introduction of exogenous nucleic acids to any particular method. Therefore, suitable methods include viral infection / transduction, conjugation, nanoparticle delivery, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, etc. The choice of method depends on the type of cells being transfected and the circumstances under which transfection occurs (i.e., in vitro, in vivo, or in vivo). A general discussion of these methods can be found in Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995, which is incorporated herein by reference.

[0096] The term "transfection agent" can encompass any compound, such as liposomes, that mediates the incorporation of DNA or RNA into host cells. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring 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. Examples of suitable transfection agents include, but are not limited to, linear or branched polyethyleneimine, nanoparticles, liposomes, lipophilic particles, solid nanoparticles, amphoteric peptides, micelles, dendritic molecules, polymer compositions, hydrogels, synthetic or naturally derived exosomes, virus-like particles, or any combination thereof.

[0097] As used herein, the term "EXOmotif" refers to the RNA sequence that controls the loading of miRNA into exosomes. In some embodiments, EXOmotif can mediate the binding of miRNA to heterogeneous ribonucleoprotein A2B1 (hnRNPA2B1), which has been described as controlling the loading of miRNA into exosomes. Such sequences include, but are not limited to, 5′-GGAG-3′ and 5′-CCCU-3′.

[0098] As used herein, the terms "nucleic acid molecule" and "polynucleotide" refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or similar molecules) of any length. Polynucleotides can have any three-dimensional structure and can perform any known or unknown function. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, shRNA, single-stranded short or long RNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, any isolated DNA sequence, control regions, any isolated RNA sequence, nucleic acid probes, and primers. Nucleic acid molecules can be linear or circular.

[0099] The term "promoter" is a DNA sequence that directs the transcription of a polynucleotide. Typically, a promoter may be located in the 5' region of the polynucleotide to be transcribed, adjacent to the transcription start site of that polynucleotide. More typically, a promoter is defined as the region upstream of the first exon; more typically, it is the first upstream region among multiple transcription start sites. Promoters are often capable of directing the transcription of genes located at the 3' position on each strand of the complementary DNA strand. In other words, many promoters exhibit bidirectionality and can direct the transcription of downstream genes in either orientation (i.e., relative to the coding region of the gene, 5' to 3' or 3' to 5'). Furthermore, promoters may also include at least one control element, such as an upstream element. Such elements include an upstream activator region (UAR), optionally, and other DNA sequences that influence the transcription of polynucleotides, such as upstream elements of synthesis.

[0100] When referring to polypeptides in this document, the terms "coding sequence" and "coding" refer to, for example, nucleic acid molecules that are transcribed (in the case of DNA) and translated (in the case of mRNA) into polypeptides when the nucleic acid is present in living cells (in vivo) and under the control of appropriate regulatory sequences (or "control elements"). The boundaries of a coding sequence typically depend on a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. Coding sequences can include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic DNA sequences from viral, eukaryotic, or prokaryotic DNA, and synthetic DNA sequences. Transcription termination sequences may be located at the 3' end of the coding sequence, and promoters may be located at the 5' end; as well as additional control sequences (if desired), such as enhancers, introns, polyadenylation sites, etc. DNA sequences encoding polypeptides can be optimized for expression in selected cells using codons selected by the cells to present a copy of the DNA encoding the desired polypeptide sequence.

[0101] As used herein, the term "barcode" or "barcode molecule" generally refers to a mark or identifier that conveys or is capable of conveying information about a molecule to which a barcode / barcode molecule is attached. Barcodes / barcode molecule can be unique. Barcodes / barcode molecule can take many different forms. For example, barcodes / barcode molecule can include polynucleotide barcodes; random nucleic acid and / or amino acid sequences; and synthetic nucleic acid and / or amino acid sequences. Barcodes / barcode molecule can be attached to molecules in a reversible or irreversible manner. Barcodes can be added to fragments of, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) samples before, during, and / or after sequencing a sample. Barcodes can allow for the identification and / or quantification of individual sequencing reads.

[0102] As used herein, the term "operably linked" refers to an arrangement of elements in which the components described in this document are configured to perform their usual functions. Thus, a given promoter operably linked to a coding sequence (e.g., a reporter expression cassette) can influence the expression of that coding sequence in the presence of a suitable enzyme. Promoters or other control elements do not need to be adjacent to the coding sequence as long as they function to direct its expression. For example, an intercalated, untranslated but transcribed sequence can exist between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0103] As used herein, the term "expression cassette" refers to any nucleic acid construct capable of directing the expression of any RNA transcription, including genes / coding target sequences and untranslated RNA such as shRNA, microRNA, siRNA, antisense RNA, and the like. Such cassettes can be constructed within "vectors," "vector constructs," "expression vectors," or "gene transfer vectors" to facilitate the transfer of the expression cassette into target cells. Therefore, the term encompasses both cloning and expression vectors, as well as viral vectors.

[0104] As used herein, the term "target cell" refers to the cell in which genetic modification is required. Target cells can be isolated (e.g., in culture) or in multicellular organisms (e.g., in the blastocyst, in the fetus, in a postnatal animal, etc.).

[0105] As used herein, the term "pharmaceuticalally acceptable carrier" refers to a diluent, adjuvant, excipient, or catalyst that is administered with the probe of this disclosure and is approved by a federal regulatory agency or state government or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals, and more particularly in humans. Such a drug carrier can be a liquid such as water and oils (including those of petroleum, animal, plant, or synthetic origin), such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Drug carriers can also be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. When administered to a patient, the probe and pharmaceutically acceptable carrier can be sterile. Water is a useful carrier when the probe is administered intravenously. Saline solutions and aqueous solutions of dextran and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable drug carriers also include excipients such as glucose, lactose, sucrose, glyceryl monostearate, sodium chloride, glycerol, propylene glycol, water, ethanol, and the like. The compositions of the present invention may also contain small amounts of wetting agents, emulsifiers, or pH buffers, if desired. The compositions of the present invention may advantageously be in the form of solutions, emulsions, sustained-release formulations, or any other form suitable for use.

[0106] The term "detectable" refers to the ability to detect a signal relative to a background signal. A detectable signal is defined as an amount sufficient to produce an acceptable image using equipment suitable for preclinical use. A detectable signal can be generated by applying the probe of this disclosure once or multiple times. The amount applied can be varied depending on factors such as individual sensitivity, age, sex, weight, individual specificity, dosimetry, and the like. The amount applied can also be varied depending on factors related to the instrument and digital processing.

[0107] As used in this article, “in vivo imaging” refers to a method or process in which the structure, function, or physiological state of a living organism can be examined without the need for the death of the organism.

[0108] As used herein, the term “non-invasive in vivo imaging” refers to a method or process in which the structure, function, or physiological state of a living organism can be examined by remote body exploration without damaging the integrity of the body’s outer (skin) or inner (accessible orifices) surfaces.

[0109] The “imaging component” can be detected externally to the subject, either as a human or non-human animal, or via the use of detectors designed for in vivo use, such as intravascular radiation detectors or optical detectors, such as endoscopes, or radiation detectors designed for intraoperative use. The imaging component is preferably, but not limited to, a reporter suitable for in vivo optical imaging.

[0110] As used in this article, the term "bioluminescence" refers to a type of chemiluminescence in which biomolecules, especially proteins, emit light. The basic conditions for bioluminescence are bound or free molecular oxygen in the presence of oxygenase, luciferase (which acts on the substrate), and luciferin (which releases energy in the form of light in the presence of molecular oxygen and converts the substrate into an excited state, and then releases energy in the form of light once it returns to a lower energy level).

[0111] As used herein, the term "luciferase" refers to an oxygenase that catalyzes a bioluminescent reaction. For example, bacterial luciferase catalyzes the oxidation of flavin mononucleotides and aliphatic aldehydes, a reaction that produces light. Another type of luciferase found in marine arthropods catalyzes the oxidation of luciferin in sea fireflies, while another type catalyzes the oxidation of luciferin in beetles. Therefore, "luciferase" refers to an enzyme or light protein that catalyzes a bioluminescent reaction. Luciferases such as firefly and sea urchin luciferases are enzymes that function catalyzed during bioluminescence and remain unchanged. Luciferase light proteins that are non-covalently bound to luciferin, such as jellyfish luciferin and obelin, are altered during bioluminescence by releasing luciferin. Luciferases are naturally occurring proteins in organisms or their variants or mutants, such as variants produced by mutagenesis that differ from naturally occurring proteins in terms of one or more properties, such as thermal stability or pH stability. Luciferases and their modified mutant or variant forms are well known. For example, the term “renilla luciferase” refers to an enzyme isolated from a member of the genus Renilla, or an equivalent molecule obtained from any other source, such as another coral or one that has been synthetically prepared.

[0112] "Bioluminescent proteins" are proteins that can act on bioluminescent initiator molecules to produce or emit bioluminescence.

[0113] "Bioluminescent initiator molecules" are molecules that can react with bioluminescent donor proteins to produce bioluminescence. Bioluminescent initiator molecules include, but are not limited to, coelentin, its analogues, and functional derivatives. Derivatives of coelentin include, but are not limited to, coelentin 400a, coelentin cp, coelentin f, coelentin fcp, coelentin h, coelentin hcp; coelentin ip, coelentin n, coelentin O, coelentin c, coelentin c, coelentin i, coelentin ICP, 2-methylcoelentin, benzyl-coelentin dideoxycoelentin, and deep blue coelentin (DBC) (described in more detail in U.S. Patents 6,020,192; 5,968,750 and 5,874,304).

[0114] Generally, coelentrin is known to emit light upon contact with various bioluminescent proteins, particularly luciferases. A useful but non-limiting coelentrin is disclosed in U.S. Patent Application Serial No. 10 / 053,482, mentioned November 2, 2001, the disclosure of which is incorporated herein by reference in its entirety. Coelentrin is available from Promega Corporation, Madison, Wis., and Molecular Probes, Inc., Eugene, Oreg. Coelentrin can also be synthesized, as described, for example, in Shimomura et al., (1989) Biochem. J. 261: 913-920; Inouye et al., (1997) Biochem. Biophys. Res. Comm. 233: 349-353, 1997; and Teranishi et al., (1997) Anal. Biochem. 249: 37-43.

[0115] As used herein, the term "survival protein" refers to a protein also known as BIRC5, which contains the baculovirus apoptosis inhibitor repeat 5, or BIRC5, and is encoded by the BIRCS gene in humans (NCBI reference sequence: NG 029069.1). Survival protein is a member of the inhibitor of apoptosis (IAP) family. Survival protein inhibits caspase activation, leading to negative regulation of apoptosis or programmed cell death. This has been shown through disruption of the survival protein-induced pathway, resulting in increased apoptosis and decreased tumor growth. Survival protein is highly expressed in most tumors and fetal tissues, but is completely absent in late-differentiated cells. Survival protein expression is also highly regulated by the cell cycle and is expressed only during the G2-M phase. Survival protein is known to localize to the mitotic spindle during mitosis by interacting with tubulin and can play a promoting role in regulating mitosis. Regulation of survival protein appears to be associated with the p53 protein. It is also a direct target gene of the Wnt pathway and is upregulated by β-catenin.

[0116] However, given that the microloops of this disclosure can utilize any tumor-specific promoter that is operatively linked to a reporter or other heterologous nucleic acid sequence desired to be expressed in target cells. For example, but not limited to, suitable promoters known in the art include: a tumor-specific CXCR4 promoter in melanoma; a tumor-specific hexokinase type II promoter in lung cancer; a TRPM4 (transient receptor potential-Melastatin 4) promoter preferentially active in prostate cancer; a breast cancer cell-specific lysosome 3 promoter (Basset et al., (1990) Nature 348: 699); a non-small cell lung cancer cell-specific surfactant protein A promoter (Smith et al., 1994) Hum. Gene Ther. 5: 29-35); a cancer-specific secretory leukocyte protease inhibitor (SLPI) promoter for expressing SLPI (Garver et al., (1994) Gene Ther. 1: 46-50); and a melanoma cell-specific tyrosinase promoter (Vile et al., (1994) Gene Ther. 1: ). 307); fibrosarcoma / tumorigenic cell-specific stress-inducible grp78 / BiP promoter (Gazit et al., (1995) Cancer Res. 55: 1660); glioblastoma cell-specific interleukin-10 promoter (Nitta et al., (1994) Brain Res. 649: 122); brain tumor cell-specific aB-crystallin / heat shock protein 27 promoter (Aoyama et al., (1993) Int. J. Cancer 55: 760); epidermal growth factor receptor promoter specific to squamous cell carcinoma, glioma, and breast tumor cells (Ishii et al., (1993) Proc. Natl. Acad. Sci. USA 90: 282); breast cancer cell-specific mucin-like glycoprotein (DF3, MUC1) promoter (Abe et al., (1993) Proc. Natl. Acad. Sci. USA). 90: 282); metastatic tumor-specific MTS 1 promoter (Tulchinsky et al., (1992) Proc. Natl. Acad. Sci. USA 89: 9146); small cell lung cancer cell-specific NSE promoter (Forss-Petter et al., (1990) Neuron 5: 187); small cell lung cancer cell-specific somatostatin receptor promoter (Bombardieri et al., (1995) Eur. J. Cancer 31A: 184; Koh et al., (1995) Int. J. Cancer 60:843); c-erbB-3 and c-erbB-2 promoters specific to breast cancer cells (Quin 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); thyroglobulin promoter specific to thyroid cancer cells (Mariotti et al., (1995) J. Clin. Endocrinol. Meth. 80: 468); alpha-fetoprotein promoter specific to liver cancer cells (Zuibel et al., (1995) J. Cell. Phys. 162: 36); chorionic villi promoter specific to gastric cancer cells (Osborn et al., (1988) Virchows Arch. A. Pathol. Anat. Histopathol. 413: 303); and albumin promoter specific to liver cancer cells (Huber, (1991)). Proc. Natl. Acad. Sci. USA 88: 8099), which is incorporated herein by reference in its entirety. Other examples of promoters are the ATP-binding box subfamily C member 4 (ABCC4) promoter, pregradient protein 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter, sealing protein 3 (CLDN3) promoter, sealing protein 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter, and collagen type I α1 chain promoter. The promoters of the following proteins are listed: COL1A1 promoter, cystatin SN (CST1) promoter, DTL promoter, family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), and laminin subunit γ.LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α promoter, Ubiquitin D promoter, Ubiquitin conjugate E2 C promoter, USH1 protein network component harmonin (USH1C), T cell activation inhibitor 1 (VTCN1) promoter containing V-set domain, Ubiquitin conjugate 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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2 S (UBE2S) promoter, Aurora kinase A and ninein interacting protein (AUNIP) promoter, Cell cycle 6 (CDC6) promoter, Centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, Deoxythymidine kinase (DTYMK) promoter, G protein regulation inducer 1 (GPRIN1) promoter, Mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, Nucleotide small ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, 2 (ATAD2) promoter containing AAA domain, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calcyclin binding protein (CBB1) promoter. Promoters for protein (CACYBP), cell cycle-associated protein 3 (CDCA3), centromere protein O (CENPO), valve-specific endonuclease 1 (FEN1), forkhead box M1 (FOXM1), cell proliferation inhibitor of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, and MYB.The following promoters are listed: proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nucleoprotein peptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, transforming acidic coiled-coil protein 3 (TACC3) promoter, TBC1 domain family member 31 (TBC1D31) promoter, thymidine kinase 1 (TK1) promoter, zinc finger protein 695 (ZNF695) promoter, aurora kinase A (AURKA) promoter, and BLM promoter. RecQ-like helicase (BLM) promoter, chromosome 17 read frame 53 (C17orf53) promoter, chromosome box 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell cycle 20 (CDC20) promoter, cell cycle 45 (CDC45) promoter, cell cycle-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-pass 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 X-type α Promoters for the following proteins: COL10A1, CSE1L, DBF4 zinc finger, GINS complex subunit 1 (GINS1), G protein-coupled receptor 19 (GPR19), kinin family member 18A (KIF18A), kinin family member 4A (KIF4A), kinin family member C1 (KIFC1), mini chromosome maintenance 10 replication initiation factor (MCM10), and mini chromosome maintenance complex component 2 (MCM2). Promoters, Miniature Chromosome Maintenance Complex Component 7 (MCM7) promoter, MRG Domain-Binding Protein (MRGBP) promoter, Methylene Tetrahydrofolate Dehydrogenase (NADP+ Dependent) 2, Methylene Tetrahydrofolate Cyclohydrolase (MTHFD2) promoter, Non-SMC Condensed Protein I Complex Subunit H (NCAPH) promoter, NDC80, Kinocele Complex Component (NDC80) promoter, Nudix Hydrolase 1 (NUDT1) promoter, Ribonuclease H2 Subunit A (RNASEH2A) promoter, RuvB-like AAAThe promoters of ATPase 1 (RUVBL1), serologically defined breast cancer antigen NY-BR-85 (SGOL1), SHC-binding and spindle-associated 1 (SHCBP1), small nucleonucleotide polypeptide G (SNRPG), permanent circadian rhythm regulator, thyroid hormone receptor interactor 13 (TRIP13), trophinin-associated protein (TROAP), ubiquitin conjugate E2C (UBE2C), WD repeat and HMG box DNA-binding protein 1 (WDHD1), their functional fragments, or any combination thereof.

[0117] Other definitions are provided in the following context. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of molecular biology. While similar or equivalent methods and materials to those described herein may be used to practice or test the contents of this disclosure, suitable methods and materials are described herein.

[0118] abbreviation

[0119] SEAP, secreted embryonic alkaline phosphatase; MRI, magnetic resonance imaging; SPECT, single-photon emission computed tomography; MC, microcircle; PP, parental plasmid; WPRE, post-transcriptional regulatory element of marmot hepatitis virus (WHP); Luc, luciferase; BLI, bioluminescence imaging; ROI, target region; AUC, area under the curve; RG, reporter gene; TS, tumor-specific; Fluc (FLUC), firefly luciferase, ROC (recipient operating characteristic curve).

[0120] introduction

[0121] Early detection of cancer can significantly improve the efficacy of available treatment strategies. However, despite decades of effort in blood-based biomarker-based cancer detection, many promising endogenous biomarkers have failed clinically due to thorny issues such as highly variable background expression from non-malignant tissues. Strategies for improved cancer diagnosis have traditionally relied on measuring endogenous molecules overexpressed in cancer cells via molecular imaging or blood-based assays. The challenge with these strategies is their often significant expression in non-cancerous tissues, leading to high background levels and confusing results. An alternative strategy is to incorporate promoters of tumor-specific (TS) proteins into exogenously delivered gene vectors to precisely drive the expression of unique reporter genes (RGs) within the tumor. For this strategy to be feasible, safety, specificity, and sensitivity are critical. While safer than viral vectors, non-viral vectors suffer from two drawbacks: low gene transfer rates and transient expression profiles. Microcircles (MCs), plasmids lacking a bacterial backbone, advantageously overcome these key issues.

[0122] This disclosure provides an alternative and advantageous detection strategy based on a safe, systemically administered, tumor-activated microloop that utilizes a pan-tumor-specific survival protein promoter to drive the expression of a secreted reporter gene, which is detectable almost exclusively in the blood of subjects with tumors. Robustness has been demonstrated in identifying mice with experimental human melanoma metastases from tumor-free subjects simply by measuring blood reporter levels over a period of up to 2 weeks following systemic administration. Cumulative changes in reporter levels also identified tumor-bearing subjects, and recipient operating characteristic curve analysis highlighted the performance of this test with an AUC of 0.918 ± 0.084. Lung tumor burden correlated with cumulative reporter levels (r² = 0.714; p < 0.05) suggests that determining disease severity is possible. Further development of our system could significantly improve tumor detectability due to the transiently controlled high reporter expression in tumors, with virtually zero background from healthy tissues being possible.

[0123] Tumor-specific nanoparticle carriers expressing secreted embryonic alkaline phosphatase (SEAP) or firefly luciferase (FLUC) have been developed and their applicability for tumor detection has been validated using blood-based and / or imaging-based assays following systemic administration. Challenges for gene vectors used for cancer screening purposes include efficient tumor delivery, achieving robust expression for maximum sensitivity, tight control of expression for tumor specificity, and minimizing safety concerns. Tumor-specific microcircular carriers overcome all these challenges, and systemic administration of tumor-specific microcircular carriers has been shown to allow for differential identification of tumor-bearing subjects from normal subjects via serum and non-invasive imaging. Importantly, the tumor-specific microcircular carriers of this disclosure are advantageously applicable to a wide range of patient populations due to the fact that their expression is driven by surviving protein promoters in tumor cells of many different tumor types. The tumor-specific microcircular carriers of this disclosure provide novel cancer management strategies that include tumor detection via initial blood-based assays, tumor localization via molecular genetic imaging, and tumor treatment using tumor-specific microcircular carriers that offer both diagnostic and therapeutic benefits.

[0124] This disclosure covers embodiments of nucleic acid nanoparticle carriers that are highly advantageous for detecting tumor cells. In practice, the microloops of this disclosure contain a tumor-specific promoter operatively linked to a nucleotide sequence desired for selective expression in tumor cells or tissues containing tumor cell populations. In some embodiments of this disclosure, the microloop carrier contains a tumor-specific promoter operatively linked to a nucleotide sequence encoding a polypeptide used as a reporter. Thus, when expressed by recipient tumor cells, the reporter can be detectable, providing information such as a visual image of the tumor cells and / or their location in tissues of a human or non-human animal subject.

