WO2024124190A1 - Engineered microorganisms for delivery of therapeutic agents - Google Patents

Engineered microorganisms for delivery of therapeutic agents Download PDF

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Publication number
WO2024124190A1
WO2024124190A1 PCT/US2023/083211 US2023083211W WO2024124190A1 WO 2024124190 A1 WO2024124190 A1 WO 2024124190A1 US 2023083211 W US2023083211 W US 2023083211W WO 2024124190 A1 WO2024124190 A1 WO 2024124190A1
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Prior art keywords
genetically engineered
engineered microorganism
gene
microorganism
plasmid
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PCT/US2023/083211
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French (fr)
Inventor
Jeffrey WAGNER
Fred H. Mermelstein
Carl D. Novina
Robert Distel
Steven NEIER
Barry Polisky
Amanda M. HUSSEY
Shinya IMADA
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Microbial Machines, Inc.
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Publication of WO2024124190A1 publication Critical patent/WO2024124190A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/28—Compounds containing heavy metals
    • A61K31/315—Zinc compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/54—Mixtures of enzymes or proenzymes covered by more than a single one of groups A61K38/44 - A61K38/46 or A61K38/51 - A61K38/53
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/67—Enzyme prodrug therapy, e.g. gene directed enzyme drug therapy [GDEPT] or VDEPT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1077—Pentosyltransferases (2.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01045—Thymidylate synthase (2.1.1.45)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
    • C12Y305/04001—Cytosine deaminase (3.5.4.1)

Definitions

  • GI diseases are the diseases involving the organs that form the GI tract, which include the mouth, esophagus, stomach, small intestine, large intestine, and rectum. GI diseases include Barrett’s esophagus, inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis), irritable bowel syndrome (IBS), precancerous syndromes, and cancer.
  • IBD inflammatory bowel disease
  • IBS irritable bowel syndrome
  • GI diseases are invasive procedures and uncomfortable for the patient, particularly for Attorney Docket No.
  • FIG. 1A–1E are schematic illustrations of payload delivery to diseased GI tissue using genetically engineered microorganisms.
  • FIG. 1A illustrates localization of cell membrane receptors in healthy cells versus diseased cells of the GI tract.
  • FIG. 1B illustrates selective binding of genetically engineered microorganisms to diseased cells via mislocalized cell membrane receptors.
  • FIG. 1C illustrates transfer of a DNA payload.
  • FIG. 1D illustrates transfer of an RNA payload.
  • FIG.1E illustrates transfer of a protein payload.
  • FIG.2 illustrates the conversion of 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) by cytosine deaminase (CD).
  • FIG.3 shows a schematic of a strain for DNA transfer.
  • the strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3).
  • the strain carries a plasmid containing a payload gene under a eukaryotic promoter and selection based on a wild-type alr gene.
  • FIG.4 shows a schematic of a strain for DNA transfer.
  • the strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv) and listeriolysin O (hlyA).
  • the strain carries a plasmid having an intron-containing gene encoding a chimeric bifunctional yeast cytosine deaminase (yCD) and the yeast uracil phosphoribosyltransferase (yUPRT) gene under a eukaryotic promoter.
  • the plasmid also carries an iRFP670 gene under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) (SEQ ID NO: 10).
  • IRS internal ribosome entry site
  • EMCV encephalomyocarditis virus
  • FIG. 5A is a bar graph demonstrating invasin- and LLO-dependent genetic transfer as assayed by the expression of iRFP670.
  • FIG.5B shows invasin- and LLO-dependent, 5-fluorocytosine-mediated cell killing.
  • FIG.6 shows a schematic of a strain for RNA transfer. The strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv) and listeriolysin O (hlyA; LLO).
  • Bottom panel shows a schematic representation of a plasmid that the strain carries, a plasmid containing an iRFP670 gene under a prokaryotic promoter and under the control of EMCV IRES.
  • FIGS. 7A and 7B demonstrate delivery of DNA and RNA by the bacterial strains disclosed herein.
  • FIG. 7A demonstrates the expression of an iRFP670 reporter under the control of a eukaryotic promoter.
  • FIG. 7B demonstrates the expression of an iRFP670 reporter expressed under a prokaryotic promoter and translated under the control of EMCV IRES.
  • FIGS.8A and 8B demonstrate that deletion of ribonuclease III (RNase III, rnc ⁇ ) improves RNA delivery as determined by flow cytometry.
  • FIG. 8A is a flow cytometry plot and
  • FIG.8B is a graph illustrating the flow cytometry data of FIG.8A.
  • FIG.9A shows a schematic representation of the mice, which develop tumors in distal colon after injection of 4-hydroxytamoxifen (right panel).
  • FIG. 9B shows a representative confocal fluorescence microscopy image of a colon tissue section showing healthy tissue and diseased tissue. Progressive induction of colorectal cancer in Apcfl/fl,VilCre mice is shown as demonstrated by confocal fluorescence microscopy images of colon tissue sections taken 3 days (FIG. 9C), 5 days (FIG.9D), 7 days (FIG.
  • FIGS.10A–10C show the bacteria disclosed herein are internalized in cells in a receptor and ligand-dependent matter.
  • FIG. 10A is a schematic representation of binding of invasin + bacteria to cells expressing ⁇ 1 integrin.
  • FIG. 10B shows that invasin is required for bacterial binding to cells.
  • FIG. 10C shows that ⁇ 1 integrin is required for bacterial binding to cells. Bacteria harboring an invasin gene were incubated in vitro with HCT116 colorectal cancer cells or HCT116 cells having the chromosomal gene for production of ⁇ 1 integrin knocked out, and internalization was measured by gentamicin protection assay.
  • FIGS. 11A and 11B show the bacteria disclosed herein can discriminate between diseased versus non-diseased tissue at 3 weeks.
  • FIG.11A is a schematic representation of the experiment. Briefly, tumorigenesis was induced in Apcfl/fl;Vil-Cre-ERT2 mice by injecting 4-hydroxytamoxifen. Three weeks later, bacteria expressing wild-type invasin (“invasin + ”) and GFP, or bacteria expressing a truncated invasin incapable of binding ⁇ 1 integrin (“invasin-”) and RFP were administered to the mice by enema.3 hours later, the mice were sacrificed, and the colon was extracted and colon tissue was subjected to epifluorescence microscopy. FIG. 11B shows internalization of invasin + bacteria but not the invasin- bacteria in diseased cells as examined by epifluorescence microscopy. FIGS.
  • FIG. 12A and 12B show the bacteria disclosed herein can discriminate between diseased versus non-diseased tissue early during tumorigenesis.
  • FIG. 12A is a schematic representation of the experiment. Briefly, tumorigenesis was induced in Apcfl/fl;Vil- Cre-ERT2 mice by injecting 4-hydroxytamoxifen. 3 to 7 days later, invasin + bacteria expressing GFP or invasin- bacteria expressing RFP were administered to the mice by enema. 3 hours later, the mice were sacrificed, and the colon was extracted and colon tissue was subjected to epifluorescence microscopy.
  • FIG. 12B illustrates epifluorescence microcopy of dysplastic and normal tissue at 5 and 7 days post induction of adenoma formation. E.
  • FIGS. 13A and 13B show the bacteria disclosed herein can discriminate between diseased versus non-diseased tissue early during tumorigenesis.
  • FIG. 13A is a schematic representation of the experiment. Briefly, tumorigenesis was induced in Apcfl/fl;Vil- Cre-ERT2 mice by injecting 4-hydroxytamoxifen.
  • FIG. 13B illustrates epifluorescence microcopy of dysplastic and normal tissue at 5 and 7 days post induction of adenoma formation.
  • EcN expressing invasin Attorney Docket No. MMS.003WO are shown in red and EcN expressing invasin trunc are shown in green. Images are at 20x magnification. Internalization of invasin + bacteria but not the invasin- bacteria in diseased cells is observed as early as 5 to 7 days post induction.
  • FIGS.14A–14C show that the bacteria disclosed herein invade only in the tumor tissue.
  • FIG.14A shows a surgical intervention to increase bacterial concentration in the colon.
  • FIG.14B is a graph showing the number of bacteria that were present in non-tumor and tumor tissue.
  • FIG. 14C shows fluorescence microscopy from different mice showing bacteria that were present in tumor tissue (right images) but not in non-tumor tissue (left images).
  • FIGS. 15A and 15B show the endocytosis of bacteria disclosed herein in an invasin-dependent manner.
  • FIG.15A shows a schematic representation of the experiment.
  • FIG. 15B is a graph that plots the number of invasin + and invasin- bacteria internalized in HCT116 colon cancer cells as a function of time.
  • FIGS.16A–16C show the endocytosis of bacteria disclosed herein in an invasin- dependent manner. Briefly, tumor bearing Apcfl/fl,VilCre mice were administered invasin + fluorescent bacteria by enema. After 2 hours, the proximal colon tissue (non-tumor tissue) and distal colon tissue (having tumors) were extracted and processed as in FIGS.16A to 16C.
  • FIG. 16A shows fluorescence microscopy and phase contrast images of colon tissue showing bacteria associated with tumor tissue but not non-tumor tissue.
  • FIG.16B shows the results of a semi-quantitative assay for bacteria that adhered to but did not invade colon tissue.
  • FIG.16C shows the results of a semi-quantitative assay for bacteria that invaded colon tissue.
  • the washed colon tissue was incubated with gentamycin followed by a treatment with 0.3% Triton-X (#2), and centrifuged at 400g for 1 minute to remove tissue fragments. The supernatant was sample #3. The tissue pellet was resuspended in PBS (sample #4).
  • FIG. 17 is a schematic representation of amplification of a payload (e.g., a nucleic acid encoding a prodrug converting enzyme and/or a detection marker) in target cells using self-amplifying RNA.
  • a payload e.g., a nucleic acid encoding a prodrug converting enzyme and/or a detection marker
  • the construct includes the functional components of RNA- dependent RNA polymerase (RdRP) or viral genome replication apparatus: nsp1, nsp2, nsp3 and nsp4, and mRNA of a payload sequence and a poly(A) tail.
  • RdRP RNA- dependent RNA polymerase
  • nsp1, nsp2, nsp3 and nsp4 mRNA of a payload sequence and a poly(A) tail.
  • FIG. 18 is a schematic representation of improvement of signal from RNA transfer of payload using self-amplifying RNA.
  • the cell on the left side shows conventional RNA transfer.
  • the cell on the right side shows amplification of the payload in target cells using self-amplifying RNA.
  • FIG. 19 is a graph showing the results of an in vitro invasion assay with mNeonGreen expressing bacteria displaying invasin or invasin trunc on HCT116 cells. Data points represent three independent biological replicates with the standard of deviation shown.
  • FIG. 20 is a graph showing the results of an in vitro gentamycin protection assay. Data represent the average of three biological replicates with the standard of deviation shown.
  • FIG.21 is a graph showing the results of an in vivo gentamycin protection assay. Data represent the average of five biological replicates with the standard of deviation shown. P values are for a paired student t-test between groups indicated.
  • FIG.22 shows a schematic of a strain for protein transfer.
  • the strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG. 22 (top panel)).
  • This strain carries a plasmid containing a payload gene under a prokaryotic promoter and selection based on a wild-type alr gene (FIG.22 (bottom panel)).
  • FIG.23 is a graph showing the conversion of 5-FC to 5-FU by engineered EcN measured by mass spectrometry over time.
  • FIG.24 is a graph showing the dose response curves for the killing of wild type HCT116 cells by engineered bacteria in the presence of varying concentrations of 5-FC. Each concentration was normalized to the total number of living cells in the wells lacking 5-FC. Data points are representative of three biological replicates with error bars denoting the standard of deviation.
  • FIG.25 is a graph showing the dose response curves for the killing of wild type HCT116 cells and ⁇ 1 integrin knockout cells by engineered bacteria in the presence of varying concentrations of 5-FC. Each concentration was normalized to the total number of living cells Attorney Docket No. MMS.003WO in the wells lacking 5-FC. Data points are representative of three biological replicates with error bars denoting the standard of deviation.
  • FIG.26 is a schematic showing a timeline of a treatment regimen for mice with induced dysplasia.
  • FIG.27 is a series of representative images showing a dissected colon. Areas of dysplasia are outlined in white.
  • FIG.28 is a graph showing the quantification of adenoma surface area in mouse cohorts. Data was generated over 3 separate experiments. Each point represents an individual animal with bars representing the average and error bars representing the standard of deviation. Asterisks indicate p-values for a one way ANOVA test with **** ⁇ 0.0001, *** ⁇ 0.001, ** ⁇ 0.01, and * ⁇ 0.05.
  • FIG.27 is a series of representative images showing a dissected colon. Areas of dysplasia are outlined in white.
  • FIG.28 is a graph showing the quantification of adenoma surface area in mouse cohorts. Data was generated over 3 separate experiments. Each point represents an individual animal with bars representing the average and error bars representing the standard of deviation. Asterisks indicate p-values for
  • FIG. 29 is a graph showing the quantification of adenoma surface area in experiments comparing engineered EcN expressing invasin or invasin trunc . Each point represents an individual animal with bars representing the average and error bars representing the standard of deviation. * indicates a p value ⁇ 0.05 by a one way ANOVA test.
  • FIG. 30 is a series of images showing representative histology for cleaved caspase 3 in mice undergoing different treatments. Arrows indicate sites of caspase 3 cleavage.
  • FIG. 31 is a graph showing the quantification of intestinal crypts showing caspase 3 cleavage as a marker for apoptosis.
  • FIG.32 is a graph showing dose response curves for the killing of ⁇ 1 integrin knockout HCT116 cells by engineered bacteria in the presence of varying concentrations of 5- FC. Each concentration was normalized to the total number of living cells in the wells lacking 5-FC. Data points are representative of three biological replicates with error bars denoting the standard of deviation.