[0125] In some embodiments, the nanoparticle vectors according to this disclosure can advantageously deliver expressible reporter genes to tumor cells. The reporter genes can be detected by non-invasive detection methods such as MRI imaging, PET imaging, SPECT imaging, luminescence imaging, and the like, within the scope of this disclosure. For example, and not intended to be limiting, MRI reporter genes encode creatine kinase; tyrosinase; transferrin receptor; ferritin; and Mag A. PET imaging reporter genes include, but are not limited to, for example, herpes simplex virus 1-thymidine kinase (HSV1-TK); hypoxanthine phosphoribosyltransferase; L-amino acid decarboxylase; dopamine 2 receptor (D2R, including the mutant D2RA80); somatostatin receptor; estrogen receptor (hERL); dopamine transporter; sodium iodide cotransporter; catecholamine transporter; and 13-galactosidase. PET / SPECT imaging reporter genes include, but are not limited to, herpes simplex virus type 1 thymidine kinase and multiple optimized mutants, such as HSV1-sr39tk; dopamine type 2 receptor; sodium iodide cotransporter; somatostatin type 2 receptor; human norepinephrine transporter; human estrogen receptor; mutants of human deoxycytidine kinase; and recombinant carcinoembryonic antigen. Bioluminescent reporter genes include, but are not limited to, firefly luciferase (FL); synthetic renin luciferase (HRL); enhanced green fluorescent protein (EGFP); red fluorescent protein (RFP); monomeric red fluorescent protein (MRFP 1); and the like. Reporter genes may also be suitable for incorporation into microrings of this disclosure to provide multimodal imaging methods. For example, and not intended to be restrictive, a suitable reporter gene for photoacoustic imaging, MRI, and PET imaging is the gene encoding human tyrosinase, as described by Qin et al., (2013), Sci. Rpts. 3: Art. No.: 1490, which is incorporated herein by reference in its entirety.

[0126] In addition to being advantageous for selectively detecting recipient tumor cells, the nanoparticles of this disclosure can also be used to reduce or eliminate targeted tumor cells from human or non-human animals in the recipient. The nucleotide sequence operatively linked to a tumor-specific promoter can encode a polypeptide for modulating the proliferative or metabolic activity of the recipient's tumor cells.

[0127] For example, and not intended to be restrictive, peptides that are beneficial for targeting and therapeutically irritating tumor cells include HSVtk; cytosine deaminase; DT flavoprotein enzyme; nitroreductase; guanine phosphoribosyltransferase; purine nucleoside phosphorylase; thymidine phosphorylase; carboxylesterase; folic acid polyglutamyl synthase; carboxypeptidase A1; carboxypeptidase G2; cytochrome P-450 (CYP2B1); and the like. The activity of these peptides in converting prodrugs into effective therapeutic compositions is described, for example, in Harrington et al., (2002) Clinical Oncology 14: 148-169, which is incorporated herein by reference in its entirety.

[0128] In other embodiments of this disclosure, it is considered that the nucleotide sequence expressed from the microcircular tumor-specific expression may not be translated into a heterologous polypeptide, but may instead be expressed as an interfering short ribonucleotide sequence (siRNA) that can interact with at least one gene regulatory element of the recipient tumor cells, thereby modulating the proliferation or metabolic activity of the recipient tumor cells. Alternatively, it is considered that the nucleotide sequence may be expressed as a microRNA sequence (miRNA) or a synthetic RNA sequence that does not correspond to any known endogenous sequence and is only used as a detectable agent (nucleic acid biomarker) by nucleic acid hybridization or amplification techniques.

[0129] Therefore, embodiments providing nucleic acid microcircular vectors (and their parental plasmids) are considered to be within the scope of this disclosure, having the ability to selectively target tumor cells cultured in vitro, or very advantageously obtain detectable signals in vivo to identify and / or locate cancer cells or tumor cell populations in a subject, and to deliver therapeutic agents (peptides, polypeptides, nucleic acids) to the targeted tumor cells.

[0130] This disclosure provides nucleic acid microcircular vectors for administration to human or non-human subjects to detect the presence of one or more targeted tumor cells, including tumor tissue. For example, the microcircular construct MC-pSurv-SEAP-WPRE-SV40 polyA (e.g.) Figure 3 As shown and having Figure 13The nucleotide sequence shown (SEQ ID NO: 1) contains a nucleic acid fragment that encodes embryonic alkaline phosphatase (SEAP), a detectable polypeptide secreted by an enzyme operatively linked to the tumor-specific promoter p-survival protein.

[0131] When delivered to cultured melanoma cells, subcutaneous melanoma xenografts, or intravenously to animals with tumor formation, the microcircular carrier of this disclosure provides a detectable signal within the microcircular carrier construct, as a serum-secreted alkaline phosphatase peptide or a bioluminescent signal, wherein SEAP has been replaced with a luciferase reporter, such as... Figure 4 As shown (and having) Figure 14 The nucleotide sequence shown is SEQ ID NO: 2. Therefore, the microcircular constructs of this disclosure have been demonstrated to be able to identify metastatic tumor cells or localized tumors in recipient animals or humans.

[0132] This disclosure further provides a method for modulating the physiology or proliferation of targeted tumor cells, the method comprising delivering microcircular nucleic acids to the tumor cells, allowing the targeted cells to express nucleotide sequences from nucleic acid sequences operatively linked to tumor-specific promoters, and allowing the expressed products to interact with the targeted cells, thereby modulating the physiological state of one or more cells.

[0133] In the first scenario, this disclosure provides an implementation scheme for nucleic acid microloops, wherein the tumor-specific promoter is, for example, but not limited to, a survival protein promoter.

[0134] Therefore, to overcome the limitations of endogenous biomarker detection, this disclosure provides an implementation scheme based on a strategy for identifying individuals with tumors using a gene-encoded reporter gene delivered exogenously via blood-based detection, wherein the reporter gene generates a tumor-driven biomarker. A key advantage of this strategy is its ability to tune biomarker expression to specific phenotypes (i.e., tumor cells), thereby reducing the number of false positives due to proteins produced from non-malignant tissues. Thus, systemic administration of a tumor-activated vector encoding a secretory reporter gene can be used to identify subjects with tumors, provided that transgene expression is transcribedly targeted to cancer cells by utilizing a tumor-specific promoter (a promoter of a gene expressing a protein present only in the tumor), such as... Figure 1As shown in the diagram. For this strategy to be translated into clinical applications, safety, specificity, sensitivity, and broad applicability are important, and each component of the system of this disclosure has been selected to provide the greatest translational potential. Specifically, this disclosure provides nonviral tumor-activated microloops (MCs) encoding reporter genes (including, but not limited to, human secreted embryonic alkaline phosphatase (SEAP)) that acquire tumor specificity through the use of tumor-specific promoters (e.g., but not limited to, the survival protein promoter (pSurv)).

[0135] Although safer than viral vectors, traditional non-viral vectors (i.e., plasmids) have two drawbacks: low gene transfer rates and short expression profiles. MCs are essentially plasmids lacking a prokaryotic backbone specifically for amplification in bacteria. MCs have been repeatedly shown to exhibit improved expression profiles (months in non-dividing cells and weeks in dividing cells) compared to their plasmid counterparts due to their smaller size and reduced promoter silencing (Darquet et al., (1997) 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). MCs also conform to the principle of regulating "plasmids without antibiotic resistance genes" (pFAR) (Marie et al., (2010) J. Gene Med. 12: 323-332), which are known to be safer for human administration than constructs containing antibiotic resistance genes. Furthermore, while the preparation of MCs has traditionally been very labor-intensive and time-consuming, recent advances in MC preparation protocols have enabled large-scale production in a relatively easy and cost-effective manner over a short period (Kay et al., (2010) Nat. Biotech. 28: 1287-1289). Finally, while integration is a safety concern in the case of many gene (especially viral) vectors, even with efficient in vivo delivery methods such as direct local injection and electroporation, the integration rate of non-viral vectors is approximately 1-3 orders of magnitude lower than the spontaneous gene inactivation mutation rate (Wang et al., (2004) Gene Therapy 11: 711-721; Nichols et al., (1995) Annals New York Acad. Sci. 772:30-39; Ledwith et al., (2000) Develop. Biologicals 104: 33-43; Ledwith et al., (2000) Intervirology 43: 258-272). Therefore, MC has become one of the most useful non-viral vector platforms in terms of translation potential, efficacy, and safety.

[0136] SEAP is a commonly used secretory reporter protein with many desirable properties. It is an artificially C-terminated secretory form of human placental alkaline phosphatase (PLAP), which is expressed only during embryogenesis; therefore, it is a unique reporter, normally absent in the blood, and should have near-zero background (Berger et al., (1988) Gene 66: 1-10). Compared to PLAP, SEAP is unusually thermostable; therefore, heating the sample to 65°C allows SEAP to be specifically measured (Bronstein et al., (1994) BioTechniques 17: 172-174, 76-177). Commercial SEAP assays are extremely sensitive in the concentration range of at least 4-log, with detection limits in the picogram / mL range. SEAP is also being considered a promising protein-based reporter for clinical use because: 1) it has demonstrated effective longitudinal surveillance of nonviral gene transfer in mice and large animals (Brown et al., (2008) Methods Mol. Biol. 423: 215-224); 2) its human origin suggests it may have attenuated or zero immunogenicity potential in patients, similar to what has been shown in immunocompetent mice with mu-SEAP (mu-SEAP) (Wang et al., (2001) Gene 279: 99-108); and 3). SEAP has been used clinically to monitor antibody levels following administration of vaccines containing HPV16 / 18 AS04-adjuvant (Kemp et al., (2008) Vaccine26: 3608-3616).

[0137] The system disclosed herein utilizes pSurv to drive SEAP expression. Survival proteins are members of the apoptosis inhibitor family that help control the mitotic process and prevent cell death, and are overexpressed in many cancers such as melanoma, liver cancer, lung cancer, breast cancer, colon cancer, and ovarian cancer, but not in healthy adult tissues (Ito et al., (2000) Hepatology 31: 1080-1085; Chen et al., (2004) Cancer Gene Therapy 11: 740-747; Lu et al., (2005) Gene Therapy 12: 330-338). Therefore, pSurv is beneficial for transcriptional targeting of tumors, as demonstrated in models of lung cancer, melanoma, colon cancer, breast cancer, ovarian cancer, and liver cancer (Lu et al., (2005) Gene Therapy 12:330-338; Li et al., (2006) J. Gene Med. 8: 1232-1242; van Houdt et al., (2006) J. Neurosurgery 104: 583-592; Ahn et al., (2011) Gene Therapy 18: 606-612; Ray et al., (2008) Mol. Therapy: J. Am. Soc. Gene Therapy 16: 1848-1856). Thus, the tumor-specific promoter-driven tumor-activated MCs of this disclosure offer broad applicability for effective cancer screening across a wide range of tumor types and patient populations.

[0138] Therefore, tumor-activated MCs have been developed for diagnostic purposes, and their ability to identify tumor-bearing subjects from healthy subjects has been tested by measuring blood levels of genetically encoded cancer biomarkers after systemic administration of MCs. For delivery, MCs were compared to non-targeted transfectants that have shown no immunogenicity (Bonnet et al., (2008) Pharmaceut. Res. 25: 2972-2982), the ability to be repeatedly administered to animals, and the ability to effectively transfect primary and metastatic tumors in mice after systemic (tail vein) administration (Yang et al., (2013) Proc. Nat. Acad. Sci. USA 110: 14717-14722; Bhang et al., (2011) Nat. Med. 17:123-129). These results suggest that the use of tumor-activated MCs is a promising and safe platform technology for cancer screening. This system is used to monitor patients at high risk of tumor recurrence, then screen high-risk groups before tumor diagnosis, and can also be beneficial for screening the general population.

[0139] The exogenous delivery strategy of gene-encoded cancer blood biomarker vectors according to this disclosure can overcome some of the inherent limitations of cancer screening targeting endogenous cancer blood biomarkers, such as high background expression in healthy tissues and stochastic fluctuations in biomarker expression over time. This disclosure provides an implementation scheme for a tumor-activated MC system that can be administered systemically to identify subjects with tumors using a simple and relatively inexpensive blood-based assay. This assay demonstrates reliable detection capability and assessment of disease severity, indicating the feasibility of tumor-activated MC as a highly robust and safe cancer screening system.

[0140] Research into cancer gene therapy has sought methods to specifically express therapeutic transgenes within tumors to avoid adverse effects in non-target cells or normal cells. To achieve this goal, several strategies have been explored, including transcriptional targeting of tumors using tumor-specific promoters (Aim et al., (2011) Gene Therapy 18: 606-612; Ye et al., (2003) Biochem. Biophys. Res. Comms. 307: 759-764; Iyer et al., (2005) Transgenic Res. 14:47-55), transcriptional silencing or repression in healthy tissues using endogenous miRNA regulation (Cawood et al., (2009) PLoS Pathogens 5: e 1 000440; Ronald et al., (2013) GeneTherapy 20: 1006-1013), and enhanced tumor tropism using viruses (transduction targets) and non-viral vectors (Chisholm et al., (2009) Cancer Res. 69: 2655-2662; Bachtarzi et al., (2008) Expert Opinion Drug Delivery 5: 1231-1240), or combinations of these strategies (Tsuruta et al., (2008) Clin. Cancer Res. 14: 3582-3588; Sugio et al., (2011) Clin. Cancer Res 17: 2807-2818). The system disclosed herein provides a tool for expressing secretory reporter genes for cancer detection. With this application of gene vectors comes the additional challenge of overcoming increased safety concerns, as the vectors, as potential screening tools, can be used on patients without any clearly visible evidence of cancer. Therefore, all components of this type of system need to be safe, including the delivery medium (if needed), the DNA vector itself, and the transgene (if expressed).

[0141] While many delivery formulations are known in the art and considered for use in the MC system of this disclosure, those with a desired safety profile (i.e., no immunostimulation) (Bonnet et al., (2008) Pharmaceut. Res. 25: 2972-2982) and in vivo transfection agents in Phase I / II clinical trials (Lisziewicz et al., (2012) PLoSONE 7:e35416) are particularly preferred. Furthermore, although non-viral vectors are far safer than viral vectors (i.e., low / near-zero integration rate, reduced immunogenicity potential), issues remain regarding immunostimulatory prokaryotic CpG motifs in the backbone of conventional plasmids. This problem is mitigated in MCs and / or nanoparticles because these vectors lack a prokaryotic backbone or have small bacterial regions (less than 500 bp). SEAP was chosen because it is of human origin and therefore should not cause an immunogenic response (Wang et al., (2001) Gene 279: 99-108) and has shown promise in clinical trials (Kemp et al., (2008) Vaccine 26: 3608-3616).

[0142] Previously, viral infection has been used to drive cancer-specific gene constructs, such as MC-OriP-IFNy (Zuo et al., (2011) PLoS ONE 6: e19407), which uses the viral OriP promoter / origin of replication to drive interferon-γ infection in Epstein-Barr virus (EBV) for nasopharyngeal carcinoma (NPC). In contrast, the MC system of this disclosure can be broadly applied to many different tumor types in addition to virally infected cells. The non-viral MC vector of this disclosure has been developed for use in cancer screening using blood-based assays.

[0143] Although vectors expressing tumor-activated reporter genes have been developed for cancer detection (Bhang et al., (2011) Nat. Med. 17: 123-129; Chaudhuri et al., (2003) Technol. In Cancer Res. & Treat. 2: 171-180; Warram et al., (2011) Mol. Imaging Biol. 13: 452-461; Warram et al., (2012) Cancer Gene Therapy 19: 545-552; Browne et al., (2011) PLoS ONE 6: e19530), the vector systems used in these cases (adenovirus, herpes simplex virus, and plasmids) have safety concerns that hinder clinical translation. Viruses are highly immunogenic, and pre-existing viral immunity in humans is a common problem (Browne et al., (2011) PLoS ONE 6: e19530; Sumida et al., (2005) J. Immunol. 174:7179-7185; Schirmbeck et al., (2008) Mol. Therapy 16: 1609-1616). Plasmids are likely immunogenic due to the unmethylated CpG sequence in the prokaryotic backbone (required only for plasmid preparation) (Tan et al., (1999) Human Gene Therapy 10:2153-2161), and the typical carrying of antibiotic resistance genes encoded in endogenous flora (Marie et al., (2010) J. Gene Med. 12: 323-332). Therefore, the tumor-activated MC of this disclosure has advantages over these other vectors and offers translational potential mainly due to its easier production practices (compared to viruses) and more promising spectrum.

[0144] The MC and / or nanoparticle systems of this disclosure can provide improved specificity through two mechanisms: 1) the biomarker is unique because no SEAP is detectable in the blood prior to MC administration; and 2) it can drive expression strictly within the tumor, thereby mitigating the signal in healthy, tumor-free subjects. The mild SEAP signal from tumor-free mice receiving MC may be due to leakage from pSurv. However, the MC systems of this disclosure are not intended to be limited to this particular promoter and alternative tumor-activated promoters, such as, but not limited to, the Idl or hTERT promoters (Warram et al., (2011) Mol. Imaging Biol. 13: 452-461; Zhang et al., (2008) Life sciences 82:1154-1161) and the like, which may be used in the MCs of this disclosure. Furthermore, the sensitivity of using endogenous biomarkers is inherently limited by the amount of biomarkers produced by the tumor (Hori & Gambhir (2011) Sci. Translational Med. 3: 109ra116). In contrast, the sensitivity of the MC system of this disclosure can be improved.

[0145] One advantage of endogenous blood biomarkers is that they can be used to determine what type of cancer an individual may have (e.g., high PSA levels may indicate prostate cancer). However, the MC system provided by this disclosure is also advantageous for screening all cancers, rather than specific tumor types. Furthermore, alternative promoters for screening patients at high risk for specific cancers (e.g., variants of the prostate-specific antigen enhancer / promoter for prostate cancer) (Iyer et al., (2005) Transgenic Res. 14: 47-55; Iyer et al., (2004) Mol. Therapy 10: 545-552; Iyer et al., (2006) Human Gene Therapy 17: 125-132) or the mucin-1 promoter for breast cancer (Huyn et al., (2009) Clin. Cancer Res. 15: 3126-3134), and the like, can be incorporated into the MC system of this disclosure.

[0146] Another limitation of exogenous biomarkers (i.e., reporters) is that they cannot pinpoint the site of origin of the biomarker in the body. The systems of this disclosure can also allow visualization of tumor localization by replacing them with imaging reporter genes or by co-expressing SEAP and imaging reporter genes (e.g., herpes simplex virus thymidine kinase 1 for positron emission tomography (PET), described in, for example, Yaghoubi SS and Gambhir SS (2006) Nat Protoc. 1(6):3069-75). Bhang et al. recently described the ability to image tumors using both BLI and single-photon emission computed tomography (SPECT) after systemic administration of a tumor-activated plasmid expressing an appropriate imaging reporter gene (Bhang et al., (2011) Nat. Med. 17: 123-129). This strategy also utilizes the SEAP-expressing viral vectors described to date, as these vectors co-express fluorescent proteins for visualization of cancer using fluorescence stereomicroscopy (Chaudhuri et al., (2003) Technol. In Cancer Res. & Treat. 2: 171-180; Warram et al., (2011) Mol. Imaging Biol. 13: 452-461; Warram et al., (2012) Cancer Gene Therapy 19: 545-552). Instead of a single vector system expressing both reporters, this strategy further considers the possibility of delivering two distinct vectors designed for a specific application; one for screening cancers expressing a secretory reporter, and another for expressing an imaging reporter to localize tumors.

[0147] Therefore, one aspect of this disclosure covers an embodiment of a recombinant nucleic acid microcircular vector comprising a nucleotide sequence operatively linked to a tumor-specific gene expression promoter and resulting in a higher level of expression by tumor cells than by non-tumor cells in the recipient.

[0148] In embodiments of this disclosure, the tumor-specific gene expression promoter may be selected from: BIRC5 (survival protein promoter), CXCR4 promoter, ATP-binding box subfamily C member 4 (ABCC4) promoter, pregradient protein 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter, sealing protein 3 (CLDN3) promoter, sealing protein 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter, collagen type I α1 chain promoter, etc. The promoters of the following proteins are listed: COL1A1 promoter, cystatin SN (CST1) promoter, DTL promoter, family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), and laminin subunit γ. LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α promoter, Ubiquitin D promoter, Ubiquitin conjugate E2C promoter, USH1 promoter, LAMC2 promoter, MISP promoter, MMP1 ...Protein network components include: harmonin (USH1C), T cell activation inhibitor 1 (VTCN1) promoter containing V-set domain, hexokinase type II promoter, TRPM4 promoter, lysosome 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible gRP78 / BiP promoter, interleukin-10 promoter, α-β-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, MTS1 promoter, NSE promoter, and somatostatin receptor. Promoters, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, alpha-fetoprotein promoter, chorionic villus promoter, albumin promoter, glycoprotein A33 promoter, B-cell specific Moronnius leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; receptor promoters containing kinase domain inserts; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugate 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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2 S (UBE2S) promoter, Aurora kinase A and ninein interacting protein (AUNIP) promoter, Cell cycle 6 (CDC6) promoter, Centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, Deoxythymidine kinase (DTYMK) promoter, G protein regulation inducer 1 (GPRIN1) promoter, Mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, Nucleotide small ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, ATAD2 promoter containing AAA domain, BUB1The promoters of mitosis checkpoint serine / threonine kinase (BUB1), calccyclin-binding protein (CACYBP) promoter, cell cycle-associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, valve-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation regulator of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, and MYB... The promoters for the following gene sequences are listed: proto-oncogene-like 2 (MYBL2), NIMA-associated kinase 2 (NEK2), RAN-binding protein 1 (RANBP1), small nucleoprotein peptides B and B1 (SNRPB), SPC24 / NDC80 kinetochore complex component (SPC24), transforming acidic coiled-coil protein 3 (TACC3), TBC1 domain family member 31 (TBC1D31), thymidine kinase 1 (TK1), zinc finger protein 695 (ZNF695), aurora kinase A (AURKA), and BLM. RecQ-like helicase (BLM) promoter, chromosome 17 read frame 53 (C17orf53) promoter, pigment box 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell cycle 20 (CDC20) promoter, cell cycle 45 (CDC45) promoter, cell cycle-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-transmembrane G receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosome protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen X-type α Promoters for the following gene sequences: COL10A1 promoter, CSE1L promoter, DBF4 zinc finger promoter, GINS complex subunit 1 (GINS1) promoter, G protein-coupled receptor 19 (GPR19) promoter, kinin family member 18A (KIF18A) promoter, kinin family member 4A (KIF4A) promoter, kinin family member C1 (KIFC1) promoter, mini chromosome maintenance 10 replication initiation factor (MCM10) promoter, and components of the mini chromosome maintenance complex.MCM2 promoter, Miniature Chromosome Maintenance Complex Component 7 (MCM7) promoter, MRG Domain-Binding Protein (MRGBP) promoter, Methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 2, Methylenetetrahydrofolate Cyclohydrolase (MTHFD2) promoter, Non-SMC Condensation Protein I Complex Subunit H (NCAPH) promoter, NDC80, Kinocele Complex Component (NDC80) promoter, Nudix Hydrolase 1 (NUDT1) promoter, Ribonuclease H2 Subunit A (RNASEH2A) promoter, RuvB-like AAA promoter ATPase 1 (RUVBL1) promoter, serum-defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nucleonucleotide ribonucleoprotein polypeptide G (SNRPG) promoter, permanent circadian rhythm regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, nutrient protein-associated protein (TROAP) promoter, ubiquitin conjugate enzyme E2C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha fetal protein (AFP) promoter, fragments thereof, or any combination thereof.