  • DETAILED DESCRIPTION The present technology relates to genetically engineered microorganisms and associated compositions and methods of use.
  • a method for treating a subject includes administering a genetically engineered microorganism to the subject.
  • the genetically engineered microorganism can include a targeting gene encoding a targeting element that binds to a surface marker on a target cell and, optionally, promotes internalization of the genetically engineered microorganism by the target cell.
  • the genetically engineered microorganism can include a payload gene encoding a therapeutic agent, such as a prodrug converting enzyme that converts an inactive prodrug to an active drug.
  • the method can further include administering the prodrug to the subject.
  • the genetically engineered microorganism can include a second payload gene encoding a detection marker.
  • the present technology can provide numerous advantages compared to conventional techniques for treating and/or diagnosing a subject having a disease such as cancer.
  • conventional techniques for diagnosing abnormally growing cells in the gastrointestinal (GI) tract are typically based upon invasive colonoscopies that are not comfortable for the patient and not always successful in detection of diseased cells.
  • the ability to visualize and remove abnormal cells and diseased tissue varies depending on the skills of the surgeon and prominence of the polyps or tumors. Certain abnormally growing cells are flat or small in number and therefore, not visualized and removed by even skilled surgeons.
  • chemotherapeutic agents used for treating cancer typically lack any mechanism for differentiating between diseased and healthy cells, and thus may produce severe side effects due to off-target activity.
  • the genetically engineered microorganisms described herein allow a therapeutic payload to be delivered to diseased cells with a high degree of specificity, thus reducing the incidence of side effects due to off-target delivery.
  • the genetically engineered microorganisms herein can differentiate between diseased and healthy cells at an early stage of cancer development, thus allowing for improved patient outcomes due to earlier detection and/or treatment.
  • the GI tract includes the mouth, esophagus, stomach, small intestine, large intestine, and rectum.
  • the GI wall surrounding the lumen of the GI tract is made up of four concentric layers, arranged from the lumen outwards: (1) mucosa, (2) submucosa, (3) muscular layer, and (4) serosa (if the tissue is intraperitoneal) or adventitia (if the tissue is retroperitoneal)
  • the characteristics of the mucosa depends on the organ.
  • the stomach mucosal epithelium is simple columnar, and is organized into gastric pits and glands to deal with secretion.
  • the small intestinal mucosa which is made of glandular epithelium intermixed with secretory cells (e.g., goblet cells and Paneth cells) and immune cells (e.g., dendritic cells and M cells of the gut-associated lymphoid tissue (GALT)) arranged into villi, creating a brush border and increasing the area for absorption.
  • secretory cells e.g., goblet cells and Paneth cells
  • immune cells e.g., dendritic cells and M cells of the gut-associated lymphoid tissue (GALT)
  • GALT gut-associated lymphoid tissue
  • the apical surface of the cells faces the GI tract lumen, and the basolateral surface sits adjacent to an internal-facing basement membrane.
  • the basement membrane is an extracellular matrix (ECM) that includes laminins, collagen IV, proteoglycans, and nidogen.
  • ECM extracellular matrix
  • the epithelial cells interact with the ECM through integrins and the transmembrane proteoglycan dystroglycan, which are integral membrane proteins that bind to ECM components as well as intracellular proteins.
  • ⁇ 1 integrins which are widely expressed in epithelial cells, have a central role in establishing their polarity.
  • the binding of integrin to ECM components activates signaling by the integrins, which influences the organization of the cytoskeleton, which contributes to cellular polarity.
  • Disruption of the polarity and barrier function can cause disease.
  • tissue polarity is lost very early during cancer progression.
  • pathogens such as enteropathogenic E. coli and Y. pseudotuberculosis may disrupt cell polarity and enable the apical migration of basolateral membrane proteins.
  • the small bile ducts fuse to form larger bile ducts.
  • the larger bile ducts in the hepatic triads coalesce to intrahepatic bile ducts that become the right and left hepatic ducts that fuse at the undersurface of the liver to become the common bile duct.
  • the cystic duct (carrying bile to and from the gallbladder) branches off to the gallbladder.
  • the common bile duct opens into the intestine.
  • the intrahepatic ducts, cystic duct, and the common bile duct are lined by a tall columnar epithelium.
  • the gallbladder stores bile excreted from the liver.
  • the columnar mucosa is arranged in folds over the lamina intestinal, allowing expansion. Beneath the lamina basement is a muscularis, and surrounding the gallbladder is a connective tissue layer and serosa.
  • the gallbladder mucosa transports out sodium in the bile, passively followed by chloride and water. Thus, bile excreted by the liver and stored in the gallbladder becomes more concentrated.
  • the muscularis of the gallbladder contracts under the influence of the hormone cholecystokinin excreted by enteroendocrine cells of the small intestine.
  • the pancreatic duct or duct of Wirsung (also known as the major pancreatic duct), is a duct joining the pancreas to the common bile duct.
  • the pancreatic duct joins the common bile duct just prior to the ampulla of Vater, after which both ducts perforate the medial side of the second portion of the duodenum at the major duodenal papilla.
  • Pancreatic ducts are lined by columnar cells with luminal microvilli and glycocalyx and small apical cytoplasmic mucin droplets.
  • epithelial cells In large pancreatic ducts, many epithelial cells also have cilia, which function to aid the downstream movement of exocrine secretions.
  • the genetically engineered microorganisms described herein can be used to detect and/or treat many different diseases of the GI tract and/or associated tissues.
  • the disease is a cancer or a precancerous syndrome, such as squamous cell carcinoma of anus, colorectal cancer (including colorectal adenocarcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer), colorectal polyposis (including Peutz-Jeghers syndrome, juvenile polyposis syndrome, MUTYH-associated polyposis, familial adenomatous polyposis/Gardner’s syndrome, and Cronkhite-Canada syndrome), carcinoid tumor, pseudomyxoma peritonei, duodenal adenocarcinoma, distal bile Attorney Docket No.
  • colorectal cancer including colorectal adenocarcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer
  • colorectal polyposis including Koz-Jeghers syndrome, juvenile polyposis syndrome, MUTYH-associated polyposi
  • MMS.003WO duct carcinomas pancreatic ductal adenocarcinomas, gastric carcinoma, signet ring cell carcinoma (SRCC), gastric lymphoma (MALT lymphoma), linitis plastic (Brinton’s disease), or squamous cell carcinoma of esophagus and adenocarcinoma.
  • Colorectal cancer is a common and often lethal cancer that originates in the large intestine. Disease recognition at an early stage is advantageous to devise suitable preventive cancer strategies. Colorectal adenoearyma is the most frequent precancerous lesion.
  • Colorectal adenoma is an asymptomatic lesion often found incidentally during colonoscopy performed for unrelated symptoms or for CRC screening. About 25% of men and 15% of women who undergo colonoscopic screening have one or more adenomas. Up to 40% of people over the age of 60 harbor colorectal adenomatous polyps as shown in the colonoscopy examinations, although not all colonic polyps are adenomas and more than 90% of adenomas do not progress to cancer. Other potentially premalignant conditions include chronic inflammatory bowel diseases and hereditary syndromes, such as familial adenomatous polyposis (FAP), Peutz- Jeghers syndrome, and juvenile polyposis. These conditions can involve different sites of the GI tract.