[0149] In some embodiments of this aspect of the disclosure, a nucleotide sequence operatively linked to a tumor-specific promoter can be expressed as a polypeptide.

[0150] In some embodiments of this aspect of the disclosure, a nucleotide sequence operatively linked to a tumor-specific promoter may encode a reporter polypeptide.

[0151] In some embodiments of this aspect of the disclosure, the reporter peptide may be an MRI reporter, a PET reporter, a SPECT reporter, a photoacoustic reporter, a bioluminescent reporter, or any combination thereof.

[0152] In some embodiments of this aspect of the disclosure, the polypeptide may be a secreted embryo alkaline phosphatase (SEAP).

[0153] In some embodiments of this aspect of the disclosure, the recombinant nucleic acid microcircular vector may have a nucleic acid sequence according to SEQ ID NO: 1.

[0154] In some embodiments of this aspect of the disclosure, the peptide may be a bioluminescent reporter.

[0155] In some embodiments of this aspect of the disclosure, the recombinant nucleic acid microcircular vector may have a nucleic acid sequence according to SEQ ID NO: 2.

[0156] In some embodiments of this aspect of the disclosure, a nucleotide sequence operatively linked to a tumor-specific promoter may be expressed as a small interfering RNA (siRNA) or a therapeutically effective peptide.

[0157] Another aspect of this disclosure covers embodiments of pharmaceutically acceptable compositions comprising a recombinant nucleic acid microcircular carrier and a pharmaceutically acceptable carrier, the recombinant nucleic acid microcircular carrier comprising a nucleotide sequence operatively linked to a tumor-specific gene expression promoter and expressed at a level higher by recipient tumor cells than by non-tumor cells, wherein: (i) the tumor-specific gene expression promoter may be selected from: survivability protein promoter (BIRC5), CXCR4 promoter, ATP-binding box subfamily C member 4 (ABCC4) promoter, pregradient protein 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA... Promoters of cell adhesion molecule 5 (CEACAM5), centromere protein F (CENPF), centrosome protein 55 (CEP55), sealant protein 3 (CLDN3), sealant protein 4 (CLDN4), collagen type XI α1 chain (COL11A1), collagen type I α1 chain (COL1A1), cystatin SN (CST1), non-dentate E3 ubiquitin protein ligase homolog (DTL), family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), and laminin subunit γ. LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin conjugate E2 promoterC (UBE2C), harmonin (USH1C), a component of the USH1 protein network, the promoter of T cell activation inhibitor 1 (VTCN1) containing the V-set domain, the hexokinase type II promoter, the TRPM4 promoter, the lysosome 3 promoter, the surfactant protein A promoter, the secretory leukocyte protease inhibitor promoter, the tyrosinase promoter, the stress-inducible gRP78 / BiP promoter, the interleukin-10 promoter, the α-β-crystallin / heat shock protein 27 promoter, the epidermal growth factor receptor promoter, the mucin-like glycoprotein promoter, the MTS1 promoter, and the NSE promoter. Somatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, alpha-fetoprotein promoter, chorionic villus promoter, albumin promoter, glycoprotein A33 promoter, B-cell specific Moronnius leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; receptor promoters containing kinase domain inserts; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugate 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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2S (UBE2S) promoter, Aurora kinase A and ninein interacting protein (AUNIP) promoter, Cell cycle 6 (CDC6) promoter, Centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, Deoxythymidine kinase (DTYMK) promoter, G protein regulation inducer 1 (GPRIN1) promoter, Mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, Nucleoside small ribonucleoprotein polypeptide A '(SNRPA1) promoter, ATPase family, AAA domain-containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calccyclin-binding protein (CACYBP) promoter, cell cycle-associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, valve-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation regulator of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nucleonucleotide ribonucleoprotein peptides B and B1 (SNRPB) promoter, SPC24 / NDC80 Promoters for the following gene groups: Kinesiosome Complex Component (SPC24), Transforming Acidic Coil-Containing Helicin 3 (TACC3), TBC1 Domain Family Member 31 (TBC1D31), Thymidine Kinase 1 (TK1), Zinc Finger Protein 695 (ZNF695), Aurora Kinase A (AURKA), BLMRecQ-like Helicase (BLM), Chromosome 17 Read Frame 53 (C17orf53), Pigment Frame 3 (CBX30), Cyclin B1 (CCNB1), Cyclin E1 (CCNE1), Cyclin F (CCNF), Cell Cycle 20 (CDC20), Cell Cycle 45 (CDC45), Cell Cycle-Associated 5 (CDCA5), Cyclin-Dependent Kinase Inhibitor 3 (CDKN3), and Cadwelling Glue Protein (EGF). LAG seven-fold transmembrane G receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosome protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen X-type α promoter.Promoters for the following proteins: COL10A1, CSE1L, DBF4 zinc finger, GINS complex subunit 1 (GINS1), G protein-coupled receptor 19 (GPR19), kinin family member 18A (KIF18A), kinin family member 4A (KIF4A), kinin family member C1 (KIFC1), mini chromosome maintenance 10 replication initiation factor (MCM10), and mini chromosome maintenance complex component 2 (MCM2). Promoters, Miniature Chromosome Maintenance Complex Component 7 (MCM7) promoter, MRG Domain-Binding Protein (MRGBP) promoter, Methylene Tetrahydrofolate Dehydrogenase (NADP+ Dependent) 2, Methylene Tetrahydrofolate Cyclohydrolase (MTHFD2) promoter, Non-SMC Condensed Protein I Complex Subunit H (NCAPH) promoter, NDC80, Kinocele Complex Component (NDC80) promoter, Nudix Hydrolase 1 (NUDT1) promoter, Ribonuclease H2 Subunit A (RNASEH2A) promoter, RuvB-like AAA The promoters of ATPase 1 (RUVBL1), serum-defined breast cancer antigen NY-BR-85 (SGOL1), SHC-binding and spindle-associated 1 (SHCBP1), small nucleoprotein polypeptide G (SNRPG), permanent circadian rhythm regulator, thyroid hormone receptor interactor 13 (TRIP13), nutrient protein-associated protein (TROAP), ubiquitin conjugate E2C (UBE2C), WD repeat and HMG box DNA-binding protein 1 (WDHD1), alpha fetal protein (AFP), fragments thereof, or any combination thereof, and (ii) nucleotide sequences operatively linked to tumor-specific promoters may be expressed as polypeptides encoding MRI, PET, SPECT, photoacoustic, bioluminescent, or any combination thereof.

[0158] In some embodiments of this aspect of the disclosure, the recombinant nucleic acid microcircular vector may have a nucleic acid sequence according to SEQ ID NO: 1 or SEQ ID NO: 2.

[0159] Another aspect of this disclosure covers an embodiment of a method for detecting tumor cells in a human or non-human subject, the method comprising the steps of: (i) delivering to a first subject, a human or non-human animal, a pharmaceutically acceptable composition comprising a recombinant nucleic acid microcircular carrier and a pharmaceutically acceptable carrier, the recombinant nucleic acid microcircular carrier comprising a nucleotide sequence operatively linked to a tumor-specific gene expression promoter and expressed at a level higher by tumor cells of the recipient than by non-tumor cells, wherein: (a) the tumor-specific gene expression promoter may be selected from: the survival protein promoter (BIRC5), the CXCR4 promoter, the ATP-binding box subfamily C member 4 (ABCC4) promoter, the pregradient protein 2, the protein disulfide isomerase family member (AGR2) promoter, the activation-induced cytidine deaminase (AICDA) promoter, the UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, the cadherin 3 (CDH3) promoter, the CEA promoter, etc. The promoters of cell adhesion molecule 5 (CEACAM5), centromere protein F (CENPF), centrosome protein 55 (CEP55), sealant protein 3 (CLDN3), sealant protein 4 (CLDN4), collagen type XI α1 chain (COL11A1), collagen type I α1 chain (COL1A1), cystatin SN (CST1), non-dentate E3 ubiquitin protein ligase homolog (DTL), family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), and laminin subunit γ LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α (TOP2A) promoter, Ubiquitin D promoter, Ubiquitin conjugate E2 C promoter (UBE2C), USH1 protein network component harmonin (USH1C), T cell activation inhibitor 1 (VTCN1) promoter containing V-set domain, hexokinase type II promoter, TRPM4 promoter, lysosome 3 promoter, surfactant proteinA promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible gRP78 / BiP promoter, interleukin-10 promoter, α-β-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, MTS1 promoter, NSE promoter, somatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin Promoters, α-fetoprotein promoter, chorionic villus promoter, albumin promoter, glycoprotein A33 promoter, B-cell specific Moroni leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; receptor promoters containing kinase domain inserts; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugate 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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 kinetochore complex component (DSN1) promoter, deoxythymidine kinase (DTYMK) promoter, inducer of G protein regulation of neurite growth 1 (GPRIN1) promoter, mitochondrial fission regulator 2 (MTFR2) promoter, RAD51-associated protein 1 (RAD51AP1) promoter, nucleoside small ribonucleoprotein polypeptide A' (SNRPA1) promoter, ATPase family, AAA domain-containing 2 (ATAD2) promoter, BUB1 The promoters of the following mitotic checkpoint serine / threonine kinases (BUB1), calcyl-cyclin-binding protein (CACYBP), cell cycle-associated 3 (CDCA3), centromere protein O (CENPO), valve-specific endonuclease 1 (FEN1), forkhead box M1 (FOXM1), cell proliferation regulator of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, and nuclear transporter subunit α.2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nucleonucleotide ribonucleoprotein peptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, converting acidic protein 3 containing coiled helix (TACC3) promoter, 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 read frame 53 (C17orf53) promoter, pigment box 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell cycle 20 (CDC20) promoter, cell cycle 45 (CDC45) promoter, cell cycle-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-transmembrane G receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosome protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen X-type α Promoters for the following proteins: COL10A1, CSE1L, DBF4 zinc finger, GINS complex subunit 1 (GINS1), G protein-coupled receptor 19 (GPR19), kinin family member 18A (KIF18A), kinin family member 4A (KIF4A), kinin family member C1 (KIFC1), mini chromosome maintenance 10 replication initiation factor (MCM10), and mini chromosome maintenance complex component 2 (MCM2). Promoters, Miniature Chromosome Maintenance Complex Component 7 (MCM7) promoter, MRG Domain-Binding Protein (MRGBP) promoter, Methylene Tetrahydrofolate Dehydrogenase (NADP+ Dependent) 2, Methylene Tetrahydrofolate Cyclohydrolase (MTHFD2) promoter, Non-SMC Condensed Protein I Complex Subunit H (NCAPH) promoter, NDC80, Kinocele Complex Component (NDC80) promoter, Nudix Hydrolase 1 (NUDT1) promoter, Ribonuclease H2 Subunit A (RNASEH2A) promoter, RuvB-like AAAATPase 1 (RUVBL1) promoter, serum-defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nucleonucleotide polypeptide G (SNRPG) promoter, permanent circadian rhythm regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, nutrient protein-associated protein (TROAP) promoter, ubiquitin conjugate E2 C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, α (i) a fetalin (AFP) promoter, a fragment thereof, or any combination thereof; and (b) a nucleotide sequence operatively linked to a tumor-specific promoter that can be expressed as a polypeptide encoding an MRI reporter, a PET reporter, a SPECT reporter, a photoacoustic reporter, a bioluminescent reporter, or any combination thereof; and (ii) detection of an expression product in the first subject, wherein the expression product is generated from a nucleotide sequence operatively linked to a tumor-specific gene expression promoter of the microcircular vector, and wherein detection of the expression product indicates the presence of tumor cells in the first subject.

[0160] In some embodiments of this aspect of the disclosure, the expression product may be a serum peptide, and step (ii) may include obtaining a serum sample from a first subject and determining the serum level of the expression product generated from the microcircular vector.

[0161] In some embodiments of this aspect of the disclosure, the expression product to be detected may be secreted embryo alkaline phosphatase (SEAP).

[0162] In some embodiments of this aspect of the disclosure, the microcircular vector may have a nucleic acid sequence according to SEQ ID NO: 1.

[0163] In some embodiments of this aspect of the disclosure, the expression product may be a bioluminescent polypeptide, and step (ii) may include generating a detectable signal derived from the expression product, measuring the level of the detectable signal generated from the microcircular carrier, and comparing the level of the signal from a first subject with the level of the signal obtained from a second subject who has never received the microcircular carrier, wherein the elevated signal level from the first subject, compared to the level obtained from the second subject, indicates that the first subject contains tumor cells or a tumor cell population.

[0164] In some embodiments of this aspect of the present disclosure, step (ii) may further include non-invasively detecting the detectable signal, converting the signal into an image, overlaying the image onto an image of a first subject, and locating the detectable signal relative to the first subject to determine the location of tumor cells or tumor cell populations in the first subject.

[0165] In some embodiments of this aspect of the disclosure, the expression product may be luciferase.

[0166] In some embodiments of this aspect of the disclosure, the microcircular vector may have a nucleic acid sequence according to SEQ ID NO: 2.

[0167] Improved synthetic biomarkers for disease diagnosis, detection, and monitoring

[0168] In some aspects, this disclosure provides a method comprising:

[0169] (a) administering a composition to a subject, wherein the composition induces in the subject the expression of a synthetic biomarker in diseased cells preferentially over the expression of the biomarker in non-diseased cells, such that the relative concentration ratio of the biomarker expressed in diseased cells to that in non-diseased cells is greater than 1.0; (b) detecting the synthetic biomarker; and (c) using the biomarker detected in (b) to detect that the subject has the diseased cells. In some embodiments, the detection has an accuracy of at least 90%.

[0170] In some cases, the composition is administered to the subject via intravenous, subcutaneous, intraventricular, intrathecal, intracerebral, percutaneous, intramuscular, oral, inhalation, nasal, rectal, intratumoral, or adjacent tumor. Adjacent tumor may refer to administration to tissues near the tumor or to areas that are predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes). Intratumoral or adjacent tumor methods may include the use of additional imaging techniques such as endoscopic ultrasound (see, for example, Shirley et al., Gastroenterol Res Pract. 2013; 2013: 207129) or via bronchoscopy (see, for example, Rojas-Solano et al., J Bronchology Interv Pulmonol. 2018 Jul; 25(3): 168–17). In some embodiments, the composition is applied to at least one of the following lymph nodes: cervical lymph nodes, medial epicondyle lymph nodes, supraclavicular lymph nodes, axillary lymph nodes, mediastinal lymph nodes, supratrochlear lymph nodes, mesenteric lymph nodes, inguinal lymph nodes, femoral lymph nodes, or popliteal lymph nodes. In some cases, lymph node-based application can be used as a method of concentrated local delivery to the tissue area.

[0171] In some cases, the detection of diseased cells can have an accuracy of at least about 50%, at least about 53%, at least about 55%, at least about 57%, at least about 60%, at least about 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%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or any range between these values. In some cases, the detection of diseased cells can have an accuracy of 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%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or any range between these values.

[0172] In some cases, the detection of diseased cells can have a sensitivity of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range between these values.

[0173] In some cases, the detection of diseased cells can have a specificity of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range between these values.

[0174] In some cases, the detection of diseased cells can have a negative predictive value (NPV) of at least approximately 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%, 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. In some cases, the detection of diseased cells can have an NPV of at least about 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%, 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.

[0175] In some cases, the detection of diseased cells can have a positive predictive value (PPV) of 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%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or any range between these values. In some cases, the detection of diseased cells can have a PPV of up to approximately 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%, or 99%, or any range between these values.

[0176] In some embodiments, the composition may comprise a vector encoding a synthesized biomarker. Suitable vectors include those suitable for administration into cells, including but not limited to microcircles, plasmids, nanoplasmids, small intron plasmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), sclerosites, phage particles, bacteriophages, and baculoviruses. Suitable vectors also include those derived from bacteriophages or plants, invertebrates or animals (including humans), and viruses, such as CELiD vectors, adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or combinations thereof, such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g., MLV, or its self-inactivating or SIN variants, or their pseudotypes), herpesvirus vectors (based on, for example, HSV- or EBV), lentiviral vectors (e.g., based on HIV, FIV, or EIAV, or their pseudotypes), or adenoviral vectors (e.g., based on Ad5, including its replication-deficient, reproducible, or helper-dependent variants). In some cases, the vector may contain attachment maintenance elements to facilitate replication in one or more target cell types, such as scaffold / matrix attachment regions (S / MAR). S / MAR elements are particularly useful for facilitating replication in the context of "naked" nucleic acid vectors, such as microloops. Exemplary suitable S / MAR elements include, but are not limited to, EμMAR from the immunoglobulin heavy chain locus, apoB MAR from the human apolipoprotein B locus, Ch-LysMAR from the chicken lysozyme locus, and huIFNβ MAR from the human IFNβ-locus. In some embodiments, the vector may be a non-viral vector.

[0177] In some cases, the composition may comprise a vector containing a sequence encoding a synthetic biomarker operatively linked to a promoter. Suitable promoters include natural pan-tumor-specific promoters, natural tissue-specific promoters, natural disease-specific / disease-activating promoters, natural constitutive promoters, and any combination thereof. The promoters can be the survival protein promoter (BIRC5), CXCR4 promoter, ATP-binding box subfamily C member 4 (ABCC4) promoter, pregradient protein 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter, sealing protein 3 (CLDN3) promoter, sealing protein 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter, collagen type I α1 chain promoter, etc. The promoters of the following proteins are listed: COL1A1 promoter, cystatin SN (CST1) promoter, DTL promoter, family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), and laminin subunit γ. LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α promoter, Ubiquitin D promoter, Ubiquitin conjugate E2 C promoter, USH1 protein network component harmonin (USH1C), T cell activation inhibitor containing V-set domain.VTCN1 promoter, hexokinase type II promoter, TRPM4 promoter, lysosome 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible gRP78 / BiP promoter, interleukin-10 promoter, α-β-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, MTS1 promoter, NSE promoter, somatostatin receptor promoter c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, alpha-fetoprotein promoter, chorionic villus promoter, albumin promoter, glycoprotein A33 promoter, B-cell specific Moronie's leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; receptor promoters containing kinase domain inserts; rad51 Recombinase promoters; TTF-1, urokinase plasminogen activator receptor promoter, ubiquitin conjugate E2T (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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 The promoters of the following proteins are listed: Kinetogranulomatous complex component (DSN1), deoxythymidine kinase (DTYMK), inducer of G protein regulation of neurite growth 1 (GPRIN1), mitochondrial fission regulator 2 (MTFR2), RAD51-associated protein 1 (RAD51AP1), small nucleonucleotide polypeptide A' (SNRPA1), ATPase family, AAA domain-containing 2 (ATAD2) promoter, BUB1 mitotic checkpoint serine / threonine kinase (BUB1) promoter, calccyclin-binding protein (CACYBP) promoter, cell cycle-associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, valvular structure-specific endonuclease 1 (FEN1) promoter, and forkhead box.M1 (FOXM1) promoter, cell proliferation regulator of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, MYB proto-oncogene-like 2 (MYBL2) promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nucleonucleotide polypeptide B and B1 (SNRPB) promoter, SPC24 / NDC80 kinosome complex component (SPC24) promoter, converting acidic coiled-coil protein 3 (TACC3) promoter, 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 read frame 53 (C17orf53) promoter, pigment box 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell cycle 20 (CDC20) promoter, cell cycle 45 (CDC45) promoter, cell cycle-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-transmembrane G receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosome protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen X-type α Promoters for the following proteins: COL10A1, CSE1L, DBF4 zinc finger, GINS complex subunit 1 (GINS1), G protein-coupled receptor 19 (GPR19), kinin family member 18A (KIF18A), kinin family member 4A (KIF4A), kinin family member C1 (KIFC1), mini chromosome maintenance 10 replication initiation factor (MCM10), and mini chromosome maintenance complex component 2 (MCM2). Promoters, Miniature Chromosome Maintenance Complex Component 7 (MCM7) promoter, MRG Domain-Binding Protein (MRGBP) promoter, Methylene Tetrahydrofolate Dehydrogenase (NADP+ Dependent) 2, Methylene Tetrahydrofolate Cyclohydrolase (MTHFD2) promoter, Non-SMC Condensed Protein I Complex Subunit H (NCAPH) promoter, NDC80, Kinocele Complex Component (NDC80) promoter, Nudix Hydrolase 1 (NUDT1) promoter, Ribonuclease H2 Subunit A (RNASEH2A) promoter, RuvB-like AAAATPase 1 (RUVBL1) promoter, serum-defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nucleonucleotide ribonucleoprotein polypeptide G (SNRPG) promoter, permanent circadian rhythm regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, nutrient protein-associated protein (TROAP) promoter, ubiquitin conjugate enzyme E2C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha fetal protein (AFP) promoter, fragments thereof, or any combination thereof.