  • the disease is polyposis (including FAP and attenuated FAP (AFAP), which are caused by pathogenic variants in the APC gene; and MUTYH-associated polyposis, which is caused by pathogenic variants in the MUTYH gene); and/or Lynch syndrome (often referred to as hereditary nonpolyposis colorectal cancer), which is caused by germline pathogenic variants in DNA MMR genes (MLH1, MSH2, MSH6, and PMS2) and EPCAM.
  • Other CRC syndromes and their associated genes include oligopolyposis (POLE, POLD1), NTHL1, juvenile polyposis syndrome (BMPR1A, SMAD4), Cowden syndrome (PTEN), and Peutz-Jeghers syndrome (STK11).
  • Lynch syndrome also known as hereditary non-polyposis colon cancer (HNPCC)
  • HNPCC hereditary non-polyposis colon cancer
  • Individuals with HNPCC have about a 75% lifetime risk of developing CRC, and are predisposed to several types of cancer.
  • Colon cancers and polyps arise in Lynch syndrome patients at a younger age than in the general population with sporadic neoplasias, and the tumors develop at a more proximal location. These cancers are often poorly differentiated and mucinous.
  • Muir-Torre syndrome is a variant of Lynch syndrome that presents additional predisposition to certain skin tumors.
  • FAP having a prevalence of 1 in 10,000 individuals, is the second most common genetic syndrome predisposing to CRC.
  • the lifetime risk of developing CRC for Attorney Docket No. MMS.003WO individuals suffering from FAP without prophylactic colectomy approaches 100%.
  • the characteristic features of FAP include the development of hundreds to thousands of colonic adenomas beginning in early adolescence.
  • the average age of CRC diagnosis (if untreated) in FAP patients is 40 years; 7% develop a tumor by the age of 20 and 95% by the age of 50.
  • AFAP is a less severe form of the disease, with an average lifetime risk of CRC of 70%.
  • approximately 30 adenomatous polyps develop in the colon, colonic neoplasms tend to be located in the proximal colon, and cancer occurs at an older age.
  • Gardner’s syndrome and Turcot’s syndrome are rare variants of FAP.
  • Gardner’s syndrome causes extra-colonic symptoms like epidermoid cysts, osteomas, dental abnormalities, and/or desmoid tumors.
  • Turcot’s syndrome causes colorectal adenomatous polyps and predisposition to developing malignant tumors of the central nervous system, such as medulloblastoma.
  • the genetic conditions MUTYH-associated polyposis, Peutz-Jeghers syndrome, and juvenile polyposis syndrome are other rarer syndromes that cause colon polyps, and predisposition to cancer.
  • Biliary tract cancers also called cholangiocarcinomas, refer to those malignancies occurring in the organs of the biliary system, including pancreatic cancer, gallbladder cancer, and cancer of bile ducts. Approximately 7,500 new cases of biliary tract cancer are diagnosed each year. These cancers include about 5,000 gallbladder cancers, and between 2,000 and 3,000 bile duct cancers.
  • Intraepithelial neoplasms are reported in the biliary tract, as biliary intraepithelial neoplasm (BilIN), and in the pancreas, as pancreatic intraepithelial neoplasm (PanIN). Both can evolve to invasive carcinomas, respectively cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC).
  • CCA cholangiocarcinoma
  • PDAC pancreatic ductal adenocarcinoma
  • BilINs are usually encountered in the epithelium lining the extrahepatic bile ducts (EHBDs), and large intrahepatic bile ducts (IHBDs), and may also be found in the gallbladder. BilINs are microscopic lesions, with a micropapillary, pseudopapillary, or flat growth pattern, involved in the process of multistep cholangiocarcinogenesis. Based on the degree of cellular and architectural atypia, BilINs have been classified into three categories: BilIN-1 (low grade dysplasia) showing the mildest changes compared to non-neoplastic Attorney Docket No.
  • MMS.003WO epithelium of the bile ducts BilIN-2 (intermediate grade dysplasia) with increased nuclear atypia and focal anomalies of cellular polarity as compared to BilIN-1; and BilIN-3 (high grade dysplasia or carcinoma in situ), which are usually identified in proximity of cholangiocarcinoma areas.
  • BilIN-2 intermediate grade dysplasia
  • BilIN-3 high grade dysplasia or carcinoma in situ
  • pancreas IPMNs
  • MCNs mucinous cystic neoplasms
  • PanINs have also been classified, according to the degree of cellular and architectural atypia, into low grade (previously classified as PanIN-1 and PanIN- 2) with mild-moderate cytological atypia and basally located nuclei, and high grade (previously classified as PanIN-3) with severe cytological atypia, loss of polarity, and mitoses.
  • the genetically engineered microorganisms disclosed herein are used to detect and/or treat an inflammatory bowel disease.
  • IBD Inflammatory bowel disease
  • Crohn’s disease Crohn’s disease
  • UC ulcerative colitis
  • IBD indeterminate colitis
  • the pathogenesis of IBD remains unclear, and it is characterized by long-lasting and relapsing intestinal inflammation.
  • the incidence of UC in the United States is estimated to be between 9 and 12 per 100,000 persons with a prevalence of 205 to 240 per 100,000 persons.
  • the etiology of UC is unknown.
  • abnormal immune responses to contents in the gut, including intestinal microbes are thought to drive disease in genetically predisposed individuals.
  • Colitis-associated colorectal cancer is one of the most serious complications of IBD, particularly in UC; it accounts for approximately 15% of all causes mortality among IBD patients. Because of worse prognosis and higher mortality in CACC than in sporadic CRC, early CACC detection is crucial.
  • Crohn’s disease is marked by inflammation of the GI tract. The inflammation can appear anywhere in the GI tract from the mouth to the anus. People with the disease often experience ups and downs in symptoms, and may even experience periods of remission. The length of diagnostic delay can represent an issue for at least a proportion of patients with Crohn’s disease. However, Crohn’s disease is a progressive disease that starts with mild Attorney Docket No. MMS.003WO symptoms and gradually gets worse.
  • IBS Irritable bowel syndrome
  • IBS-C constipation
  • IBS-D diarrhoea
  • IBS- M mixture of both conditions
  • the genetically engineered microorganisms disclosed herein are used to detect and/or treat Barrett’s esophagus.
  • Barrett’s esophagus is a condition in which tissue that is similar to the lining of intestine replaces tissue lining the esophagus. People with Barrett’s esophagus may develop esophageal adenocarcinoma.
  • the exact cause of Barrett’s esophagus is unknown, but gastroesophageal reflux disease (GERD) increases the risk of developing Barrett’s esophagus.
  • GSD gastroesophageal reflux disease
  • the present technology provides genetically engineered microorganisms that can be used to detect and/or treat a disease in a subject, such as a disease characterized by the presence of diseased GI tissue.