[0178] In some cases, the synthesized biomarker may be a peptide or a nucleic acid biomarker. Peptides include any reporter peptides described herein. Nucleic acids include natural or engineered miRNAs, RNA hairpins, and RNA aptamers, or barcoded variations thereof. When the nucleic acid is a miRNA, it can be detected, for example, by standard library generation techniques such as annealing and ligation based on degenerate primers, poly(A) polymerase labeling followed by RT or ligation, or sequential adaptor ligation to q-PCR, sequencing, or electrophoresis detection methods. When the biomarker is a peptide, it may contain an N-terminal secretory signal sequence (e.g., an N-terminal signal peptide from CD33 or CD8a).

[0179] By assigning unique markers to unique members within a larger group, barcoding provides the opportunity to identify and quantify a member (e.g., the expression of a reporter under the control of a specific cancer-specific promoter) within a larger and more complex mixture of many members (e.g., multiple promoter-reporter constructs expressed within the same cell), and also provides the opportunity to isolate individual members from complex mixtures. For example, in the case of nucleic acid-based barcoding, hybridization based on base pair complementarity can be used for capture and isolation, or otherwise reduce the complexity of the mixture through said capture event. For peptide-based barcoding, unique characteristics (including immune capture or interaction of ligands and receptors) can be used for capture and isolation, or otherwise reduce the complexity of the mixture through said capture event.

[0180] When the nucleic acid is an engineered miRNA, the nucleic acid can be a Sec-miR or miR-neg construct as described in Ronald et al. (Ronald et al., PLoS ONE 11(7): e0159369). Such a construct comprises: (a) a coding sequence that is not endogenously expressed and has no known vertebrate targets (e.g., Sec-miR 5′-AAAUGUACUGCGCGUGGAGAC-3′); (b) a miR backbone sequence that provides treatment of the pre-miRNA to mature the miRNA flanking the coding sequence (e.g., miR-155 or miR-130 backbone sequence); and (c) an EXOmotif (e.g., GGAG) that enhances loading into the exosome. Such a miRNA construct can be expressed in, for example, the 3'-UTR of a gene encoding a reporter polypeptide, or from the 3'-UTR of a gene encoding a suitable non-toxic protein (e.g., an endogenous structural protein such as actin or tubulin, or a highly expressed protein such as ubiquitin). In some implementations, multiple (e.g., at least 2, at least 4) copies of the engineered miRNA can be provided in tandem.

[0181] In some cases, the synthetic biomarker may be a peptide biomarker detectable by a non-invasive imaging method performed on a subject, and / or the method includes detecting the synthetic biomarker by non-invasive imaging. Such non-invasive imaging methods include MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescence imaging. Synthetic biomarkers detectable by MRI imaging include peptide contrast agents, such as ferritin (or its mutants, such as *C. fibrillosa*). Pyrococcus furioususFerritin mutants (L55P, F57S, or F123S) or lanthanide-binding proteins (or their engineered fusions, such as the LBT-ubiquitin fusions described in Daughtry et al., ChemBioChem 2012, 13, 2567–2574). Synthetic biomarkers detectable by PET or SPECT imaging include human sodium iodide cotransporters (e.g., in combination with PET-applied active iodine / iodine isotopes, see, for example, Penheiter et al., Curr Gene Ther. 2012 Feb; 12(1): 33–47), HSV-tk or mutants of it such as HSV-sr39tk (e.g., in combination with PET-applied positron-labeled acycloguanosine or pyrimidine analogues such as [18F]FHBG, see, Yaghoubi SS et al., Nat Protoc. 2006; 1(6): 3069-75), and dopamine D2 receptors or mutants of them such as D2R80A or D2R194A (e.g., in combination with positron-labeled D2 binders such as 3-(2'-[18F]-fluoroethyl)-spiropepidermobenzene). Synthetic biomarkers detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal administration), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants). Synthetic biomarkers detectable by bioluminescence imaging include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renal luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396 (2010): 290-297). In some embodiments, the synthetic biomarker may be a contrast agent, an enzyme that produces a detectable molecule, or a transporter that drives the accumulation of the detectable molecule. The synthetic biomarker can be measured in situ in a subject.

[0182] In cases where the synthesized biomarker is a peptide biomarker detectable by a non-invasive imaging method, the method includes administering a composition to a subject that induces expression of the synthesized biomarker in diseased cells. The method may further include (d) localizing the diseased cells in the subject's body. Localization may be associated with a specific resolution, such as 10 mm to 10 cm, at least 10 mm, or at most 10 cm. Localization may also be associated with a specific minimum detectable tumor size, such as a tumor size of 3 mm. 3 Up to 5 cm 3 In some cases, a specific minimum size range can be 1 cm. 3 up to 5cm 3 or 900 mm 3 Up to 1 cm 3 or 800 mm 3 Up to 900 mm 3 or 700 mm 3 Up to 800 mm 3 or 600 mm 3 Up to 700 mm 3 or 500 mm 3 Up to 600 mm 3 or 400 mm 3 Up to 500 mm 3 or 300 mm 3 Up to 400 mm 3 or 200 mm 3 Up to 300 mm 3 or 100 mm 3 Up to 200 mm 3 or 50 mm 3 Up to 100 mm 3 or 10 mm 3 Up to 50 mm 3 or 3 mm 3 Up to 10mm 3 In some cases, localization occurs during non-invasive imaging scans (e.g., PET, MRI, SPECT, etc.). In other cases, localization occurs during on-site surgical intervention, such as through visual examination (in cases where the absorbance report is visible) or through visual examination combined with fluorescence excitation.

[0183] In some cases, the additional localization steps described above may be followed by a surgical step to eliminate the detected and / or localized diseased cells. The surgical step may be performed on the same or different sites as where the composition encoding the biomarker is applied and / or localized to the diseased cells. The surgical step may be surgical removal of the diseased cells or the tumor associated with the diseased cells. Surgical or non-surgical elimination steps may include minimally invasive killing techniques, such as radiosurgery (including, but not limited to, Gamma Knife, Reflexion, CyberKnife, and related techniques that utilize targeted ionizing radiation to kill diseased cells).

[0184] In some cases, synthetic biomarkers can be detected in biological samples from subjects who have been administered a composition that induces the expression of the synthetic biomarker. In other cases, synthetic biomarkers are detected in vivo and used to pinpoint the location of diseased cells.

[0185] In some cases, the composition administered to the subject may contain a transfection agent. Suitable transfection agents include, but are not limited to, linear or branched polyethyleneimine, nanoparticles, lipophilic particles, peptides, micelles, dendritic molecules, hydrogels, synthetic or naturally derived exosomes, polymer compositions, virus-like particles, and any combination thereof.

[0186] In some cases, the composition may further comprise a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers include, but are not limited to, water, peanut oil, soybean oil, mineral oil, sesame oil, saline solution, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, hydrated dextrose, glycerol solution, glucose, lactose, sucrose, glyceryl monostearate, sodium chloride solution, propylene glycol or ethanol, or any combination thereof.

[0187] Biological samples can be samples collected from subjects using non-invasive methods. Exemplary non-invasive samples include, but are not limited to, saliva, sputum, sweat, urine, feces, semen, cervical and vaginal secretions, breast milk, inflammatory secretions, tears, and buccal epithelial swabs. Biological samples can also be samples collected from subjects using minimally invasive methods. Exemplary minimally invasive samples include, but are not limited to, blood samples (e.g., obtained via venipuncture or capillary), pleural fluid samples (e.g., obtained via thoracentesis), amniotic fluid samples (e.g., obtained via amniocentesis), and gastric fluid samples (e.g., obtained via gastric lavage). Biological samples can be samples obtained through biopsy, such as skin biopsy samples (e.g., obtained through perforation, scraping, discoid surgery, wedge, incision, or excision biopsy), bone marrow samples (e.g., obtained through aspiration biopsy), lymph node or breast biopsy samples (e.g., obtained through fine-needle aspiration, core needle biopsy, vacuum-assisted biopsy, or image-guided biopsy), surgical biopsy samples (e.g., internal organ samples obtained through excision or incision biopsy), or mouth, gastrointestinal tract, lung, bladder, or urethra biopsy samples (e.g., obtained through endoscopy).

[0188] In some cases, biological samples can be obtained at a certain time after administration of the composition for inducing the expression of the synthetic biomarker. Biological samples can be obtained at least about 15 minutes, 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 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 about 5 months, or at least about 6 months after administration of the composition for inducing the expression of the synthetic biomarker. Biological samples can be obtained at most approximately 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of the composition inducing the expression of the synthetic biomarker. In some embodiments, biological samples can be obtained after administration of the composition inducing the expression of the synthetic biomarker, and any biomarker detection protocol can be performed multiple times (e.g., to monitor synthetic biomarker levels over time). Biological samples can be obtained at least approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 times after administration of the composition inducing the expression of the synthetic biomarker. Biological samples can be obtained weekly or monthly after administration of the composition inducing the expression of the synthetic biomarker.

[0189] In some cases, diseased cells may be cancer cells, cells indicating autoimmune diseases (such as self-directing T cells or lymphocytes, or normal cells impaired by autoimmunity), cells indicating neurodegenerative diseases (such as cells with or near toxic amyloid), or cells that may have altered gene expression profiles due to the subject from whom the cells originate having or being diagnosed with a disease. A cell population containing cells with altered gene expression profiles can be described as transcriptionally altered cells (TACs). In some cases, diseased cells may be cancer cells. Exemplary cancers include, but are not limited to, carcinomas, sarcomas, lymphomas, leukemias, and adenomas. Carcinomas may arise in cells covering internal and external parts of the body, such as the lungs, breast, and colon. Sarcomas may arise in cells located in bone, cartilage, fat, connective tissue, muscle, and other supporting tissues. Lymphomas may arise in lymph nodes and immune system tissues. Leukemia may arise in the bone marrow and accumulate in the bloodstream. Adenomas may arise in the thyroid gland, pituitary gland, adrenal gland, and other glandular tissues.Specific exemplary examples of cancer types suitable for detection using the methods according to this disclosure include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic gliomas, breast cancer, bronchial adenoma, Burkitt lymphoma, cancers of unknown primary origin, central nervous system lymphomas, and cerebellar astrocytoma. Cytokine tumor, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer, such as non-small cell lung cancer and small cell lung cancer, lymphoma Tumors, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer of unknown primary origin, oral cancer, multiple endocrine tumor syndrome, myelodysplastic syndrome, myeloid leukemia, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cells, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma Cell tumors, pineal germ cell tumors, pituitary adenomas, pleural pulmonary blastomas, plasmacytoma formation, primary central nervous system lymphomas, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell skin cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumors (pregnancy), cancers of unknown primary origin, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0190] In some cases, diseased cells may be virus-infected cells. Exemplary viruses include, but are not limited to, HIV, hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesvirus, Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus type III.

[0191] In some cases, diseased cells may indicate an autoimmune disease. Exemplary autoimmune diseases include, but are not limited to, achalasia, Addison's disease, adult-onset Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axononeuropathy (AMAN), Baló's disease, Behçet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castleman's disease (CD), steatorrhea, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS), or Eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, herpetic dermatitis, dermatomyositis, Devic disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, idiopathic mixed cryoglobulinemia, Evans syndrome, Fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpassuu syndrome, granulomatous polyangiitis, Grave's disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (acne paradox), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eton syndrome, leukocyte rupture vasculitis, lichen planusLichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren'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 neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, pars plana cyclitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, recurrent polychondritis, restless legs syndrome (RL) S), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), aortitis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0192] In some cases, diseased cells may indicate neurodegenerative diseases. Neurodegenerative diseases include, but are not limited to, multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), or neurodegeneration caused by infection with the following viruses: Herpesviridae, Polyomaviridae, Bornaviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Picornaviridae, or Retroviridae (see Zhou et al., Virol J. 2013; 10: 172).

[0193] Gene / DNA-based therapies for diseased cells

[0194] In some aspects, this disclosure provides a method of treating a subject who has or is suspected of having a disease, the method comprising administering a composition to the subject, the composition inducing in the subject disease-associated disease cells to express a therapeutic efficacy preferentially to non-disease cells, such that the relative concentration of the therapeutic efficacy expressed by the disease-associated cells compared to the non-disease cells is greater than 1.0, the therapeutic efficacy treating the subject with at least 10% therapeutic efficacy, as determined by a reduction in the cell population of the disease-associated cells.

[0195] In some cases, the composition is administered to the subject intravenously, subcutaneously, intraventricularly, intrathecally, intracerebrally, percutaneously, intramuscularly, or orally, by inhalation, nasally, or transrectally, intratumorally, or adjacent to a tumor. Adjacent to a tumor may mean administration to tissues near the tumor or to areas that are predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes). Methods involving intratumoral or adjacent tumors may include the use of additional imaging techniques such as endoscopic ultrasound (see, for example, Shirley et al., Gastroenterol Res Pract. 2013; 2013: 207129) or bronchoscopy (see, for example, Rojas-Solano et al., J Bronchology Interv Pulmonol. 2018 Jul; 25(3):168–175). In some embodiments, the composition is applied to at least one of the following lymph nodes: cervical lymph nodes, medial epicondyle lymph nodes, supraclavicular lymph nodes, axillary lymph nodes, mediastinal lymph nodes, supratrochlear lymph nodes, mesenteric lymph nodes, inguinal lymph nodes, femoral lymph nodes, or popliteal lymph nodes. In some cases, lymph node-based application can be used as a method of concentrated local delivery to the tissue area.

[0196] In some cases, compositions administered to treat subjects with or suspected of having a disease may contain a promoter operatively linked to a nucleotide sequence encoding a therapeutic agent. The promoter may be a cancer-specific promoter. Suitable promoters include natural pan-tumor-specific promoters, natural tissue-specific promoters, natural disease-specific / disease-activating promoters, natural constitutive promoters, and any combination thereof. The promoters can be the survival protein promoter (BIRC5), CXCR4 promoter, ATP-binding box subfamily C member 4 (ABCC4) promoter, pregradient protein 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter, sealing protein 3 (CLDN3) promoter, sealing protein 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter, collagen type I α1 chain promoter, etc. The promoters of the following proteins are listed: COL1A1 promoter, cystatin SN (CST1) promoter, DTL promoter, family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), and laminin subunit γ. LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α promoter, Ubiquitin D promoter, Ubiquitin conjugate E2C promoter, USH1 promoter, LAMC2 promoter, MISP promoter, MMP1 ... Protein network components include harmonin (USH1C), T cell activation inhibitor 1 (VTCN1) promoter containing V-set domain, hexokinase type II promoter, TRPM4 promoter, lysosome 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, and stress-inducible protein.GRP78 / BiP promoter, interleukin-10 promoter, α-β-crystallin / heat shock protein 27 promoter, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, MTS1 promoter, NSE promoter, somatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, α-fetoprotein promoter, chorionic villus promoter, albumin promoter, glycoprotein A33 promoter, B-cell specific Moronnius leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; receptor promoters containing kinase domain inserts; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugate 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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 The promoters of the kinetochore complex component (DSN1), deoxythymidine kinase (DTYMK), inducer of G protein regulation of neurite growth 1 (GPRIN1), mitochondrial fission regulator 2 (MTFR2), RAD51-associated protein 1 (RAD51AP1), small nucleonucleotide polypeptide A' (SNRPA1), ATPase family, AAA domain-containing 2 (ATAD2) promoter, and BUB1. The promoters of mitosis checkpoint serine / threonine kinase (BUB1), calccyclin-binding protein (CACYBP), cell cycle-associated 3 (CDCA3), centromere protein O (CENPO), valve-specific endonuclease 1 (FEN1), forkhead box M1 (FOXM1), cell proliferation regulator of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, and MYB proto-oncogene-like genes.MYBL2 promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nucleonucleotide ribonucleoprotein peptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinosome complex component (SPC24) promoter, converting acidic coiled-coil protein 3 (TACC3) promoter, 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 read frame 53 (C17orf53) promoter, pigment frame 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF) promoter. Cell cycle 20 (CDC20) promoter, cell cycle 45 (CDC45) promoter, cell cycle-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-transmembrane G receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosome protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen X-type α promoter. Promoters for the following proteins: COL10A1 (COL10A1), CSE1L (CSE1L), DBF4 zinc finger, GINS complex subunit 1 (GINS1), G protein-coupled receptor 19 (GPR19), kinin family member 18A (KIF18A), kinin family member 4A (KIF4A), kinin family member C1 (KIFC1), mini chromosome maintenance 10 replication initiation factor (MCM10), mini chromosome maintenance complex component 2 (MCM2), mini chromosome maintenance complex component 7 (MCM7), MRG domain-binding protein (MRGBP), methylenetetrahydrofolate dehydrogenase (NADP+ dependent), methylenetetrahydrofolate cyclohydrolase (MTHFD2), non-SMC condensed protein I complex subunit H (NCAPH), and NDC80. Kinetic-granulocyte complex component (NDC80) promoter, nudix hydrolase 1 (NUDT1) promoter, ribonuclease H2 subunit A (RNASEH2A) promoter, RuvB-like AAA promoterATPase 1 (RUVBL1) promoter, serum-defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nucleonucleotide ribonucleoprotein polypeptide G (SNRPG) promoter, permanent circadian rhythm regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, nutrient protein-associated protein (TROAP) promoter, ubiquitin conjugate enzyme E2C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha fetal protein (AFP) promoter, fragments thereof, or any combination thereof.

[0197] In some cases, a promoter operatively linked to a nucleotide sequence encoding a therapeutic agent may be present on a vector, which may be a component of a composition administered to a subject. Suitable vectors include those suitable for intracellular administration, including but not limited to microcircles, plasmids, nanoparticles, small intron plasmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), granules, phage particles, bacteriophages, and baculoviruses. Suitable vectors also include those derived from bacteriophages or plants, invertebrates or animals (including humans), and viruses, such as CELiD vectors, adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or combinations thereof such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g., MLV, or its self-inactivating or SIN variants, or their pseudotypes), herpesviruses (based on, for example, HSV- or EBV), lentiviral vectors (e.g., based on HIV, FIV, or EIAV, or their pseudotypes), or adenoviral vectors (e.g., based on Ad5, including its replication-deficient, reproducible, or helper-dependent variants). In some cases, the vector may contain attachment maintenance elements to facilitate replication in one or more target cell types, such as scaffold / matrix attachment regions (S / MAR). S / MAR elements are particularly useful for facilitating replication in the context of “naked” nucleic acid vectors such as microloops. Exemplary suitable S / MAR elements include, but are not limited to, EμMAR from the immunoglobulin heavy chain locus, apoB MAR from the human apolipoprotein B locus, Ch-LysMAR from the chicken lysozyme locus, and huIFNβ MAR from the human IFNβ-locus. In some embodiments, the vector may be a non-viral vector.

[0198] In some cases, the therapeutic efficacy may comprise a specific class of therapeutic agents. Exemplary classes of therapeutic agents suitable for use with the methods according to this disclosure include, but are not limited to, therapeutically effective peptides (e.g., therapeutic antibodies, fragments or derivatives thereof; cytokines; growth factors; engineered or alternative metabolic / catabolistic enzymes, engineered short peptide agonists or antagonists or prodrug activators), small activated RNA (saRNA), microRNA (miRNA), small interfering RNA (siRNA), or any combination thereof. In some cases, the therapeutic efficacy may be a prodrug activator. Exemplary prodrug activators include, but are not limited to, HSVtk, cytosine deaminase, DT xanthine, nitroreductase, guanine phosphoribosyltransferase, purine nucleoside phosphorylase, thymidine phosphorylase, carboxylesterase, folic acid polyglutamyl synthase, carboxypeptidase A1, carboxypeptidase G2, and cytochrome P-450. In cases where the therapeutic efficacy is a prodrug activator, the method may additionally administer the drug via any of the routes described herein. When the therapeutic agent is a peptide, the peptide may contain an N-terminal secretory signal sequence (e.g., an N-terminal signal peptide from CD33 or CD8a).

[0199] Improved synthetic biomarker constructs and methods for normalizing transfection rates across subjects

[0200] In some aspects, this disclosure provides a composition comprising a first nucleic acid sequence encoding a first polypeptide or nucleic acid biomarker, and a second nucleic acid sequence encoding a second polypeptide or second nucleic acid biomarker, wherein the composition is configured such that, when the composition is in a cell: the amount of the second polypeptide or nucleic acid biomarker expressed reflects at least the delivery of the first and second nucleic acids to the cell, and the first polypeptide or nucleic acid biomarker is differentially expressed in diseased cells relative to unaffected cells. In some cases, (i) the cells induce expression of the first nucleic acid sequence in diseased cells preferentially over expression in unaffected cells, wherein the first polypeptide is a detectable biomarker or therapeutic agent; and (ii) the cells induce equivalent expression of the second nucleic acid sequence in diseased and unaffected cells, and the second nucleic acid sequence produces the second polypeptide, which is not a detectable biomarker or therapeutic agent, such that the expression level of the second polypeptide provides a control for assessing the relative levels of the nucleic acid sequences in the cells. In some cases, the first nucleic acid sequence encoding the first polypeptide and the second nucleic acid sequence encoding the second polypeptide may be on separate gene constructs. In some cases, the sequences comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide may be on a separate gene construct. In some cases, the vector comprises: (a) a first promoter operatively linked to the first nucleic acid sequence, wherein the promoter induces expression of the first nucleic acid sequence in diseased cells preferentially over expression of the first nucleic acid sequence in non-diseased cells; and (b) a second promoter sequence that induces equal expression in diseased and non-diseased cells and is operatively linked to the second nucleic acid.

[0201] In some cases, the first polypeptide can serve as both a detectable biomarker and a therapeutic agent. In some cases, the first polypeptide is a therapeutic antibody, a therapeutic antibody fragment or derivative, or a prodrug activating enzyme. Exemplary prodrug activating enzymes include, but are not limited to, HSVtk, cytosine deaminase, DT flavotransferase, nitroreductase, guanine phosphoribosyltransferase, purine nucleoside phosphorylase, thymidine phosphorylase, carboxylesterase, folic acid polyglutamyl synthase, carboxypeptidase A1, carboxypeptidase G2, and cytochrome P-450. The polypeptide may contain an N-terminal secretory signal sequence (e.g., an N-terminal signal peptide from CD33 or CD8a).

[0202] In some cases, the first and / or second nucleic acid can be on a vector. Suitable vectors include those suitable for administration into cells, including but not limited to microcircles, plasmids, nanoplasmids, small intron plasmids, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), serosomes, phage particles, bacteriophages, and baculoviruses. Suitable vectors also include those derived from bacteriophages or plants, invertebrates or animals (including humans), and viruses, such as CELiD vectors, adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or combinations thereof such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g., MLV, or its self-inactivating or SIN variants, or their pseudotypes), herpesviruses (based on, for example, HSV- or EBV), lentiviral vectors (e.g., based on HIV, FIV, or EIAV, or their pseudotypes), or adenoviral vectors (e.g., based on Ad5, including its replication-deficient, reproducible, or helper-dependent variants). In some cases, the vector may contain attachment maintenance elements to facilitate replication in one or more target cell types, such as scaffold / matrix attachment regions (S / MAR). S / MAR elements are particularly useful for facilitating replication in the context of “naked” nucleic acid vectors such as microloops. Exemplary suitable S / MAR elements include, but are not limited to, EμMAR from the immunoglobulin heavy chain locus, apoB MAR from the human apolipoprotein B locus, Ch-LysMAR from the chicken lysozyme locus, and huIFNβ MAR from the human IFNβ-locus. In some embodiments, the vector may be a non-viral vector.

[0203] In some cases, the cells delivering the first and second nucleic acids may be diseased cells. In some cases, diseased cells may be cancer cells, cells indicating autoimmune diseases (e.g., self-directing T cells or lymphocytes, or normal cells impaired by autoimmunity), TACs, or cells indicating neurodegenerative diseases (e.g., cells carrying or adjacent to toxic amyloid). Exemplary cancers, autoimmune diseases, and neurodegenerative diseases that such cells may indicate include any cancers, autoimmune diseases, and neurodegenerative diseases described herein. In some cases, diseased cells may be virally infected cells. Exemplary viruses include, but are not limited to, HIV, hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesvirus, Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus type III.

[0204] In some cases, the first or second nucleic acid can be a detectable nucleic acid biomarker. Exemplary detectable nucleic acids include, but are not limited to, natural or engineered miRNAs, RNA hairpins, and RNA aptamers, or barcoded variations thereof. When the nucleic acid is a miRNA, the miRNA can be detected, for example, by standard library generation techniques such as annealing and ligation based on degenerate primers, poly(A) polymerase labeling followed by RT or ligation, or sequential adaptor ligation to q-PCR, sequencing, or electrophoresis detection methods. When the biomarker is a peptide, the peptide may contain an N-terminal secretory signal sequence (e.g., an N-terminal signal peptide from CD33 or CD8a).

[0205] When the nucleic acid is an engineered miRNA, the nucleic acid can be a Sec-miR or miR-neg construct as described in Ronald et al. (Ronald et al., PLoS ONE 11(7): e0159369). Such a construct comprises: (a) a coding sequence that is not endogenously expressed and has no known vertebrate targets (e.g., Sec-miR 5′-AAAUGUACUGCGCGUGGAGAC-3′); (b) a miR backbone sequence that provides treatment of the pre-miRNA to mature the miRNA flanking the coding sequence (e.g., miR-155 or miR-130 backbone sequence); and (c) an EXOmotif (e.g., GGAG) that enhances loading into the exosome. Such a miRNA construct can be expressed in, for example, the 3'-UTR of a gene encoding a reporter polypeptide, or from the 3'-UTR of a gene encoding a suitable non-toxic protein (e.g., an endogenous structural protein such as actin or tubulin, or a highly expressed protein such as ubiquitin). In some implementations, multiple (e.g., at least 2, at least 4) copies of the engineered miRNA can be provided in tandem.

[0206] In some cases, the second or first peptide may be detectable by a non-invasive imaging method performed on the subject. Such non-invasive imaging methods include MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescence imaging. Synthetic biomarkers detectable by MRI imaging include peptide contrast agents, such as ferritin (or mutants thereof, such as the *Porcine ferritin* mutants L55P, F57S, or F123S) or lanthanide-binding proteins (or engineered fusions thereof, such as the LBT-ubiquitin fusion described in Daughtry et al., ChemBioChem 2012, 13, 2567–2574). Synthetic biomarkers detectable by PET or SPECT imaging include human sodium iodide cotransporters (e.g., in combination with PET-applied active iodine / iodine isotopes, see, for example, Penheiter et al., Curr Gene Ther. 2012 Feb; 12(1): 33–47), HSV-tk or mutants of it such as HSV-sr39tk (e.g., in combination with PET-applied positron-labeled acycloguanosine or pyrimidine analogues such as [18F]FHBG, see, Yaghoubi SS et al., Nat Protoc. 2006; 1(6): 3069-75), and dopamine D2 receptors or mutants of them such as D2R80A or D2R194A (e.g., in combination with positron-labeled D2 binding agents such as 3-(2'-[18F]-fluoroethyl)-spiropepidermobenzene). Synthetic biomarkers detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal administration), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants).Synthetic biomarkers detectable by bioluminescence imaging include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renal luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396 (2010): 290-297). In some embodiments, the synthetic biomarker may be a contrast agent, an enzyme that produces a detectable molecule, or a transporter that drives the accumulation of the detectable molecule. The synthetic biomarker can be measured in situ in a subject.

[0207] In some cases, this disclosure provides a method for detecting diseased cells in a subject, the method comprising administering a composition to the subject, wherein the composition comprises: a first nucleic acid sequence encoding a first polypeptide or nucleic acid biomarker, and a second nucleic acid sequence encoding a second polypeptide or second nucleic acid biomarker, wherein the composition is configured such that, when the composition is in cells: the amount of the second polypeptide or nucleic acid biomarker expressed reflects at least the delivery of the first and second nucleic acids to the cells, and the first polypeptide or nucleic acid biomarker is differentially expressed in diseased cells relative to non-diseased cells. In some cases, (i) the cells induce expression of the first nucleic acid sequence in diseased cells preferentially over expression of the first nucleic acid sequence in non-diseased cells, wherein the first polypeptide is a detectable biomarker or 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 produces the second polypeptide, which is not a detectable biomarker or therapeutic agent, such that the expression level of the second polypeptide provides a control for assessing the relative levels of the nucleic acid sequence in the cells. In some cases, the first nucleic acid sequence encoding the first polypeptide and the second nucleic acid sequence encoding the second polypeptide may be on separate gene constructs. In some cases, the sequences comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide may be on a separate gene construct. In some cases, the vector comprises: (a) a first promoter operatively linked to the first nucleic acid sequence, wherein the promoter induces expression of the first nucleic acid sequence in diseased cells preferentially over expression of the first nucleic acid sequence in non-diseased cells; and (b) a second promoter sequence that induces equivalent expression in diseased and non-diseased cells and is operatively linked to the second nucleic acid. In some cases, the method may include detecting a first polypeptide or nucleic acid biomarker and / or a second polypeptide or nucleic acid biomarker. In some cases, the method is a non-invasive imaging approach performed on a subject. Such non-invasive imaging approaches include MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescence imaging.

[0208] In cases where the synthesized biomarker is a peptide biomarker detectable by a non-invasive imaging method, the method may further include locating diseased cells in the subject's body. Localization may be related to a specific resolution, such as 10 mm to 10 cm, at least 10 mm, or at most 10 cm. Localization may also be related to a specific minimum detectable tumor size, such as a tumor size of 3 mm. 3 Up to 10 cm 3 In some cases, a specific minimum size range can be 1 cm. 3Up to 10cm 3 or 900 mm 3 Up to 1 cm 3 or 800 mm 3 Up to 900 mm 3 or 700 mm 3 Up to 800 mm 3 or 600 mm 3 Up to 700 mm 3 or 500 mm 3 Up to 600 mm 3 or 400 mm 3 Up to 500 mm 3 or 300 mm 3 Up to 400 mm 3 or 200 mm 3 Up to 300 mm 3 or 100 mm 3 Up to 200 mm 3 or 50 mm 3 Up to 100 mm 3 or 10 mm 3 Up to 50 mm 3 or 3 mm 3 Up to 10mm 3 In some cases, localization occurs during non-invasive imaging scans (e.g., PET, MRI, SPECT, etc.). In other cases, localization occurs during on-site surgical intervention, such as through visual examination (in cases where the absorbance report is visible) or through visual examination combined with fluorescence excitation.

[0209] In some cases, the additional localization steps described above may be followed by a surgical step to eliminate the detected and / or localized diseased cells. The surgical step may be performed on the same or different sites as where the composition encoding the biomarker is applied and / or localized to the diseased cells. The surgical step may be surgical removal of the diseased cells or the tumor associated with the diseased cells. Surgical or non-surgical elimination steps may include minimally invasive killing techniques, such as radiosurgery (including, but not limited to, Gamma Knife, Reflexion, CyberKnife, and related techniques that utilize targeted ionizing radiation to kill diseased cells).

[0210] In some cases, non-invasive imaging methods can be performed at a time after the administration of a composition inducing the expression of a biomarker. Non-invasive imaging methods can be performed at least about 15 minutes, 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 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 about 5 months, at least about 6 months, or at least about 1 year after the administration of a composition containing the first and second nucleic acids. Non-invasive imaging methods can be performed for up to about 15 minutes, up to about 30 minutes, up to about 1 hour, 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 about 36 hours, up to about 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, 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, up to about 6 months, or up to about 1 year after the application of the composition containing the first and second nucleic acids. In some embodiments, the non-invasive imaging method can be performed multiple times after the application of the composition containing the first and second nucleic acids (e.g., to monitor the level of synthetic biomarkers over time). The non-invasive imaging method can be performed at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 times after the application of the composition containing the first and second nucleic acids. The non-invasive imaging method can be performed weekly or monthly after the application of the composition containing the first and second nucleic acids.

[0211] In some cases, a first polypeptide or nucleic acid biomarker and / or a second polypeptide or nucleic acid biomarker can be detected in biological samples from the subject. Biological samples can be samples collected from the subject using non-invasive methods. Exemplary non-invasive samples include, but are not limited to, saliva, sputum, sweat, urine, feces, semen, cervical and vaginal secretions, breast milk, inflammatory secretions, tears, and buccal epithelial swabs. Biological samples can also be samples collected from the subject using minimally invasive methods. Exemplary minimally invasive samples include, but are not limited to, blood samples (e.g., obtained via venipuncture or capillary), pleural fluid samples (e.g., obtained via thoracentesis), amniotic fluid samples (e.g., obtained via amniocentesis), and gastric fluid samples (e.g., obtained via gastric lavage). Biological samples can be obtained through biopsies, such as skin biopsies (e.g., obtained through perforation, scraping, discoidal surgery, wedge-shaped, incision, or excision biopsies), bone marrow samples (e.g., obtained through aspiration biopsies), lymph node or breast biopsies (e.g., obtained through fine-needle aspiration, core-needle biopsies, vacuum-assisted biopsies, or image-guided biopsies), surgical biopsies (e.g., internal organ samples obtained through excision or incision biopsies), or biopsies of the mouth, gastrointestinal tract, lungs, bladder, or urethra (e.g., obtained through endoscopy). In some cases, biological samples may be obtained at a time after administration of a composition that induces the expression of a synthetically produced biomarker. Biological samples can be obtained at least about 15 minutes, 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 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 about 5 months, or at least about 6 months after administration of the composition containing the first and second nucleic acids. Biological samples can be obtained at most about 15 minutes, at most 30 minutes, at most 1 hour, at most 2 hours, at most 4 hours, at most 8 hours, at most 16 hours, at most 24 hours, at most 36 hours, at most 48 hours, at most 3 days, at most 4 days, at most 5 days, at most 6 days, at most 7 days, at most 8 days, at most 9 days, at most 10 days, at most 11 days, at most 12 days, at most 13 days, at most 14 days, at most 15 days, at most 1 month, at most 2 months, at most 3 months, at most 4 months, at most 5 months, or at most 6 months after administration of the composition containing the first and second nucleic acids.In some embodiments, biological samples can be obtained after administration of a composition containing the first and second nucleic acid sequences, and any biomarker detection protocol can be performed multiple times (e.g., to monitor synthetic biomarker levels over time). Biological samples can be obtained at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 times after administration of the composition containing the first and second nucleic acid sequences. Biological samples can be obtained weekly or monthly after administration of the composition containing the first and second nucleic acid sequences.

[0212] In some cases, the method may include detecting a first or second nucleic acid biomarker using a specific nucleic acid detection method. The first or second nucleic acid biomarker can be detected by sequencing. Sequencing methods may include: next-generation sequencing, high-throughput sequencing, pyrosequencing, classical Sanger sequencing, ligation sequencing, synthesis sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule synthesis sequencing (SMSS) (Helicos), Ion Torrent sequencing machines (Life Technologies / Thermo-Fisher), massively parallel sequencing, cloning single-molecule arrays (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, and primer walking.

[0213] In some cases, the first or second nucleic acid biomarker can be detected using a “real-time amplification” method (also known as quantitative PCR (qPCR)) or TaqMan (see, for example, US Patent Nos. Gelfand 5,210,015, Livak et al. 5,538,848, and Haaland 5,863,736, 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., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, KJ, et al., PCR Methods and Applications 357-362 (1995)). This method for monitoring the formation of amplified products is based on the use of double-labeled fluorescent oligonucleotide probes to continuously measure the accumulation of PCR products. The probes used in this assay are typically short (approximately 20-25 bases) polynucleotides labeled with two different fluorescent dyes. The 5' end of the probe is typically attached to a reporter dye, and the 3' end to a quenching dye. The probe is designed to have a sequence that is at least substantially complementary to the site on the target mRNA or nucleic acid. Upstream and downstream PCR primers that bind to the flanking regions of the locus are also added to the reaction mixture. When the probe is intact, energy transfer occurs between the two fluorophores, and the quencher quenches the emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase, such as Taq polymerase, thereby releasing the reporter from the polynucleotide-quencher and resulting in an increase in reporter emission intensity, which can be measured by an appropriate detector. The recorded values ​​can then be used to continuously calculate the normalized increase in reporter emission intensity and ultimately quantify the amount of amplified mRNA.

[0214] In some implementations, for qPCR or Taqman assays, an RT-PCR step may be performed first to generate cDNA from cellular RNA. This RT-PCR amplification can be general (e.g., amplification with partially / completely degenerate oligonucleotide primers) or targeted (e.g., amplification with oligonucleotide primers targeting a specific gene to be analyzed in subsequent steps).

[0215] In some implementations, qPCR or Taqman can be used immediately after the reverse transcriptase reaction of isolated cellular mRNAs; this variation is used to quantify the levels of various mRNAs during qPCR.

[0216] In some implementations, for qPCR or Taqman detection or RNA sequencing, a "pre-amplification" step may first be performed on cDNA transcribed from cellular RNA. This is used to amplify the signal when the native levels of the RNA / cDNA to be detected are very low. Suitable methods for pre-amplification include, but are not limited to, LM-PCR, PCR with random oligonucleotide primers (e.g., random hexamer PCR), PCR with polyA-specific primers, and any combination thereof. Pre-amplification can be general or targeted, performed in the same manner as the reverse transcription reaction described above.

[0217] RNA levels can also be measured without amplification by hybridization with probes, such as branched nucleic acid probes (e.g., the QuantiGene® Reagent System from Panomics).

[0218] Biomarker design based on heterodimer synthesis

[0219] In some aspects, this disclosure provides a composition comprising a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, wherein the composition is configured such that, 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 configured to combine to form a heterodimeric protein. In some cases, the first polypeptide and the second polypeptide may be on separate gene constructs.

[0220] In some cases, heterodimeric proteins can be naturally occurring heterodimers, derivatives of natural enzymes, or autofluorescent proteins split into two complementary peptide halves. Examples of such systems include, but are not limited to, FRB / FKBP12 heterodimers, split luciferase proteins, or split GFP proteins.

[0221] In some cases, when the heterodimeric protein may be a derivative of a naturally occurring heterodimer (e.g., the FRB / FKBP12 pair), each half of the heterodimeric protein is linked to the complementary half of an enzyme or detection pair, thereby dimerizing the heterodimer to activate the enzyme or allow detection of the detection pair. In some cases, each half of the heterodimeric protein may be linked to a detached recombinase, such as Cre recombinase, thereby activating its expression when the activity of another element (e.g., a synthetic biomarker or therapeutic molecule) is reconstituted through the dimerization of the heterodimer. In some cases, each half of the heterodimeric protein may be linked to one of two autofluorescent proteins forming the FRET pair, so that FRET can be detected when the heterodimer forms.

[0222] In some cases, a first nucleic acid sequence and a second nucleic acid sequence can be operatively linked to a first gene element and a second gene element, wherein both the first and second gene elements can be selectively activated to express the first and second polypeptides in the same diseased cell type. The first or second gene element can be a promoter, an enhancer, or a miRNA binding site. Exemplary promoters include, but are not limited to, the survival protein promoter (BIRC5), CXCR4 promoter, ATP-binding box subfamily C member 4 (ABCC4) promoter, pregradient protein 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter, sealing protein 3 (CLDN3) promoter, sealing protein 4 (CLDN4) promoter, and collagen type XI α. COL11A1 promoter, collagen type I α1 chain (COL1A1) promoter, cystatin SN (CST1) promoter, toothless E3 ubiquitin protein ligase homolog (DTL) promoter, family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), laminin subunit γ LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α (TOP2A) promoter, ubiquitin D (UBD) promoter, ubiquitin conjugate E2 promoter C (UBE2C), harmonin (USH1C), T cell activation inhibitor 1 (VTCN1) promoter containing V-set domain, hexokinase type II promoter, TRPM4 promoter, lysosome 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible gRP78 / BiP promoter, interleukin-10 promoter, α-β-crystallin / heat shock protein27 promoters, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, MTS1 promoter, NSE promoter, somatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, alpha-fetoprotein promoter, chorionic villus promoter, albumin promoter, glycoprotein A33 promoter, B-cell specific Moronie's leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; receptor promoters containing kinase domain inserts; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugate 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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 The promoters of the kinetochore complex component (DSN1), deoxythymidine kinase (DTYMK), inducer of G protein regulation of neurite growth 1 (GPRIN1), mitochondrial fission regulator 2 (MTFR2), RAD51-associated protein 1 (RAD51AP1), small nucleonucleotide ribonucleoprotein polypeptide A' (SNRPA1), ATPase family, ATAD2 (ATAD2) containing the AAA domain, and BUB1 The promoters of mitosis checkpoint serine / threonine kinase (BUB1), calccyclin-binding protein (CACYBP), cell cycle-associated 3 (CDCA3), centromere protein O (CENPO), valve-specific endonuclease 1 (FEN1), forkhead box M1 (FOXM1), a cell proliferation regulator of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, and MYB proto-oncogene-like genes.MYBL2 promoter, NIMA-associated kinase 2 (NEK2) promoter, RAN-binding protein 1 (RANBP1) promoter, small nucleoprotein peptides B and B1 (SNRPB) promoter, SPC24 / NDC80 kinetochore complex component (SPC24) promoter, converting acidic coiled-coil protein 3 (TACC3) promoter, 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 read frame 53 (C17orf53) promoter, pigment box 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell cycle 20 (CDC20) promoter, cell cycle 45 (CDC45) promoter, cell cycle-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-transmembrane G receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosome protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen X-type α Promoters for the following proteins: COL10A1, CSE1L, DBF4 zinc finger, GINS complex subunit 1 (GINS1), G protein-coupled receptor 19 (GPR19), kinin family member 18A (KIF18A), kinin family member 4A (KIF4A), kinin family member C1 (KIFC1), mini chromosome maintenance 10 replication initiation factor (MCM10), and mini chromosome maintenance complex component 2 (MCM2). Promoters, Miniature Chromosome Maintenance Complex Component 7 (MCM7) promoter, MRG Domain-Binding Protein (MRGBP) promoter, Methylene Tetrahydrofolate Dehydrogenase (NADP+ Dependent) 2, Methylene Tetrahydrofolate Cyclohydrolase (MTHFD2) promoter, Non-SMC Condensed Protein I Complex Subunit H (NCAPH) promoter, NDC80, Kinocele Complex Component (NDC80) promoter, Nudix Hydrolase 1 (NUDT1) promoter, Ribonuclease H2 Subunit A (RNASEH2A) promoter, RuvB-like AAAThe promoters of ATPase 1 (RUVBL1), serologically defined breast cancer antigen NY-BR-85 (SGOL1), SHC-binding and spindle-associated 1 (SHCBP1), small nucleonucleotide polypeptide G (SNRPG), permanent circadian rhythm regulator, thyroid hormone receptor interactor 13 (TRIP13), nutrient protein-associated protein (TROAP), ubiquitin conjugate enzyme E2C (UBE2C), WD repeat and HMG box DNA-binding protein 1 (WDHD1), alpha fetal protein (AFP), fragments thereof, or any combination thereof. Exemplary miRNA binding sites include, but are not limited to, at least one miR-15, miR-16, let-7, miR-122, or miR-34 binding sequence.