  • diagnosis, and specifically early diagnosis can be advantageous for preventing mortality and morbidity in individuals suffering from precancerous lesions, cancers of the GI tract and/or associated tissues (e.g., CRC, biliary tract cancer, pancreatic cancer), IBD (e.g., Crohn’s disease, ulcerative colitis), IBS, Barrett’s esophagus, and/or other diseases described herein.
  • the genetically engineered microorganisms of the present disclosure can be used to detect the presence of target cells (e.g., diseased and/or abnormal cells) that are indicative of the disease at an early stage with a high level of sensitivity and specificity, thus allowing for prophylactic measures and/or providing improved treatment outcomes.
  • target cells e.g., diseased and/or abnormal cells
  • the genetically engineered microorganisms herein include one or more of the following: a targeting gene that encodes a targeting element that allows the microorganism to bind to and enter a target cell (e.g., as described in Section II.A below), a lysis gene that encodes a lysis element that allows the microorganism and/or payload to enter the cytoplasmic space of the target cell (e.g., as described in Section II.B below), an auxotrophic mutation (e.g., as described in Section II.C below), a selection mechanism (e.g., as described in Section II.D below), and/or a payload gene encoding a therapeutic agent to be delivered to the target cell, such as a prodrug converting enzyme (e.g., as described in Section III below).
  • a targeting gene that encodes a targeting element that allows the microorganism to bind to and enter a target cell
  • a lysis gene that encodes a lysis element that allows the microorganism and
  • the genetically engineered microorganism of the present technology may be derived from any non-pathogenic microorganism, such as the non-pathogenic microorganisms that are normal flora of human GI tract or the microorganisms that are generally recognized as safe for human consumption via foods like yogurts, cheeses, breads and the like.
  • the genetically engineered microorganism of any one of the embodiments disclosed herein may be derived from a microorganism selected from Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Lactococcus, Pediococcus, Leuconostoc, Bacillus, or Escherichia coli.
  • Illustrative species that are suitable for the genetically engineered microorganism of any one of the embodiments disclosed herein include Bacillus coagulans, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium essencis, Bifidobacterium faecium, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium longum subsp.
  • infantis Bifidobacterium pseudolungum, Lactobacillus acidophilus, Lactobacillus boulardii, Lactobacillus breve, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus delbrueckii ssp.
  • the genetically engineered microorganism of any one of the embodiments disclosed herein may be derived from a probiotic Escherichia coli strain, such as Escherichia coli Nissle 1917, Escherichia coli Symbioflor2 (DSM 17252), Escherichia coli strain A034/86, or Escherichia coli O83 (Colinfant).
  • a probiotic Escherichia coli strain such as Escherichia coli Nissle 1917, Escherichia coli Symbioflor2 (DSM 17252), Escherichia coli strain A034/86, or Escherichia coli O83 (Colinfant).
  • the genetically Attorney Docket No. MMS.003WO engineered microorganism of any one of the embodiments disclosed herein is Escherichia coli Nissle 1917 or a derivative thereof.
  • Escherichia coli Nissle 1917 contains two naturally occurring, stable, cryptic plasmids pMUT1 (GenBank Accession No. MW240712) and pMUT2 (GenBank Accession No. CP023342).
  • the Escherichia coli Nissle 1917 or the derivative thereof harbors a plasmid pMUT1 and/or a plasmid pMUT2, and/or one or more derivatives thereof.
  • the Escherichia coli Nissle 1917 or the derivative thereof is cured of the plasmid pMUT1 and/or the plasmid pMUT2.
  • the Escherichia coli Nissle 1917 or the derivative thereof includes an exogenous plasmid (e.g., a plasmid based on pUC18/19, a pBR322, R6K, 15A or a pSC10).
  • an Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT1 having a wild type alr gene as a selection mechanism.
  • the Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT1 having a wild type alr gene as a selection mechanism, and genes encoding invasin and/or listeriolysin, or a mutant derivative thereof.
  • the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of plasmid pMUT1 having a wild type alr gene under its own promoter as a selection mechanism, and optionally, genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof.
  • an Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT2 having a wild type alr gene under the control of its own promoter as a selection mechanism. In some embodiments, the Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT2 having wild type alr gene under the control of its own promoter as a selection mechanism, and genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof.
  • the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of plasmid pMUT2 having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof.
  • an Escherichia coli Nissle 1917 derivative harbors an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism.
  • the exogenous plasmid may be based on any plasmid that replicates in the microorganism (e.g., a plasmid based on pUC18/19, a pBR322, R6K, 15A or a pSC101).
  • the Escherichia coli Nissle 1917 derivative harbors a derivative of the exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and genes encoding invasin and/or listeriolysin O, or a mutant derivative Attorney Docket No. MMS.003WO thereof.
  • the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of the exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof.
  • the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of the exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and, optionally, comprising genes encoding a payload (e.g., a detection marker and/or a therapeutic agent).
  • a payload e.g., a detection marker and/or a therapeutic agent
  • an Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of pMUT1, pMUT2, or an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, a gene encoding a payload (e.g., a detection marker and/or a therapeutic agent).
  • a payload e.g., a detection marker and/or a therapeutic agent
  • the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of pMUT1, pMUT2, or an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, a gene encoding invasin and/or listeriolysin O, or a mutant derivative thereof.
  • the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of pMUT1, pMUT2, or an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, genes encoding one or more of a payload (e.g., a detection marker and/or a therapeutic agent), invasin, listeriolysin O, or a mutant derivative thereof.
  • a payload e.g., a detection marker and/or a therapeutic agent
  • the targeting gene e.g., a gene encoding a surface protein such as invasin
  • a lysis gene e.g., a gene encoding a lysin such as listeriolysin O
  • the target gene and the lysis gene are integrated at a single genomic site, optionally, the single genomic site is an integration site of a bacteriophage.
  • the bacteriophage is bacteriophage Lambda.
  • the target gene and/or lysis gene are inserted into a plasmid, which is optionally a naturally occurring plasmid.
  • the one or more payload genes may be inserted on a natural endogenous plasmid from Escherichia coli Nissle 1917 (e.g., pMUT1, pMUT2, and/or a derivative thereof).
  • the plasmid includes a selection Attorney Docket No. MMS.003WO mechanism (e.g., an auxotrophic marker such as alr as described).
  • the targeting gene is inserted on a plasmid.
  • the lysis gene is inserted on the plasmid.
  • the genetically engineered microorganism includes at least one targeting gene encoding a targeting element that, when expressed, causes the microorganism to specifically interact with a target cell (e.g., a diseased and/or abnormal cell).
  • a target cell e.g., a diseased and/or abnormal cell
  • the targeting element can promote binding of the microorganism to the target cell and/or entry of the microorganism into the target cell.
  • the targeting element can be selected so that the microorganism has little or no interaction with non-target cells (e.g., non-diseased and/or normal cells).
  • the targeting element is a protein or peptide expressed on the surface of the microorganism that specifically interacts with one or more surface markers on the target cell.
  • the surface marker can be a protein (e.g., a cell membrane receptor), peptide, glycoprotein, carbohydrate, or any other cell surface molecule that is expressed by or is otherwise characteristic of the target cell.
  • the surface marker can be found on the target cell but not on non-target cells.
  • the surface marker can be found on both target and non-target cells, but can be localized differently in target and non-target cells such that the surface marker is accessible to the microorganism on the target cell but not accessible to the microorganism on the non-target cell.
  • the surface marker can be a mislocalized and/or aberrantly expressed protein found in cancerous tissues and/or precancerous lesions (e.g., polyps or adenomas), tears and erosions (Barrett’s Esophagus), and/or cells indicative of inflammatory diseases.
  • the targeting gene is an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptors.
  • the cell membrane receptor is not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, or common bile duct, etc., but is exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, or common bile duct, etc., in the subject suffering from a disease.
  • the surface protein can promote binding and invasion of the microorganism in the diseased epithelial cells, but not binding and invasion into normal cells.
  • the genetically engineered microorganism may Attorney Docket No.
  • the genetically engineered microorganism specifically binds to one or more of oral epithelial cells, buccal epithelial cells of the tongue, pharyngeal epithelial cells, mucosal epithelial cells, endothelial cells of the stomach, intestinal epithelial cells, colon epithelial cells, etc.
  • the targeting element is or includes an invasin, or a fragment thereof.
  • the invasin can be selected from Yersinia enterocolitica invasin, Yersinia pseudotuberculosis invasin, Salmonella enterica PagN, Candida albicans Als3.
  • the targeting element is or includes an intimin, or a fragment thereof.
  • the intimin can be selected from Escherichia albertii intimin (e.g., NCBI accession no. WP_113650696.1), Escherichia coli intimin (e.g., NCBI accession no. WP_000627885), or Citrobacter rodentium intimin (e.g., NCBI accession no. WP_012907110.1).
  • the targeting element is or includes an adhesin, or a fragment thereof.
  • the targeting element is or includes a flagellin, or a fragment thereof.
  • the targeting element includes one or more of the following: invasin, YadA (Yersinia enterocolitica plasmid adhesion factor), Rickettsia invasion factor RickA (actin polymerization protein), Legionella RaIF (guanine exchange factor), one or more Neisseria invasion factors (e.g., NadA (Neisseria adhesion/invasion factor), OpA, and/or OpC (opacity-associated adhesions)), Listeria InlA and/or InlB, one or more Shigella invasion plasmid antigens (e.g., IpaA, IpaB, IpaC, IpgD, IpaB-IpaC complex, VirA, and/or IcsA), one or more Salmonella invasion factors (e.g., SipA, SipC, SpiC, SigD, SopB, SopE, SopE2, and/or SptP), Staphylococcus F
  • the targeting element is or includes a type III secretion system or a component thereof.
  • the targeting element is or includes a peptide or protein that specifically binds to the surface of target cells, such as a leptin, an antibody, or a fragment thereof (e.g., sdAb (also known as a Nanobody®) or an scFv fragment).
  • the targeting element is or includes a fusion protein of any two or more of the aforementioned proteins or fragments thereof.
  • the targeting element can be or include a fusion protein of invasin and intimin.
  • the targeting element includes an active fragment of one or more of invasin, YadA, RickA, RaIF, NadA, OpA, OpC, InlA, InlB, IpaA, IpaB, IpaC, IpgD, IpaB-IpaC, VirA, Attorney Docket No. MMS.003WO IcsA, SipA, SipC, SpiC, SigD, SopB, SopE, SopE2, SptP, FnBPA, FnBPB, ACP, Fba, F2, Sfb1, Sfb2, SOF, PFBP, or FimB.
  • the fragment(s) are expressed on the surface of the engineered microorganism disclosed herein, e.g., on an adhesion scaffold.
  • Representative examples of targeting elements and their associated polynucleotide sequences are provided in Table 1 below.
  • Table 1 Targeting Elements Attorney Docket No. MMS.003WO
  • the genetically engineered microorganism produces a targeting element having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1.
  • the genetically engineered microorganism includes a targeting element of SEQ ID NO: 1.
  • the genetically engineered microorganism produces a targeting element that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or the coding region thereof (e.g., residues 256–3213).
  • the genetically engineered microorganism includes a targeting element that is encoded by the sequence of SEQ ID NO: 2 or the coding region thereof.
  • the genetically engineered microorganism includes a single targeting gene encoding a targeting element that facilitates both binding to the target cell and entry into the target cell.
  • the genetically engineered microorganism can include multiple targeting genes expressing multiple targeting elements, such as a first targeting gene encoding a first targeting element that facilitates binding to the target cell, and a second targeting gene encoding a second targeting element that facilitates entry into the target cell.
  • the multiple targeting elements can be expressed as multiple separate proteins, or can be expressed as a single fusion protein.
  • the genetically engineered microorganism can include a targeting gene encoding a targeting element that facilitates binding to the target cell, without any targeting elements to facilitate entry into the target cell.
  • the targeting gene(s) can be incorporated in a genomic site of the genetically engineered microorganism (e.g., a chromosome), a non-genomic site of the genetically engineered microorganism (e.g., a plasmid such as an episome, which may be a natural endogenous plasmid or an exogenous plasmid), or suitable combinations thereof.
  • one or more targeting genes can be integrated into a genomic site Attorney Docket No. MMS.003WO of the Escherichia coli Nissle 1917 or derivative thereof, which can be the integration site of a bacteriophage (e.g., bacteriophage Lambda).
  • one or more targeting genes can be inserted into a plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the targeting gene(s) can be incorporated at the same site or different site as the other genetic elements described herein (e.g., lysis gene(s), payload gene(s), and/or selection mechanism(s)).
  • the targeting gene(s) are integrated at a first genomic site, and the lysis gene(s) are integrated at the first genomic site or a second, different genomic site.
  • the target gene(s) and the lysis gene(s) are integrated at a single genomic site, which can be an integration site of a bacteriophage (e.g., bacteriophage Lambda).
  • the targeting gene(s) are integrated at a first genomic site; the lysis gene(s) are integrated at the first genomic site or a second, different genomic site; and the payload gene(s) are integrated at the first genomic site, the second genomic site, or a third, different genomic site.
  • the target gene(s), the lysis gene(s), and the payload gene(s) are integrated at a single genomic site, which can be an integration site of a bacteriophage (e.g., bacteriophage Lambda).
  • the targeting gene(s) are integrated at a first genomic site, the lysis gene(s) are integrated at the first genomic site or a second, different genomic site, and the payload gene(s) is inserted in a plasmid.
  • the targeting gene(s) are integrated at a first genomic site, the lysis gene(s) are integrated at the first genomic site or a second, different genomic site, and the payload gene(s) and selection mechanism(s) are inserted in a plasmid.
  • the plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the plasmid may be a single copy of a multi-copy plasmid.
  • the targeting gene(s) are integrated at a first genomic site, and the lysis gene(s) are inserted into a first plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the first plasmid may be a single copy of a multi-copy plasmid.