[0223] In some cases, gene constructs encoding the first and second polypeptides can be mounted on a vector. Exemplary vectors include any of the vectors described herein.

[0224] In some aspects, this disclosure provides a method for detecting or treating diseased cells, the method comprising administering a composition comprising: a first nucleic acid sequence encoding a first polypeptide and a second nucleic acid sequence encoding a second polypeptide, wherein the composition is configured such that 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 configured to combine to form a heterodimeric protein. In some cases, the composition is administered to a subject intravenously, subcutaneously, intravenously, intrathecally, percutaneously, intramuscularly, orally, by inhalation, nasally, rectally, intratumorally, or adjacent to a tumor. Adjacent to a tumor may refer to administration to tissue near a tumor or to an area that is predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes). Methods involving intratumoral or adjacent tumors may include the use of additional imaging techniques such as endoscopic ultrasound (see, for example, Shirley et al., Gastroenterol Res Pract. 2013; 2013: 207129) or bronchoscopy (see, for example, Rojas-Solano et al., J Bronchology Interv Pulmonol. 2018 Jul; 25(3): 168–175). In some embodiments, the composition is applied to at least one of the following lymph nodes: cervical lymph nodes, medial epicondyle lymph nodes, supraclavicular lymph nodes, cervical lymph nodes, axillary lymph nodes, mediastinal lymph nodes, supratrochlear lymph nodes, mesenteric lymph nodes, inguinal lymph nodes, femoral lymph nodes, or popliteal lymph nodes. In some cases, lymph node-based application may be used as a method of concentrated local delivery to the tissue area. In some cases, the method may further include the detection of heterodimeric proteins.

[0225] In some cases, the detection includes performing a non-invasive detection method on the subject. Exemplary non-invasive detection methods (e.g., for autofluorescent or luminescent proteins) include, but are not limited to, SPECT imaging and bioluminescence imaging. The imaging method may be performed at least about 15 minutes, 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 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 about 5 months, at least about 6 months, or at least about 1 year after administration of the composition encoding the heterodimeric protein. Imaging methods can be performed at most about 15 minutes, at most about 30 minutes, at most about 1 hour, at most about 2 hours, at most about 4 hours, at most about 8 hours, at most about 16 hours, at most about 24 hours, at most about 36 hours, at most about 48 hours, at most 3 days, at most about 4 days, at most about 5 days, at most about 6 days, at most about 7 days, at most about 8 days, at most about 9 days, at most about 10 days, at most about 11 days, at most about 12 days, at most about 13 days, at most about 14 days, at most about 15 days, at most about 1 month, at most about 2 months, at most about 3 months, at most about 4 months, at most about 5 months, at most about 6 months, or at most about 1 year after administration of the composition encoding the heterodimeric protein. In some embodiments, imaging methods can be performed multiple times after administration of the composition encoding the heterodimeric protein (e.g., to monitor the level of synthesized biomarkers over time). The imaging method can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 times after the application of the composition encoding the heterodimeric protein. The imaging method can be performed weekly or monthly after the application of the composition encoding the heterodimeric protein.

[0226] In some cases, the test may include detecting heterodimeric proteins from a biological sample taken from the subject. The biological sample may be a sample collected from the subject using non-invasive methods. Exemplary non-invasive samples include, but are not limited to, saliva, sputum, sweat, urine, feces, semen, cervical and vaginal secretions, breast milk, inflammatory secretions, tears, and buccal epithelial swabs. The biological sample may also be a sample collected from the subject using minimally invasive methods. Exemplary minimally invasive samples include, but are not limited to, blood samples (e.g., obtained via venipuncture or capillary), pleural fluid samples (e.g., obtained via thoracentesis), amniotic fluid samples (e.g., obtained via amniocentesis), and gastric fluid samples (e.g., obtained via gastric lavage). Biological samples can be samples obtained through biopsy, such as skin biopsy samples (e.g., obtained through perforation, scraping, discoid surgery, wedge, incision, or excision biopsy), bone marrow samples (e.g., obtained through aspiration biopsy), lymph node or breast biopsy samples (e.g., obtained through fine-needle aspiration, core needle biopsy, vacuum-assisted biopsy, or image-guided biopsy), surgical biopsy samples (e.g., internal organ samples obtained through excision or incision biopsy), or mouth, gastrointestinal tract, lung, bladder, or urethra biopsy samples (e.g., obtained through endoscopy).

[0227] In some cases, biological samples can be obtained at a time after administration of the composition encoding the induced biomarker expression. Biological samples can be obtained at least about 15 minutes, 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 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 about 5 months, or at least about 6 months after administration of the composition encoding the heterodimeric protein. Biological samples can be obtained at most approximately 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of the composition encoding the heterodimeric protein. In some embodiments, biological samples can be obtained after administration of the composition encoding the heterodimeric protein, and any biomarker detection protocol can be performed multiple times. Biological samples can be obtained at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 times after administration of the composition encoding the heterodimeric protein. Biological samples can also be obtained weekly or monthly after administration of the composition encoding the heterodimeric protein. The heterodimeric protein in the biological samples can be determined by fluorescence assay, FRET assay, TR-FRET assay, or luminescence assay.

[0228] Alternatively or additionally, heterodimeric proteins can be detected in heterodimer-specific immunoassays. Several methods and apparatuses are well known for determining protein levels, including immunoassays, described, for example, in U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524, and 5,480,792. These assays include various sandwich, competitive, or non-competitive assay formats to generate a signal related to the presence or amount of the target protein analyte. Any suitable immunoassay can be used, such as lateral flow, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, or any combination thereof.

[0229] In vitro constructs and methods for synthetic biomarkers used in disease detection, monitoring, or diagnosis

[0230] In some aspects, this disclosure provides a composition comprising a non-naturally occurring recombinant gene construct comprising a sequence encoding a polypeptide or nucleic acid sequence, wherein the sequence comprises a first promoter that, when transduced into cells in vitro, selectively drives the expression of the polypeptide or nucleic acid biomarker sequence in a variety of different types of diseased cells isolated from a subject.

[0231] In some cases, the composition may comprise cells transduced with the recombinant gene construct. In some cases, the various cell types are diseased or defective cells. In some cases, the cells are blood cells, lymphocytes, leukocytes, epithelial cells, gastrointestinal cells, placental cells, amniotic fluid cells, lung epithelial cells, urinary epithelial cells, or kidney cells.

[0232] In some cases, diseased or impaired cells may be cancer cells, cells indicating autoimmune diseases (such as self-directing T cells or lymphocytes, or normal cells impaired by autoimmunity), TACs, or cells indicating neurodegenerative diseases (such as cells with or near toxic amyloid). Exemplary cancers, autoimmune diseases, and neurodegenerative diseases include any of the diseases described herein. In some cases, diseased or impaired cells may be virally infected cells. Exemplary viral infections include, but are not limited to, those caused by HIV, hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesvirus, Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus type III.

[0233] In some cases, the first promoter can be a promoter that is activated in the cell when the cell is in a diseased state. The first promoter can be a pan-tumor-specific promoter. In some cases, the first promoter is a cancer-specific promoter. In some cases, the first promoter is the survival protein promoter (BIRC5), CXCR4 promoter, ATP-binding box subfamily C member 4 (ABCC4) promoter, pregradient protein 2, protein disulfide isomerase family member (AGR2) promoter, activation-induced cytidine deaminase (AICDA) promoter, UDP-GlcNAc:βGal β-1,3-N-acetylglucosamine transferase 3 (B3GNT3) promoter, cadherin 3 (CDH3) promoter, CEA cell adhesion molecule 5 (CEACAM5) promoter, centromere protein F (CENPF) promoter, centrosome protein 55 (CEP55) promoter, sealing protein 3 (CLDN3) promoter, sealing protein 4 (CLDN4) promoter, collagen type XI α1 chain (COL11A1) promoter, collagen type I α1 chain promoter, etc. The promoters of the following proteins are listed: COL1A1 promoter, cystatin SN (CST1) promoter, DTL promoter, family 111 member B (FAM111B) promoter with sequence similarity, forkhead box A1 (FOXA1) promoter, kinin family member 20A (KIF20A), and laminin subunit γ. LAMC2 promoter, MISP promoter, MMP1 promoter, MMP12 promoter, MMP13 promoter, MSLN promoter, MUC1 promoter, PLA2G2D promoter, G protein signaling regulator 13 promoter, SCGB2A1 promoter, topoisomerase II α promoter, Ubiquitin D promoter, Ubiquitin conjugate E2C promoter, USH1 promoter, LAMC2 promoter, MISP promoter, MMP1 ... Protein network components include harmonin (USH1C), T cell activation inhibitor 1 (VTCN1) promoter containing V-set domain, hexokinase type II promoter, TRPM4 promoter, lysosome 3 promoter, surfactant protein A promoter, secretory leukocyte protease inhibitor promoter, tyrosinase promoter, stress-inducible gRP78 / BiP promoter, interleukin-10 promoter, and α-β-crystallin / heat shock protein.27 promoters, epidermal growth factor receptor promoter, mucin-like glycoprotein promoter, MTS1 promoter, NSE promoter, somatostatin receptor promoter, c-erbB-3 promoter, c-erbB-2 promoter, c-erbB4 promoter, thyroglobulin promoter, alpha-fetoprotein promoter, chorionic villus promoter, albumin promoter, glycoprotein A33 promoter, B-cell specific Moronnius leukemia virus insertion site 1 promoter, cyclooxygenase-2 promoter, fibroblast growth factor promoter; human epidermal growth factor receptor 2, human telomerase reverse transcriptase promoter; receptor promoters containing kinase domain inserts; rad51 recombinase promoter; TTF-1, urokinase-type plasminogen activator receptor promoter, ubiquitin conjugate 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 synthase 1 (FLAD1) promoter, Mg2+ / Mn2+ dependent protein phosphatase 1G (PPM1G) promoter, ubiquitin conjugate E2S (UBE2S) promoter, aurora kinase A and ninein interacting protein (AUNIP) promoter, cell cycle 6 (CDC6) promoter, centromere protein L (CENPL) promoter, DNA replication helicase / nuclease 2 (DNA2) promoter, DSN1 homolog, MIS12 The promoters of the kinetochore complex component (DSN1), deoxythymidine kinase (DTYMK), inducer of G protein regulation of neurite growth 1 (GPRIN1), mitochondrial fission regulator 2 (MTFR2), RAD51-associated protein 1 (RAD51AP1), small nucleonucleotide polypeptide A' (SNRPA1), ATPase family, AAA domain-containing 2 (ATAD2) promoter, and BUB1. The promoters of mitosis checkpoint serine / threonine kinase (BUB1), calccyclin-binding protein (CACYBP) promoter, cell cycle-associated 3 (CDCA3) promoter, centromere protein O (CENPO) promoter, valve-specific endonuclease 1 (FEN1) promoter, forkhead box M1 (FOXM1) promoter, cell proliferation regulator of protein phosphatase 2A (KIAA1524) promoter, kinin family member 2C (KIF2C) promoter, nuclear transporter subunit α2 (KPNA2) promoter, and MYB...The promoters for the following gene sequences are listed: proto-oncogene-like 2 (MYBL2), NIMA-associated kinase 2 (NEK2), RAN-binding protein 1 (RANBP1), small nucleoprotein peptides B and B1 (SNRPB), SPC24 / NDC80 kinetochore complex component (SPC24), transforming acidic coiled-coil protein 3 (TACC3), TBC1 domain family member 31 (TBC1D31), thymidine kinase 1 (TK1), zinc finger protein 695 (ZNF695), aurora kinase A (AURKA), and BLM. RecQ-like helicase (BLM) promoter, chromosome 17 read frame 53 (C17orf53) promoter, pigment box 3 (CBX30) promoter, cyclin B1 (CCNB1) promoter, cyclin E1 (CCNE1) promoter, cyclin F (CCNF), cell cycle 20 (CDC20) promoter, cell cycle 45 (CDC45) promoter, cell cycle-associated 5 (CDCA5) promoter, cyclin-dependent kinase inhibitor 3 (CDKN3) promoter, cadherin EGF LAG seven-transmembrane G receptor 3 (CELSR3) promoter, centromere protein A (CENPA) promoter, centrosome protein 72 (CEP72) promoter, CDC28 protein kinase regulatory subunit 2 (CKS2) promoter, collagen X-type α Promoters for the following proteins: COL10A1, CSE1L, DBF4 zinc finger, GINS complex subunit 1 (GINS1), G protein-coupled receptor 19 (GPR19), kinin family member 18A (KIF18A), kinin family member 4A (KIF4A), kinin family member C1 (KIFC1), mini chromosome maintenance 10 replication initiation factor (MCM10), and mini chromosome maintenance complex component 2 (MCM2). Promoters, Miniature Chromosome Maintenance Complex Component 7 (MCM7) promoter, MRG Domain-Binding Protein (MRGBP) promoter, Methylene Tetrahydrofolate Dehydrogenase (NADP+ Dependent) 2, Methylene Tetrahydrofolate Cyclohydrolase (MTHFD2) promoter, Non-SMC Condensed Protein I Complex Subunit H (NCAPH) promoter, NDC80, Kinocele Complex Component (NDC80) promoter, Nudix Hydrolase 1 (NUDT1) promoter, Ribonuclease H2 Subunit A (RNASEH2A) promoter, RuvB-like AAAATPase 1 (RUVBL1) promoter, serum-defined breast cancer antigen NY-BR-85 (SGOL1) promoter, SHC-binding and spindle-associated 1 (SHCBP1) promoter, small nucleonucleotide ribonucleoprotein polypeptide G (SNRPG) promoter, permanent circadian rhythm regulator promoter, thyroid hormone receptor interactor 13 (TRIP13) promoter, nutrient protein-associated protein (TROAP) promoter, ubiquitin conjugate enzyme E2C (UBE2C) promoter, WD repeat and HMG box DNA-binding protein 1 (WDHD1) promoter, alpha fetal protein (AFP) promoter, fragments thereof, or any combination thereof.

[0234] In some cases, recombinant gene constructs used for in vitro detection may contain retroviral, lentiviral, or adenoviral packaging elements or long terminal repeat sequences. Recombinant gene constructs may be CELID vectors. Recombinant gene constructs may be vectors derived from bacteriophages or plants, invertebrates or animals (including humans), or viruses, such as adeno-associated virus vectors (e.g., AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or their pseudotype combinations such as AAV2 / 5, AAV2 / 2, AAV-DJ, or AAV-DJ8), retroviral vectors (e.g., MLV, or their self-inactivated or SIN variants, or their pseudotype variants), herpesviruses (based on, for example, HSV- or EBV), lentiviral vectors (e.g., based on HIV, FIV, or EIAV, or their pseudotype variants), or adenoviral vectors (e.g., based on Ad5, including its replication-deficient, reproducible, or helper-dependent variants). Recombinant gene constructs may also be packaging vectors compatible with any of these viral systems.

[0235] The vector can be a non-viral vector. The non-viral vector can be a microcircular vector. The microcircle can be a self-replicating microcircle. The self-replicating microcircle can contain S / MAR elements. The non-viral vector can be a nanoparticle or a small intronic plasmid (MIP). The MIP places the bacterial origin of replication and any optional markers as introns within the transgene expression cassette. Furthermore, the MIP can maintain the 5' and 3' ends of the transgene expression cassette juxtaposed within the microcircle (see, for example, Lu et al., a mini-intronic plasmid (MIP): a novel robust transgene expression vector in vivo and in vitro, mol. Ther. 2013 May; 21(5): 954-963).

[0236] In some cases, the polypeptide or nucleic acid sequence selectively expressed when transduced in vivo can be selected from photoacoustic reporters, bioluminescent reporters, autofluorescent reporters, chemiluminescent reporters, luminescent reporters, colorimetric reporters, quantifiable nucleic acids, and any combination thereof. Autofluorescent reporters include GFP, mCherry, or derivatives thereof. Colorimetric reporters include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal administration) and tyrosinase. Bioluminescent, chemiluminescent, or luminescent reporters include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renida luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396(2010): 290-297). Reporter proteins detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants). Quantifiable nucleic acids can be ribozymes, self-splicing introns, RNA hairpins, microRNAs or their barcoded variations, or other types of quantifiable RNA. Quantifiable nucleic acids can contain unique sequences detectable by quantitative PCR or hybridization-based techniques. When the polypeptide or nucleic acid is a polypeptide, the polypeptide can contain an N-terminal secretory signal sequence (e.g., an N-terminal signal peptide from CD33 or CD8a).

[0237] In some cases, the composition may have a diagnostic efficiency, wherein the diagnostic efficiency is measured by the expression of the biomarker in the subject preferentially over in the diseased cells, such that the relative ratio of the biomarker expressed in the diseased cells to that in the diseased cells is greater than 1.0; (b) detecting the biomarker; and (c) using the biomarker detected in (b) to determine with at least 90% accuracy that the subject has the diseased cells.

[0238] In some cases, compositions applied to cells in vitro may contain a second polypeptide or nucleic acid that modulates the proliferation of diseased or impaired cells. The second polypeptide may be under the control of a second promoter that selectively drives the expression of the second polypeptide or nucleic acid in diseased or impaired cells. The second promoter may be a pan-cancer-specific promoter. The second promoter may be a cancer-specific promoter. The promoter may be any specific promoter described herein. The second polypeptide may contain a transforming agent or a growth factor. A transforming agent may include telomerase or SV40 large T antigen. A growth factor may be, for example, EGF, PDGF, FGF, HGH, or IGF-1.

[0239] In some aspects, this disclosure provides a method for detecting diseased or impaired cells in vitro, the method comprising delivering a non-naturally occurring recombinant gene construct to a cell population isolated from a subject in vitro, wherein the non-naturally occurring recombinant gene construct comprises: a sequence encoding a polypeptide or nucleic acid sequence, wherein the sequence comprises a first promoter that, when transduced into cells, selectively drives the expression of the polypeptide or nucleic acid sequence in a variety of different cell types isolated from the subject.

[0240] In some aspects, this disclosure provides a method for detecting a disease or the absence of a disease in a subject, the method comprising contacting one or more cells of the subject in vitro with a gene construct, wherein the gene construct comprises a disease-activated promoter operatively linked to a barcode molecule, and the disease-activated promoter drives the expression of the barcode molecule in cells suffering from the disease; quantifying the expression level of the barcode molecule; and detecting the disease or its absence based on the expression level.

[0241] In some cases, methods for detecting diseased or obstructed cells in vitro can detect a specific number of diseased cells against a background of normal cells. In some embodiments, the method can detect approximately 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 diseased cells per 5 million normal cells. In some embodiments, the normal cells are blood cells (e.g., PBMCs).

[0242] In some cases, the method may include separating a biological sample containing cells from the subject. The biological sample may be a sample collected from the subject using non-invasive methods. Exemplary non-invasive samples include, but are not limited to, samples consisting of naturally shed bodily material or non-destructive scrapings from externally accessible tissue, such as saliva, sputum, sweat, urine, feces, semen, mucus, cervical and vaginal secretions, breast milk, inflammatory secretions, tears, and buccal epithelial swabs. The biological sample may also be a sample collected from the subject using minimally invasive methods. Exemplary minimally invasive samples include, but are not limited to, blood samples or fractions thereof (e.g., obtained via venipuncture or capillary), pleural fluid samples (e.g., obtained via thoracentesis), amniotic fluid samples (e.g., obtained via amniocentesis), and gastric fluid samples (e.g., obtained via gastric lavage). Biological samples can be samples obtained through biopsy, such as skin biopsy samples (e.g., obtained through perforation, scraping, discoid surgery, wedge, incision, or excision biopsy), bone marrow samples (e.g., obtained through aspiration biopsy), lymph node or breast biopsy samples (e.g., obtained through fine-needle aspiration, core needle biopsy, vacuum-assisted biopsy, or image-guided biopsy), surgical biopsy samples (e.g., internal organ samples obtained through excision or incision biopsy), or mouth, gastrointestinal tract, lung, bladder, or urethra biopsy samples (e.g., obtained through endoscopy).

[0243] In some cases, the method may include culturing a cell population for a certain period of time after the recombinant gene construct is delivered to the cells. The cell population may be cultured for at least about 15 minutes, 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 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, or at least about 1 month after the recombinant gene construct is delivered to the cells. Cell populations can be cultured for up to 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 approximately 3 days, up to approximately 4 days, up to approximately 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 approximately 12 days, up to approximately 13 days, up to approximately 14 days, up to approximately 15 days, or up to approximately 1 month after the recombinant gene construct is delivered to the cells.

[0244] In some cases, the method may include detecting a peptide or nucleic acid sequence. This detection may occur before or after culturing the cell population. The detection may include photoacoustic, bioluminescent, fluorescent reporter, chemiluminescent, luminescent, colorimetric, or nucleic acid assays. The detection may also include immunoassays. Immunoassays include, for example, those described in U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524, and 5,480,792. Immunoassays include various sandwich, competitive, or non-competitive assay forms that produce a signal related to the presence or amount of the target protein analyte. Any suitable immunoassay can be used, such as lateral flow, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, and the like.

[0245] The detection method may include sequencing. Sequencing methods may include: next-generation sequencing, high-throughput sequencing, pyrosequencing, classic Sanger sequencing, ligation sequencing, synthesis sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule synthesis sequencing (SMSS) (Helicos), Ion Torrent sequencing machine (Life Technologies / Thermo-Fisher), massively parallel sequencing, cloning single-molecule array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, and primer walking.

[0246] Detection can include “real-time amplification” methods (also known as quantitative PCR (qPCR)) or Taqman (see, for example, U.S. Patent Nos. Gelfand 5,210,015, Livak et al. 5,538,848, and Haaland 5,863,736, 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., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, KJ, et al., PCR Methods and Applications 357-362 (1995)). This method for monitoring the formation of amplified products is based on the use of double-labeled fluorescent oligonucleotide probes to continuously measure the accumulation of PCR products. The probes used in this assay are typically short (approximately 20-25 bases) polynucleotides labeled with two different fluorescent dyes. The 5' end of the probe is typically attached to a reporter dye, and the 3' end to a quenching dye. The probe is designed to have a sequence that is at least substantially complementary to the site on the target mRNA or nucleic acid. Upstream and downstream PCR primers that bind to the flanking regions of the locus are also added to the reaction mixture. When the probe is intact, energy transfer occurs between the two fluorophores, and the quencher quenches the emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase, such as Taq polymerase, thereby releasing the reporter from the polynucleotide-quencher and resulting in an increase in reporter emission intensity, which can be measured by an appropriate detector. The recorded values ​​can then be used to continuously calculate the normalized increase in reporter emission intensity and ultimately quantify the amount of amplified mRNA.

[0247] In some implementations, for qPCR or Taqman assays, an RT-PCR step may be performed first to generate cDNA from cellular RNA. This RT-PCR amplification can be general (e.g., amplification with partially / completely degenerate oligonucleotide primers) or targeted (e.g., amplification with oligonucleotide primers targeting a specific gene to be analyzed in subsequent steps).

[0248] In some implementations, qPCR or Taqman can be used immediately after the reverse transcriptase reaction of isolated cellular mRNAs; this variation is used to quantify the levels of various mRNAs during qPCR.

[0249] In some implementations, for qPCR or Taqman detection or RNA sequencing, a "pre-amplification" step may first be performed on cDNA transcribed from cellular RNA. This is used to amplify the signal when the native levels of the RNA / cDNA to be detected are very low. Suitable methods for pre-amplification include, but are not limited to, LM-PCR, PCR with random oligonucleotide primers (e.g., random hexamer PCR), PCR with polyA-specific primers, and any combination thereof. Pre-amplification can be general or targeted, performed in the same manner as the reverse transcription reaction described above.

[0250] Improved biomarkers, construct designs, and methods for indicating disease staging

[0251] In some aspects, this disclosure provides a composition comprising a vector, wherein the vector comprises a plurality of different promoters operatively linked to a plurality of different nucleic acid sequences, wherein each of the promoters drives the expression of the plurality of nucleic acid sequences in a cell to produce a plurality of polypeptide or nucleic acid biomarker sequences, wherein the level of each polypeptide or nucleic acid biomarker sequence among the plurality of nucleic acid sequences indicates a stage of disease in the cell. In some cases, the stage of disease in the cell is diseased, disease-free, or intermediate. In some cases, the plurality of different promoters may be included on a plurality of independent gene constructs or vectors administered simultaneously or separately. In some embodiments, the plurality of independent gene constructs administered separately are administered at intervals of 8, 16, 24, 36, 48, 60, or 72 hours from each other. In some embodiments, the stage of disease can be assessed by the dissemination of cancer cells away from their originating tissue via metastasis to distant tissues. In such cases, the plurality of different promoters may include at least one promoter having high cancer specificity at the initial tissue site (e.g., the breast, when breast cancer is staged), and an active promoter having high specificity at common metastatic sites different from the initial site (e.g., the lung, spleen, liver). In some cases, multiple different promoters may include at least one promoter with high cancer specificity at the initial tissue site (e.g., the breast, when breast cancer is staged), and multiple active promoters with high specificity at multiple different metastatic sites (e.g., the lung, spleen, liver). Therefore, such a system can provide activation of more different promoters as cancer metastasizes from its original site to metastatic sites (read out via downstream biomarkers operatively linked to them), thereby providing an assessment of how extensively the tumor has metastasized. In some embodiments, one of the active promoters with high specificity at common metastatic sites is MMP-2, which is highly expressed in all stages of lung cancer but is not overexpressed in breast cancer.

[0252] In some cases, the disease may be cancer, an autoimmune disease (such as T cells or lymphocytes with self-directing activity, or normal cells damaged by autoimmunity), or a neurodegenerative disease (such as cells containing or adjacent to toxic amyloid). Exemplary cancers include, but are not limited to, carcinomas, sarcomas, lymphomas, leukemias, and adenomas. Carcinomas may arise in cells covering internal and external parts of the body, such as the lungs, breasts, and colon. Sarcomas may arise in cells located in bone, cartilage, fat, connective tissue, muscle, and other supporting tissues. Lymphomas may arise in lymph nodes and immune system tissues. Leukemia may arise in the bone marrow and accumulate in the bloodstream. Adenomas may arise in the thyroid gland, pituitary gland, adrenal glands, and other glandular tissues.Specific exemplary examples of cancer types suitable for detection using the methods according to this disclosure include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt lymphoma, cancer of unknown primary origin, and central nervous system lymphoma. Cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer, such as non-small cell lung cancer and small... Cellular lung cancer, lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma, neuroblastoma, melanoma, mesothelioma, metastatic squamous neck cancer of unknown primary origin, oral cancer, multiple endocrine tumor syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cells, paranasal sinus and nasal cavity cancer, parathyroid carcinoma, penile cancer, pharyngeal cancer. Pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pituitary adenoma, pleural pulmonary blastoma, plasmacytoma formation, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, 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, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (pregnancy), cancer of unknown primary origin, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0253] In some cases, the disease may be caused by viral infection of cells. Exemplary viral infections include, but are not limited to, those caused by HIV, hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesvirus, Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus type III.

[0254] In some cases, when the disease is cancer, multiple different promoters may include a first promoter that is activated in the early stages of cancer. In some cases, multiple different promoters may include a second promoter that is activated in the middle stages of cancer. In some cases, multiple different promoters may include a third promoter that is activated in the late stages of cancer.

[0255] In some cases, the disease may be an autoimmune disease. Exemplary autoimmune diseases include, but are not limited to, achalasia, Addison's disease, adult-onset Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid antibody syndrome, autoimmune angioedema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axononeuropathy (AMAN), Baló's disease, Behçet's disease, benign mucosal pemphigoid, bullous pemphigoid, Castle Man's disease (CD), steatorrhea, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, herpetic dermatitis, dermatomyositis, Devic disease (neuromyelitis optica), discoid lupus, Dressler syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, idiopathic mixed cryoglobulinemia, Evans disease. Syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Gudpassutia syndrome, granulomatous polyangiitis, Grave's disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (abnormal acne), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosis, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile Type I myositis (JM), Kawasaki disease, Lambert-Eton syndrome, leukocytolytic vasculitis, lichen planus, lichen sclerosus, woody conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren'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 neuritis, relapsing rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD)Paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars plana cyclitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome type I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, recurrent polychondritis, restless legs syndrome (RL). S), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), aortitis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada disease.

[0256] In some cases, the disease may be a neurodegenerative disease. Neurodegenerative diseases include, but are not limited to, multiple sclerosis (MS), Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), or neurodegeneration caused by infection with the following viruses: Herpesviridae, Polyomaviridae, Bonaviridae, Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Flaviviridae, Picornaviridae, or Retroviridae (see Zhou et al., Virol J. 2013; 10:172).

[0257] In some cases, nucleic acid biomarkers may be, for example, natural or engineered miRNAs, RNA hairpins, RNA aptamers, or barcoded variations thereof.

[0258] In some cases, the carrier provided in the composition used to detect a stage of disease can be any carrier described herein.

[0259] In some cases, at least one of a variety of peptides may include a peptide detectable by non-invasive imaging. Such non-invasive imaging methods include MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescence imaging. Synthetic biomarkers detectable by MRI imaging include, but are not limited to, peptide contrast agents, such as ferritin (or mutants thereof, such as *Porcine ferritin* mutants L55P, F57S, or F123S) or lanthanide-binding proteins (or engineered fusions thereof, such as the LBT-ubiquitin fusion described in Daughtry et al., ChemBioChem 2012, 13, 2567–2574). Synthetic biomarkers detectable by PET or SPECT imaging include human sodium iodide cotransporters (e.g., in combination with PET-applied active iodine / iodine isotopes, see, for example, Penheiter et al., Curr Gene Ther. 2012 Feb; 12(1): 33–47), HSV-tk or mutants of it such as HSV-sr39tk (e.g., in combination with PET-applied positron-labeled acycloguanosine or pyrimidine analogues such as [18F]FHBG, see, Yaghoubi SS et al., Nat Protoc. 2006; 1(6): 3069-75), and dopamine D2 receptors or mutants of them such as D2R80A or D2R194A (e.g., in combination with positron-labeled D2 binding agents such as 3-(2'-[18F]-fluoroethyl)-spiropepidermobenzene). Synthetic biomarkers detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal administration), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants).Synthetic biomarkers detectable by bioluminescence imaging include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renal luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396 (2010): 290-297). In some embodiments, the synthetic biomarker may be a contrast agent, an enzyme that produces a detectable molecule, or a transporter that drives the accumulation of the detectable molecule. The synthetic biomarker can be measured in situ in a subject.

[0260] In some cases, the barcode molecule may be a peptide or nucleic acid detectable in a biological sample from a subject. When the barcode molecule is a peptide, the peptide may contain an N-terminal secretory signal sequence (e.g., an N-terminal signal peptide from CD33 or CD8a). Exemplary peptide biomarkers include, but are not limited to, photoacoustic reporters, bioluminescent reporters, autofluorescent reporters, chemiluminescent reporters, luminescent reporters, colorimetric reporters, and any combination thereof. Autofluorescent reporters include GFP, mCherry, or derivatives thereof. Colorimetric reporters include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal administration) and tyrosinase. Bioluminescent, chemiluminescent, or luminescent reporters include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renida luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396(2010):290-297). Reporter proteins detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants). When the barcoded molecule is a nucleic acid sequence, the detectable nucleic acid sequence can include, but is not limited to, ribozymes, self-splicing introns, RNA hairpins, microRNAs or their barcoded variations, or other types of quantifiable RNA. Quantifiable nucleic acid sequences can contain unique sequences detectable by quantitative PCR or hybridization-based techniques.

[0261] By assigning unique markers to unique members within a larger group, barcoding provides the opportunity to identify and quantify a member (e.g., reporter gene expression under the control of a specific cancer-specific promoter) within a larger and more complex mixture of many members (e.g., multiple promoter-reporter gene constructs expressed within the same cell), and also provides the opportunity to isolate individual members from complex mixtures. For example, in the case of nucleic acid-based barcoding, hybridization based on base pair complementarity can be used for capture and separation, or otherwise to reduce the complexity of the mixture through said capture event. For peptide-based barcoding, unique characteristics (including immune capture or interaction of ligands and receptors) can be used for capture and separation, or otherwise to reduce the complexity of the mixture through said capture event.

[0262] In some aspects, this disclosure provides a method for detecting a stage of disease, the method comprising administering to a subject a composition comprising a vector, wherein the vector comprises: a plurality of different promoters operatively linked to a plurality of different nucleic acid sequences, wherein each of the promoters drives the expression of the plurality of nucleic acid sequences in cells to produce a plurality of polypeptides or synthetic nucleic acid sequences, wherein the level of each polypeptide of the plurality of nucleic acid sequences indicates a stage of disease in the cells. In some cases, the stage of disease in the cells may be diseased, non-diseased, or an intermediate state. In some aspects, this disclosure provides a method for detecting different types of cancer, the method comprising administering to a subject a composition comprising a vector, wherein in cells the vector comprises a plurality of different promoters operatively linked to a plurality of different nucleic acid sequences to produce a plurality of polypeptides or synthetic nucleic acid sequences, wherein the level of each polypeptide of the plurality of nucleic acid sequences indicates a different type of cancer in vivo. In some cases, cancer detected in the body can originate from, but is not limited to, tissues, blood or blood components of the breast, liver, colon, brain, lungs, kidneys, pancreas, testes, ovaries, bones, stomach, eyes, endocrine or neuroendocrine tissues, head and neck, gastrointestinal tract, musculoskeletal system, skin, respiratory system, nerves or genitourinary system, or cancer originating from other parts of the body.

[0263] In some cases, the composition is administered to the subject intravenously, subcutaneously, intraventricularly, intrathecally, intracerebrally, percutaneously, intramuscularly, or orally, by inhalation, nasally, or transrectally, intratumorally, or adjacent to a tumor. Adjacent to a tumor may mean administration to tissues near the tumor or to areas that are predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes). Methods involving intratumoral or adjacent tumors may include the use of additional imaging techniques such as endoscopic ultrasound (see, for example, Shirley et al., Gastroenterol Res Pract. 2013; 2013: 207129) or bronchoscopy (see, for example, Rojas-Solano et al., J Bronchology Interv Pulmonol. 2018 Jul; 25(3): 168–175). In some embodiments, the composition is applied to at least one of the following lymph nodes: cervical lymph nodes, medial epicondyle lymph nodes, supraclavicular lymph nodes, axillary lymph nodes, mediastinal lymph nodes, supratrochlear lymph nodes, mesenteric lymph nodes, inguinal lymph nodes, femoral lymph nodes, or popliteal lymph nodes. In some cases, lymph node-based application can be used as a method of concentrated local delivery to the tissue area.

[0264] In some cases, when the disease is cancer, multiple different promoters may include a first promoter that is activated in the early stages of cancer. In some cases, multiple different promoters may include a second promoter that is activated in the middle stages of cancer. In some cases, multiple different promoters may include a third promoter that is activated in the late stages of cancer. In some cases, the method may identify tissue masses or lesions in a subject as precancerous, benign, dysplastic, or metastatic in nature.

[0265] In some cases, the method may include separating biological samples from the subject. Biological samples may be samples collected from the subject using non-invasive methods. Exemplary non-invasive samples include, but are not limited to, samples consisting of naturally shed bodily material or non-destructive scrapings from externally accessible tissues, such as saliva, sputum, sweat, urine, feces, semen, mucus, cervical and vaginal secretions, breast milk, inflammatory secretions, tears, and buccal epithelial swabs. Biological samples may also be samples collected from the subject using minimally invasive methods. Exemplary minimally invasive samples include, but are not limited to, blood samples or fractions thereof (e.g., obtained via venipuncture or capillary), pleural fluid samples (e.g., obtained via thoracentesis), amniotic fluid samples (e.g., obtained via amniocentesis), and gastric fluid samples (e.g., obtained via gastric lavage). Biological samples can be obtained through biopsies, such as skin biopsies (e.g., obtained through perforation, scraping, discoidal surgery, wedge-shaped, incision, or excision biopsies), bone marrow samples (e.g., obtained through aspiration biopsies), lymph node or breast biopsies (e.g., obtained through fine-needle aspiration, core-needle biopsies, vacuum-assisted biopsies, or image-guided biopsies), surgical biopsies (e.g., internal organ samples obtained through excision or incision biopsies), or mouth, gastrointestinal tract, lung, bladder, or urethra biopsies (e.g., obtained through endoscopy). In some cases, biological samples may be collected at a time after the composition has been administered to the subject.

[0266] The cell population can be cultured for at least approximately 15 minutes, at least approximately 30 minutes, at least approximately 1 hour, at least approximately 2 hours, at least approximately 4 hours, at least approximately 8 hours, at least approximately 16 hours, at least approximately 24 hours, at least approximately 36 hours, at least approximately 48 hours, at least approximately 3 days, at least approximately 4 days, at least approximately 5 days, at least approximately 6 days, at least approximately 7 days, at least approximately 8 days, at least approximately 9 days, at least approximately 10 days, at least approximately 11 days, at least approximately 12 days, at least approximately 13 days, at least approximately 14 days, at least approximately 15 days, or at least approximately 1 month after the gene construct is delivered to the cells. Cell populations can be cultured for up to 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 approximately 3 days, up to approximately 4 days, up to approximately 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 approximately 12 days, up to approximately 13 days, up to approximately 14 days, up to approximately 15 days, or up to approximately 1 month after the gene construct is delivered to the cells.

[0267] In some cases, the method may include detecting a peptide or nucleic acid sequence. This detection may occur before or after culturing the cell population. The detection may include photoacoustic, bioluminescent, fluorescent reporter, chemiluminescent, luminescent, colorimetric, or nucleic acid assays. The detection may also include immunoassays. Immunoassays include, for example, those described in U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524, and 5,480,792. Immunoassays include various sandwich, competitive, or non-competitive assay forms that produce a signal related to the presence or amount of the target protein analyte. Any suitable immunoassay can be used, such as lateral flow, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, and the like.

[0268] The detection method may include sequencing. Sequencing methods may include: next-generation sequencing, high-throughput sequencing, pyrosequencing, classic Sanger sequencing, ligation sequencing, synthesis sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule synthesis sequencing (SMSS) (Helicos), Ion Torrent sequencing machine (Life Technologies / Thermo-Fisher), massively parallel sequencing, cloning single-molecule array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, and primer walking.

[0269] Detection can include “real-time amplification” methods (also known as quantitative PCR (qPCR)) or Taqman (see, for example, U.S. Patent Nos. Gelfand 5,210,015, Livak et al. 5,538,848, and Haaland 5,863,736, 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., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, KJ, et al., PCR Methods and Applications 357-362 (1995)). This method for monitoring the formation of amplified products is based on the use of double-labeled fluorescent oligonucleotide probes to continuously measure the accumulation of PCR products. The probes used in this assay are typically short (approximately 20-25 bases) polynucleotides labeled with two different fluorescent dyes. The 5' end of the probe is typically attached to a reporter dye, and the 3' end to a quenching dye. The probe is designed to have a sequence that is at least substantially complementary to the site on the target mRNA or nucleic acid. Upstream and downstream PCR primers that bind to the flanking regions of the locus are also added to the reaction mixture. When the probe is intact, energy transfer occurs between the two fluorophores, and the quencher quenches the emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase, such as Taq polymerase, thereby releasing the reporter from the polynucleotide-quencher and resulting in an increase in reporter emission intensity, which can be measured by an appropriate detector. The recorded values ​​can then be used to continuously calculate the normalized increase in reporter emission intensity and ultimately quantify the amount of amplified mRNA.

[0270] In some implementations, for qPCR or Taqman assays, an RT-PCR step may be performed first to generate cDNA from cellular RNA. This RT-PCR amplification can be general (e.g., amplification with partially / completely degenerate oligonucleotide primers) or targeted (e.g., amplification with oligonucleotide primers targeting a specific gene to be analyzed in subsequent steps).

[0271] In some implementations, qPCR or Taqman can be used immediately after the reverse transcriptase reaction of isolated cellular mRNAs; this variation is used to quantify the levels of various mRNAs during qPCR.

[0272] In some implementations, for qPCR or Taqman detection or RNA sequencing, a "pre-amplification" step may first be performed on cDNA transcribed from cellular RNA. This is used to amplify the signal when the native levels of the RNA / cDNA to be detected are very low. Suitable methods for pre-amplification include, but are not limited to, LM-PCR, PCR with random oligonucleotide primers (e.g., random hexamer PCR), PCR with polyA-specific primers, and any combination thereof. Pre-amplification can be general or targeted, performed in the same manner as the reverse transcription reaction described above.

[0273] Improved design and methods for synthetic biomarkers to reduce expression leakage

[0274] In some aspects, this disclosure provides a composition comprising an engineered nucleic acid encoding an expressible reporter gene that exhibits about 10% or less expression in normal cells relative to diseased cells when compared to a recombinant nucleic acid comprising a reporter gene containing the nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.

[0275] In some cases, engineered nucleic acids may contain pan-tumor-specific promoters operatively linked to an expressible reporter gene. In some cases, pan-tumor-specific promoters may contain transcriptional response elements. Transcriptional response elements may contain modified p53 response elements. Modifications within modified p53 response elements may result in decreased promoter activity relative to diseased cells in normal cells. Modifications within modified p53 response elements may result in increased promoter activity relative to normal cells in diseased cells.

[0276] In some cases, the engineered nucleic acid encoding the expressible reporter gene can be any vector described herein.

[0277] In some cases, reporter genes can encode detectable peptides or detectable nucleic acids. Detectable nucleic acid biomarkers can be ribozymes, self-splicing introns, RNA hairpins, microRNAs, RNA aptamers or their barcoded variations, or other types of quantifiable RNA. Quantifiable nucleic acids can contain unique sequences detectable by quantitative PCR or hybridization-based techniques.

[0278] Reporter genes can encode photoacoustic reporters, bioluminescent reporters, autofluorescent reporters, chemiluminescent reporters, luminescent reporters, colorimetric reporters, and any combination thereof. Autofluorescent reporters include GFP, mCherry, or derivatives thereof. Colorimetric reporters include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal application) and tyrosinase. Bioluminescent, chemiluminescent, or luminescent reporters include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renida luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396(2010): 290-297). Reporter proteins detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants). Detectable nucleic acid biomarkers can be ribozymes, self-splicing introns, RNA hairpins, microRNAs or their barcoded variations, or other types of quantifiable RNA. Quantifiable nucleic acid sequences can contain unique sequences detectable by quantitative PCR or hybridization-based techniques. Reporter genes can encode peptides detectable by non-invasive imaging methods. Such non-invasive imaging methods include MRI imaging, PET imaging, SPECT imaging, photoacoustic imaging, and bioluminescence imaging. Peptides detectable by MRI imaging include peptide contrast agents such as ferritin (or its mutants, such as the *C. fibrillosa* ferritin mutants L55P, F57S, or F123S) or lanthanide-binding proteins (or their engineered fusions, such as the LBT-ubiquitin fusion described in Daughtry et al., ChemBioChem 2012, 13, 2567–2574).Peptides detectable by PET or SPECT imaging include human sodium iodide cotransporters (e.g., in combination with PET-applied active iodine / iodine isotopes, see, for example, Penheiter et al., Curr Gene Ther. 2012 Feb; 12(1): 33–47), HSV-tk or mutants of it such as HSV-sr39tk (e.g., in combination with PET-applied positron-labeled acycloguanosine or pyrimidine analogues such as [18F]FHBG, see, Yaghoubi SS et al., Nat Protoc. 2006; 1(6): 3069-75), and dopamine D2 receptors or mutants of them such as D2R80A or D2R194A (e.g., in combination with positron-labeled D2 binding agents such as 3-(2'-[18F]-fluoroethyl)-spiropepidermobenzene). Peptides detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants). Peptides detectable by bioluminescence imaging include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renal luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396 (2010): 290-297). In some embodiments, the peptide may be a contrast agent, an enzyme that produces a detectable molecule, or a transporter that drives the accumulation of a detectable molecule.