  • the payload gene(s) can be integrated into the first genomic site, or a second, different genomic site; or can be inserted into the first plasmid or a second, different plasmid.
  • the second plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the second plasmid may be a single copy of a multi-copy plasmid.
  • Attorney Docket No. MMS.003WO the lysis gene(s) are integrated at a first genomic site, and the targeting gene(s) are inserted into a first plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the first plasmid may be a single copy of a multi-copy plasmid.
  • the payload gene(s) can be integrated into the first genomic site or a second, different genomic site; or can be inserted into the first plasmid or a second, different plasmid.
  • the second plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the second plasmid may be a single copy of a multi-copy plasmid.
  • the targeting gene(s) are inserted into a first plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the first plasmid may be a single copy of a multi-copy plasmid.
  • the lysis gene(s) can be inserted into the first plasmid or a second, different plasmid.
  • the second plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the second plasmid may be a single copy of a multi-copy plasmid.
  • the payload gene(s) can be inserted into the first plasmid or the second plasmid.
  • the first plasmid and/or the second plasmid can include the selection mechanism(s).
  • the targeting gene(s), lysis gene(s), payload gene(s), and selection mechanism(s) are all inserted in the same plasmid.
  • the genetically engineered microorganism upon binding to the target cell, is endocytosed (e.g., phagocytosed) by the target cell and thus becomes internalized within an endocytic vacuole (e.g., phagosome) the target cell.
  • the genetically engineered microorganism can include at least one lysis gene encoding a lysis element that, when expressed, lyses the endocytic vacuole to allow the genetically engineered microorganism and/or payload to escape into the intracellular space.
  • the lysis element can be a lysin that lyses the endocytotic vacuole, and thereby contributes to pore formation, breakage, and/or degradation of the phagosome.
  • the lysin is a cholesterol-dependent cytolysin.
  • the lysin is selected from one or more of listeriolysin O, ivanolysin O, streptolysin, perfringolysin, botulinolysin, or leukocidin, or fragment or a mutant derivative thereof.
  • the lysin is listeriolysin O or a mutant derivative thereof (e.g., a derivative lacking a periplasmic secretion signal).
  • the genetically engineered microorganism produces a lysis element having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4.
  • the genetically engineered microorganism includes a lysis element of any one of SEQ ID NO: 3 or SEQ ID NO: 4.
  • the genetically engineered microorganism includes a single lysis gene encoding a single lysis element.
  • the genetically engineered microorganism can include multiple lysis genes expressing multiple lysis elements, such as a first targeting gene encoding a first lysin and a second targeting gene encoding a second, different lysin.
  • the multiple lysis elements can be expressed as multiple separate proteins, or can be expressed as a single fusion protein.
  • the lysis gene(s) can be incorporated in a genomic site of the genetically engineered microorganism (e.g., a chromosome), a non-genomic site of the genetically engineered microorganism (e.g., a plasmid such as an episome, which may be a natural endogenous plasmid or an exogenous plasmid), or suitable combinations thereof.
  • a genomic site of the genetically engineered microorganism e.g., a chromosome
  • a non-genomic site of the genetically engineered microorganism e.g., a plasmid such as an episome, which may be a natural endogenous plasmid or an exogenous plasmid
  • one or more lysis genes can be integrated into a genomic site of the Escherichia coli Nissle 1917 or derivative thereof, which can be the integration site of a bacteriophage (e.g., bacteriophage Lambda).
  • a bacteriophage e.g., bacteriophage Lambda
  • one or more lysis genes can be inserted into a plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid.
  • the lysis gene(s) can be incorporated at the same site or different site as the other genetic elements described herein (e.g., targeting gene(s), payload gene(s), and/or selection mechanism(s)).
  • the lysis gene(s) are integrated at a first genomic site
  • the targeting gene(s) are integrated at the first genomic site or a second, different genomic site
  • the payload gene(s) and selection mechanism(s) are inserted in a plasmid.
  • the lysis gene(s), targeting gene(s), payload gene(s), and selection mechanism(s) can all be inserted in the same plasmid.
  • the lysis gene(s) are constitutively expressed.
  • the lysis gene(s) can be conditionally expressed. For instance, expression of the lysis gene(s) can be induced after endocytosis, e.g., using a promoter that responds to changes in the environment indicative of endosomal localization, such as pH, temperature, and/or addition of a small molecule. Expression of the lysis gene(s) can occur from a plasmid (e.g., an episome) or from a genomic site (e.g., a chromosome, at the same location as the targeting gene(s) or at a different location). In other embodiments, however, the lysis gene(s) are optional and may be omitted. C.
  • the genetically engineered microorganism harbors at least one auxotrophic mutation, such as a nutritional auxotrophic mutation.
  • the auxotrophic mutation can be used to inhibit proliferation of the genetically engineered microorganism after administration to the subject (e.g., for containment purposes).
  • the auxotrophic mutation can be selected from one or more of dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF.
  • the mutation can be a partial or complete deletion of the gene, or any other modification that causes inactivation of the gene.
  • the genetically engineered microorganism includes a single auxotrophic mutation (e.g., a partial or complete deletion of a single gene), while in other embodiments, the genetically engineered Attorney Docket No. MMS.003WO microorganism can include multiple auxotrophic mutations (e.g., a partial or complete deletion of multiple genes).
  • the auxotrophic mutation includes a deletion, inactivation, or decreased expression or activity of at least one gene involved in the synthesis of a metabolite (e.g., a non-genetically encoded amino acid) required for cell wall synthesis.
  • the gene can be required for synthesis of D-alanine or diaminopimelic acid.
  • the engineered microorganism has an inactivation of the dapA gene, which can be caused by a partial or complete deletion.
  • the engineered microorganism can have an inactivation of the alr and dadX genes, which can be caused by a partial or complete deletion.
  • the engineered microorganism harbors a combination of dapA, alr, and dadX auxotrophic mutations.
  • the auxotrophic mutation facilitates lysis of the microorganism inside the target cell upon invasion, which can enhance efficacy of payload delivery to the intracellular space of the target cell.
  • the dapA auxotrophic mutation causes a defect in cell wall synthesis that can facilitate lysis of the microorganism inside the target cell upon invasion.
  • an auxotrophic mutation that facilitates lysis of the microorganism is optional and may be omitted.
  • the auxotrophic mutation can be used as a selection mechanism (e.g., an antibiotic-free selection mechanism).
  • the genetically engineered microorganism can include a plasmid that is selected by complementation of the at least one auxotrophic mutation (e.g., alr and dadX mutations) by at least one corresponding functional gene present on the plasmid (e.g., a functional alr gene).
  • the plasmid can also incorporate any of the other genetic elements described herein such as the targeting gene(s), lysis gene(s) and/or payload gene(s).
  • the targeting gene(s), lysis gene(s) and/or payload gene(s) Representative examples of polynucleotide sequences for selection markers for complementation of an auxotrophic mutation are provided in Table 3 below. Table 3: Selection Markers Attorney Docket No.