[0279] In some cases, a disease that invades diseased cells may be cancer. Exemplary cancers include, but are not limited to, carcinoma, sarcoma, lymphoma, leukemia, and adenoma. Carcinoma may arise in cells covering internal and external parts of the body, such as the lungs, breast, and colon. Sarcoma may arise in cells located in bone, cartilage, fat, connective tissue, muscle, and other supporting tissues. Lymphoma may arise in lymph nodes and immune system tissues. Leukemia may arise in the bone marrow and accumulate in the bloodstream. Adenoma may arise in the thyroid gland, pituitary gland, adrenal gland, and other glandular tissues.Specific exemplary examples of cancer types suitable for detection using the methods according to this disclosure include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, visual pathway and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt lymphoma, cancer of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, etc. Cytokine tumor, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing sarcoma, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer, liposarcoma, liver cancer, lung cancer, such as non-small cell lung cancer and small cell lung cancer, lymphoma Tumors, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma, medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer of unknown primary origin, oral cancer, multiple endocrine tumor syndrome, myelodysplastic syndrome, myeloid leukemia, nasal and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cells, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma Cell tumors, pineal germ cell tumors, pituitary adenomas, pleural pulmonary blastomas, plasmacytoma formation, primary central nervous system lymphomas, prostate cancer, rectal cancer, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell skin cancer, small bowel cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, laryngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumors (pregnancy), cancers of unknown primary origin, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0280] In some aspects, this disclosure provides a method for detecting a disease in a subject, comprising an engineered nucleic acid encoding an expressible reporter gene that, when expressed in normal cells relative to diseased cells from the subject, exhibits about 10% or less expression compared to a recombinant nucleic acid containing a reporter gene comprising the nucleic acid sequence SEQ ID NO: 1 or SEQ ID NO: 2. The subject may be suspected of having cancer. The disease may be cancer or any subtype mentioned herein.

[0281] In some cases, engineered nucleic acids can be administered to a subject via intravenous, subcutaneous, intracardiac, intrathecal, intraventricular, percutaneous, intramuscular, oral, inhalation, nasal, rectal, intratumoral, or adjacent tumor sites. Adjacent tumor sites can refer to administration to tissues near a tumor or to areas that are predicted to be accessible to the tumor via the lymphatic system (e.g., adjacent lymph nodes). Intratumoral or adjacent tumor sites can include the use of additional imaging techniques such as endoscopic ultrasound (see, for example, Shirley et al., Gastroenterol Res Pract. 2013; 2013: 207129) or via bronchoscopy (see, for example, Rojas-Solano et al., J Bronchology Interv Pulmonol. 2018 Jul; 25(3): 168–175). In some implementations, engineered nucleic acids can be applied to at least one of the following lymph nodes: cervical lymph nodes, medial epicondyle lymph nodes, supraclavicular lymph nodes, axillary lymph nodes, mediastinal lymph nodes, supratrochlear lymph nodes, mesenteric lymph nodes, inguinal lymph nodes, femoral lymph nodes, or popliteal lymph nodes. In some cases, lymph node-based application can be used as a method for centralized local delivery to the tissue area.

[0282] In some cases, the method may include separating biological samples from the subject. Biological samples may be samples collected from the subject using non-invasive methods. Exemplary non-invasive samples include, but are not limited to, samples consisting of naturally shed bodily material or non-destructive scrapings from externally accessible tissues, such as saliva, sputum, sweat, urine, feces, semen, mucus, cervical and vaginal secretions, breast milk, inflammatory secretions, tears, and buccal epithelial swabs. Biological samples may also be samples collected from the subject using minimally invasive methods. Exemplary minimally invasive samples include, but are not limited to, blood samples or fractions thereof (e.g., obtained via venipuncture or capillary), pleural fluid samples (e.g., obtained via thoracentesis), amniotic fluid samples (e.g., obtained via amniocentesis), and gastric fluid samples (e.g., obtained via gastric lavage). Biological samples can be obtained through biopsies, such as skin biopsies (e.g., obtained through perforation, scraping, discoidal surgery, wedge-shaped, incision, or excision biopsies), bone marrow samples (e.g., obtained through aspiration biopsies), lymph node or breast biopsies (e.g., obtained through fine-needle aspiration, core-needle biopsies, vacuum-assisted biopsies, or image-guided biopsies), surgical biopsies (e.g., internal organ samples obtained through excision or incision biopsies), or mouth, gastrointestinal tract, lung, bladder, or urethra biopsies (e.g., obtained through endoscopy). In some cases, biological samples may be collected at a time after the composition has been administered to the subject.

[0283] The cell population can be cultured for at least approximately 15 minutes, at least approximately 30 minutes, at least approximately 1 hour, at least approximately 2 hours, at least approximately 4 hours, at least approximately 8 hours, at least approximately 16 hours, at least approximately 24 hours, at least approximately 36 hours, at least approximately 48 hours, at least approximately 3 days, at least approximately 4 days, at least approximately 5 days, at least approximately 6 days, at least approximately 7 days, at least approximately 8 days, at least approximately 9 days, at least approximately 10 days, at least approximately 11 days, at least approximately 12 days, at least approximately 13 days, at least approximately 14 days, at least approximately 15 days, or at least approximately 1 month after the gene construct is delivered to the cells. Cell populations can be cultured for up to 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 approximately 3 days, up to approximately 4 days, up to approximately 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 approximately 12 days, up to approximately 13 days, up to approximately 14 days, up to approximately 15 days, or up to approximately 1 month after the gene construct is delivered to the cells.

[0284] In some cases, the method may include detecting a peptide or nucleic acid sequence. This detection may include photoacoustic, bioluminescent, fluorescent reporter, chemiluminescent, luminescent, colorimetric, or nucleic acid assays. The detection may also include immunoassays. Immunoassays include, for example, those described in U.S. Patents 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524, and 5,480,792. Immunoassays include various sandwich, competitive, or non-competitive assay formats that produce a signal related to the presence or amount of the target protein analyte. Any suitable immunoassay can be used, such as lateral flow, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), competitive binding assay, and the like.

[0285] The detection method may include sequencing. Sequencing methods may include: next-generation sequencing, high-throughput sequencing, pyrosequencing, classic Sanger sequencing, ligation sequencing, synthesis sequencing, hybridization sequencing, RNA-Seq (Illumina), digital gene expression (Helicos), next-generation sequencing, single-molecule synthesis sequencing (SMSS) (Helicos), Ion Torrent sequencing machine (Life Technologies / Thermo-Fisher), massively parallel sequencing, cloning single-molecule array (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, and primer walking.

[0286] Detection can include “real-time amplification” methods (also known as quantitative PCR (qPCR)) or Taqman (see, for example, U.S. Patent Nos. Gelfand 5,210,015, Livak et al. 5,538,848, and Haaland 5,863,736, 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., Proc. Natl. Acad. Sci. USA 88:7276-7280, (1991); and Livak, KJ, et al., PCR Methods and Applications 357-362 (1995)). This method for monitoring the formation of amplified products is based on the use of double-labeled fluorescent oligonucleotide probes to continuously measure the accumulation of PCR products. The probes used in this assay are typically short (approximately 20-25 bases) polynucleotides labeled with two different fluorescent dyes. The 5' end of the probe is typically attached to a reporter dye, and the 3' end to a quenching dye. The probe is designed to have a sequence that is at least substantially complementary to the site on the target mRNA or nucleic acid. Upstream and downstream PCR primers that bind to the flanking regions of the locus are also added to the reaction mixture. When the probe is intact, energy transfer occurs between the two fluorophores, and the quencher quenches the emission from the reporter. During the extension phase of PCR, the probe is cleaved by the 5' nuclease activity of a nucleic acid polymerase, such as Taq polymerase, thereby releasing the reporter from the polynucleotide-quencher and resulting in an increase in reporter emission intensity, which can be measured by an appropriate detector. The recorded values ​​can then be used to continuously calculate the normalized increase in reporter emission intensity and ultimately quantify the amount of amplified mRNA.

[0287] In some implementations, for qPCR or Taqman assays, an RT-PCR step may be performed first to generate cDNA from cellular RNA. This RT-PCR amplification can be general (e.g., amplification with partially / completely degenerate oligonucleotide primers) or targeted (e.g., amplification with oligonucleotide primers targeting a specific gene to be analyzed in subsequent steps).

[0288] In some implementations, qPCR or Taqman can be used immediately after the reverse transcriptase reaction of isolated cellular mRNAs; this variation is used to quantify the levels of various mRNAs during qPCR.

[0289] In some implementations, for qPCR or Taqman detection or RNA sequencing, a "pre-amplification" step may first be performed on cDNA transcribed from cellular RNA. This is used to amplify the signal when the native levels of the RNA / cDNA to be detected are very low. Suitable methods for pre-amplification include, but are not limited to, LM-PCR, PCR with random oligonucleotide primers (e.g., random hexamer PCR), PCR with polyA-specific primers, and any combination thereof. Pre-amplification can be general or targeted, performed in the same manner as the reverse transcription reaction described above.

[0290] Design and methods of synthetic biomarkers using miRNA binding sites to modulate biomarker expression

[0291] In some aspects, this disclosure provides a composition that exhibits about 10% or less expression in normal cells compared to diseased cells and comprises a recombinant nucleic acid containing a nucleic acid sequence encoding a reporter gene, wherein the reporter gene contains one or more miRNA binding sequences in its 3′ untranslated region.

[0292] In some cases, the binding or lack thereof of miRNAs expressed in diseased cells to at least one of one or more miRNA binding sequences may lead to differential translation or half-life of the mRNA encoding the reporter gene. In some cases, the binding of miRNAs expressed in diseased cells to at least one of one or more miRNA binding sequences may lead to reduced translation of the reporter gene or a decreased half-life of the mRNA encoding the reporter gene. In some cases, the reporter gene may exhibit increased expression in cancer cells due to the downregulation of at least one miRNA expressed in cancer cells.

[0293] In some cases, diseased cells may be cancer cells, cells indicating autoimmune diseases (such as self-directing T cells or lymphocytes, or normal cells damaged by autoimmunity), or cells indicating neurodegenerative diseases (such as cells with or near toxic amyloid). Cancer, neurodegenerative diseases, and autoimmune diseases include any of the diseases described herein. In some cases, diseased cells may be virally infected cells. Exemplary viruses include, but are not limited to, HIV, hepatitis C virus, hepatitis B virus, hepatitis D virus, herpesvirus, Epstein-Barr virus, cytomegalovirus, and human T-lymphotropic virus type III.

[0294] In some cases, the composition may contain more than one miRNA binding sequence in the 3' untranslated region of the reporter gene. The composition may contain at least two miRNA binding sequences in the 3' untranslated region of the reporter gene, wherein the two miRNA binding sequences have substantially identical nucleotide sequences capable of binding to the same miRNA. The composition may contain at least two miRNA binding sequences in the 3' untranslated region of the reporter gene, wherein the at least two miRNA binding sequences have different nucleotide sequences, each capable of binding to a different miRNA. The composition may contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 miRNA binding sequences, or combinations of miRNAs, capable of binding to the same miRNA.

[0295] In some cases, recombinant nucleic acids can contain DNA. When recombinant nucleic acids contain DNA, the recombinant nucleic acid can be part of any vector described herein.

[0296] Recombinant nucleic acids can be synthetic or in vitro transcribed mRNA.

[0297] One or more miRNA binding sequences may include at least one miR-15, miR-16, let-7, miR-122, or miR-34 binding sequence.

[0298] In some cases, reporter genes can encode detectable peptides or detectable nucleic acids. Detectable nucleic acids can be ribozymes, self-splicing introns, RNA hairpins, microRNAs, RNA aptamers or their barcoded variations, or other types of quantifiable RNA. Quantifiable nucleic acids can contain unique sequences detectable by quantitative PCR or hybridization-based techniques.

[0299] Reporter genes may encode photoacoustic reporters, bioluminescent reporters, autofluorescent reporters, chemiluminescent reporters, luminescent reporters, colorimetric reporters, or any combination thereof. Autofluorescent reporters include GFP, mCherry, or derivatives thereof. Colorimetric reporters include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal application) and tyrosinase. Bioluminescent, chemiluminescent, or luminescent reporters include luciferases (e.g., in combination with coelenterate as described herein), including Gaussian luciferase, Renida luciferase, and Photinus luciferase (e.g., including engineered Ppy RE8 and RE9 variants, described in Branchini et al., Anal. Biochem. 396(2010): 290-297). Reporter proteins detectable by photoacoustic imaging include pigment-producing enzymes, such as β-galactosidase (e.g., in combination with X-gal), as well as tyrosinases, autofluorescent proteins (e.g., GFP, mCherry, or derivatives thereof), nonfluorescent GFP-like proteins (e.g., aeCP597 and cjBlue and their derivatives), near-infrared fluorescent proteins based on bacterial phytochromes (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 proteins that are reversibly photo-switched (e.g., Dronpa, Dronpa-M159T, and BphP1 or their variants). Detectable nucleic acids can be ribozymes, self-splicing introns, RNA hairpins, microRNAs or their barcoded variations, or other types of quantifiable RNA. Quantifiable nucleic acid sequences can contain unique sequences detectable by quantitative PCR or hybridization-based techniques.

[0300] Reporter genes can encode peptides detectable by non-invasive imaging methods. These non-invasive imaging methods include MRI imaging, PET...

Claims

1. Use of the composition in the preparation of a reagent for detecting cancer in a subject, wherein the cancer is lung cancer or liver cancer, and wherein: The composition for preparing the reagent for detecting the lung cancer in the subject comprises a first circular nucleic acid molecule, the first circular nucleic acid molecule comprising a first cancer-specific promoter operatively linked to a first nucleotide sequence encoding a first biomarker, wherein the first cancer-specific promoter comprises two or more promoter elements derived from two or more cancer-specific genes, the cancer-specific genes including family 111 member B (FAM111B), CEA cell adhesion molecule 5 (CEACAM5), or kinin family member 20A (KIF20A) having sequence similarity; or The composition for preparing the reagent for detecting the liver cancer in the subject comprises a second circular nucleic acid molecule containing a second cancer-specific promoter operatively linked to a second nucleotide sequence encoding a second biomarker, wherein the second cancer-specific promoter comprises two or more promoter elements derived from two or more cancer-specific genes, including family 111 member B (FAM111B) or kinin family member 20A (KIF20A) with sequence similarity; and The first or second cancer-specific promoter respectively induces an increase in the expression of the first or second biomarker in the lung cancer cells or liver cancer cells of the subject relative to the expression of the first or second biomarker in non-cancer cells, such that the relative ratio of the first or second biomarker expressed in the lung cancer cells or liver cancer cells of the subject to the expression of the first or second biomarker in the non-cancer cells is greater than 1.

0.

2. The use as described in claim 1, wherein the relative ratio is a concentration ratio.

3. The use as claimed in claim 1, wherein the first or second biomarker is detected in a biological sample from the subject.

4. The use as described in claim 3, wherein the biological sample is bodily fluid from the subject.

5. The use as claimed in claim 4, wherein the biological sample is blood from the subject or a blood-based sample.

6. The use as claimed in claim 3, wherein the biological sample is a gaseous sample from the subject.

7. The use as claimed in claim 6, wherein the gaseous sample is the exhaled breath of the subject.

8. The use as described in claim 3, wherein the biological sample is measured in situ within the subject.

9. The use as claimed in claim 1, wherein the first or second circular nucleic acid molecule independently comprises a mini R6K origin and an RNA-out optional marker.

10. The use as claimed in claim 1, wherein the first or second cancer-specific promoter drives the expression of the first or second biomarker in multiple different types of cancer cells in the subject, wherein the multiple different types of cancer cells are obtained from lung cancer or liver cancer.

11. The use as claimed in claim 10, wherein the first or second cancer-specific promoter respectively drives increased expression of the first or second biomarker in the multiple different types of cancer cells in the subject relative to the first or second biomarker in the non-cancer cells.

12. The use as claimed in claim 1, wherein the first or second biomarker is independently selected from MRI reporter, PET reporter, SPECT reporter, photoacoustic reporter, fluorescent reporter, quantifiable nucleic acid biomarker, and any combination thereof.

13. The use as described in claim 12, wherein the quantifiable nucleic acid biomarker is an engineered miRNA.

14. The use as claimed in claim 1, wherein the first or second biomarker is independently a luminescent reporter.

15. The use as described in claim 14, wherein the luminescent reporter is selected from bioluminescent reporters, chemiluminescent reporters, and any combination thereof.

16. The use as claimed in claim 1, wherein the first or second biomarker is independently a colorimetric reporter.

17. The use as claimed in claim 3, wherein detection of the first or second biomarker determines the location of the cancer cells.

18. The use as claimed in claim 3, wherein the first or second biomarker is independently detectable in the subject's body sample by non-invasive imaging.

19. The use as claimed in claim 1, wherein the first or second biomarker is detected using a blood-based assay.

20. The use as claimed in claim 1, wherein the composition further comprises a transfection agent.

21. The use as claimed in claim 20, wherein the transfection agent is linear or branched polyethyleneimine, nanoparticles, lipophilic particles, peptides, micelles, dendritic molecules, hydrogels, synthetic or naturally derived exosomes, virus-like particles, or any combination thereof.

22. The use as described in claim 21, wherein the nanoparticles are solid nanoparticles.

23. The use as claimed in claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier.

24. The use as described in claim 23, wherein the pharmaceutically acceptable carrier is selected from water, peanut oil, soybean oil, mineral oil, sesame oil, brine, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, hydrated dextrose, glycerol solution, glucose, lactose, sucrose, glyceryl monostearate, sodium chloride solution, propylene glycol, cocoa butter, or ethanol.

25. The use as claimed in claim 1, wherein the cancer cells in the subject are metastatic cancer cells.

26. The use as claimed in claim 25, wherein the metastatic cancer cells in the subject are lung cancer cells or liver cancer cells.

27. Use of the composition in the preparation of an agent for treating cancer in a subject, wherein said cancer is lung cancer or liver cancer, and wherein: The composition for preparing the reagent for treating the lung cancer in the subject comprises a first circular nucleic acid molecule, the first circular nucleic acid molecule comprising a first cancer-specific promoter operatively linked to a first nucleic acid sequence encoding a first therapeutic agent, wherein the first cancer-specific promoter comprises two or more promoter elements derived from two or more cancer-specific genes, the cancer-specific genes including family 111 member B (FAM111B), CEA cell adhesion molecule 5 (CEACAM5), or kinin family member 20A (KIF20A) having sequence similarity; or The composition for preparing the reagent for treating the liver cancer in the subject comprises a second circular nucleic acid molecule, the second circular nucleic acid molecule comprising a second cancer-specific promoter operatively linked to a second nucleic acid sequence encoding a second therapeutic agent, wherein the second cancer-specific promoter comprises two or more promoter elements derived from two or more cancer-specific genes, the cancer-specific genes including family 111 member B (FAM111B) or kinesin family member 20A (KIF20A) having sequence similarity. When the composition is administered to the subject, the expression of the first or second therapeutic agent in the lung cancer cells or liver cancer cells in the subject increases relative to the expression of the first or second therapeutic agent in non-cancer cells, such that the relative concentration of the first or second therapeutic agent expressed in the lung cancer cells or liver cancer cells of the subject compared to the expression in non-cancer cells is greater than 1.0, wherein the treatment of the lung cancer or liver cancer in the subject by the first or second therapeutic agent is determined by observing a reduction of at least 10% in the lung cancer cells or liver cancer cells in the subject or in a sample obtained from the subject after the composition is administered to the subject.

28. The use as described in claim 27, wherein the first or second therapeutic agent is independently selected from antibodies or fragments thereof, cytokines, enzymes, growth factors, engineered short peptide agonists, and engineered short peptide antagonists.

29. The use as described in claim 28, wherein the enzyme is selected from HSVtk, cytosine deaminase, DT yellow transaminase, nitroreductase, guanine phosphoribosyltransferase, purine nucleoside phosphorylase, thymidine phosphorylase, carboxylesterase, folic acid polyglutamyl synthase, carboxypeptidase A1, carboxypeptidase G2 and cytochrome P-450.

30. The use as claimed in claim 27, wherein the cancer cells in the subject are metastatic cancer cells.

31. The use as described in claim 30, wherein the metastatic cancer cells in the subject are lung cancer cells or liver cancer cells.

32. The use as claimed in claim 27, wherein the composition further comprises a transfection agent.

33. The use as described in claim 32, wherein the transfection agent is linear or branched polyethyleneimine, nanoparticles, lipophilic particles, peptides, micelles, dendritic molecules, hydrogels, synthetic or naturally derived exosomes, virus-like particles, or any combination thereof.

34. The use as described in claim 33, wherein the nanoparticles are solid nanoparticles.

35. The use as described in claim 27, wherein the composition further comprises a pharmaceutically acceptable carrier.

36. The use as described in claim 35, wherein the pharmaceutically acceptable carrier is selected from water, peanut oil, soybean oil, mineral oil, sesame oil, brine, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, hydrated dextrose, glycerol solution, glucose, lactose, sucrose, glyceryl monostearate, sodium chloride solution, propylene glycol, cocoa butter, or ethanol.

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