  • the genetically engineered microorganism includes a selection marker that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or the coding region thereof (e.g., residues 191–1270).
  • the genetically engineered microorganism includes a selection marker that is encoded by the sequence of SEQ ID NO: 5 or the coding region thereof.
  • D. Selection Mechanisms In some embodiments, the genetically engineered microorganism includes one or more plasmids having a selection mechanism.
  • the selection mechanism can be used to ensure that the plasmid(s) having one or more of the genetic elements described herein (e.g., payload gene(s), targeting gene(s), and/or lysis gene(s)) are maintained within the genetically engineered microorganism.
  • the plasmid(s) can include an endogenous plasmid (e.g., pMUT1 and/or pMUT2 for Escherichia coli Nissle 1917), an exogenous plasmid, or suitable combinations thereof.
  • the genetically engineered microorganism includes a single selection mechanism, while in other embodiments, Attorney Docket No. MMS.003WO the genetically engineered microorganism can include multiple selection mechanisms.
  • selection mechanisms examples include, but are not limited to, an antibiotic resistance marker, a toxin-antitoxin system, a marker causing complementation of a mutation in an essential gene (e.g., complementation of an auxotrophic mutation as described in Section II.C above), or a cis acting genetic element, or a combination of any two or more thereof.
  • the selection mechanism is a resistance marker to an antibiotic, such as an antibiotic that is not used or is rarely used in humans or animals for therapy.
  • the selection mechanism is an antibiotic resistance marker selected from a kanamycin resistance gene or a tetracycline resistance gene, or a combination thereof. In some embodiments, the selection mechanism may not require an antibiotic for plasmid maintenance.
  • the selection mechanism can be a toxin-antitoxin system selected from a hok/sok system of plasmid system of plasmid RK2, ccdAB of F plasmid, flmAB of F plasmid, kis/kid system of plasmid R1, XCV2162- ptaRNA1 of Xanthomonas campestris, ataT-ataR of enterohemorragic E.
  • the selection mechanism can be an essential gene encoding an enzyme involved in biosynthesis of an essential nutrient or a substrate (e.g., an amino acid).
  • the essential nutrient or substrate can be required for cell wall synthesis and/or a house- keeping function.
  • amino acids required for cell wall synthesis include D-alanine and diaminopimelic acid.
  • the essential gene is selected from dapA, dapD, murA, alr, dadX, murI, dapE, or thyA, or a combination of any two or more thereof.
  • the essential genes are a combination of alr and dadX (both of which encode for alanine racemases).
  • the essential genes are a combination of alr and dadX, and the plasmid(s) are selected using a functional alr gene (alr + , e.g., a wild type alr gene) as a selection marker.
  • the plasmid(s) are selected by complementation of the alr and dadX mutations by a functional alr gene present on the plasmid and/or the second plasmid.
  • the house-keeping function is selected from infA, a gene encoding a subunit of an RNA polymerase, a DNA polymerase, an rRNA, a tRNA, a cell division protein, or a chaperon protein, or a combination of any two or more thereof.
  • the genetically engineered microorganism includes a selection marker that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or the coding region thereof.
  • the genetically engineered microorganism includes a selection marker that is encoded by the sequence of SEQ ID NO: 5 or the coding region thereof.
  • the selection mechanism can be a cis acting genetic element, such as ColE1 cer locus or par from pSC101.
  • the selection mechanism can be incorporated in the same plasmid(s) as the other genetic elements described herein (e.g., targeting gene(s), payload gene(s), and/or selection mechanism(s)).
  • the targeting gene(s) and/or lysis gene(s) are inserted in a first plasmid
  • the payload gene(s) are inserted in a second plasmid
  • the selection mechanism is inserted in both the first and second plasmids.
  • the lysis gene(s), targeting gene(s), payload gene(s), and selection mechanism(s) can all be inserted in the same plasmid. III.
  • the genetically engineered microorganisms of the present disclosure deliver at least one payload to a target cell (e.g., a diseased and/or abnormal cell).
  • the payload can be a nucleic acid (e.g., DNA, RNA), a peptide, a protein, or a combination thereof.
  • the payload can be delivered to the target cell in order to treat and/or detect a disease, such as any of the diseases described herein.
  • the payload can facilitate treatment and/or detection of a disease characterized by the presence of diseased GI tissue in subject in need thereof, such as precancerous lesions (e.g., adenomas), GI tract cancers (e.g., CRC), IBD (e.g., UC, Crohn’s disease), IBS, or Barrett’s esophagus.
  • a disease characterized by the presence of diseased GI tissue in subject in need thereof such as precancerous lesions (e.g., adenomas), GI tract cancers (e.g., CRC), IBD (e.g., UC, Crohn’s disease), IBS, or Barrett’s esophagus.
  • FIGS. 1A–1E are schematic illustrations of a method for delivering a payload to diseased GI tissue using genetically engineered microorganisms, in accordance with embodiments of the present technology.
  • certain types of GI diseases can be characterized by the presence of diseased GI epithelium having mislocalized and/or aberrantly expressed cell surface molecules.
  • certain types of cell membrane receptors e.g., integrins such as ⁇ 1 integrins
  • integrins such as ⁇ 1 integrins
  • cell membrane receptors are present only on the basolateral surfaces of healthy GI epithelial cells, and are not present on the apical surfaces facing the GI tract lumen.
  • diseased GI epithelial cells at Attorney Docket No. MMS.003WO least some of the cell membrane receptors can be mislocalized and displayed on the apical surface facing the GI tract lumen.
  • diseased GI epithelial cells can also exhibit novel mammalian membrane receptors, which may be receptors that are normally not found in healthy cells or receptors that are formed by translocations and other genomic rearrangement.
  • the genetically engineered microorganisms of the present disclosure e.g., genetically engineered Escherichia coli Nissle 1917
  • the genetically engineered microorganisms can be introduced into the lumen of the GI tract of the subject and thus be exposed to diseased and healthy cells of the GI epithelium.
  • the genetically engineered microorganisms can include one or more targeting genes encoding one or more targeting elements that facilitate binding and/or entry into the diseased GI cells, while avoiding binding and/or entry into the healthy GI cells, as described herein.
  • the targeting element(s) expressed by the genetically engineered microorganism bind to the diseased cells via the mislocalized and/or aberrantly expressed molecules on the apical surfaces of the diseased cells, and undergo internalization.
  • the genetically engineered microorganisms can express invasin, which can bind to mislocalized integrins on the apical surfaces of the diseased cells and can facilitate endocytosis of the microorganisms by the diseased cells.
  • the genetically engineered microorganisms can exhibit little or no binding affinity to the healthy cells due to lack of mislocalized and/or aberrantly expressed cell surface molecules on the apical surfaces of the healthy cells (cell surface molecules present on the basolateral surfaces of the healthy cells may be inaccessible to microorganisms within the GI tract lumen).
  • the genetically engineered microorganism upon internalization, can initially be sequestered in a phagosome of the diseased cell.
  • the genetically engineered microorganism can undergo lysis due to an auxotrophic mutation (e.g., a dapA attenuation mutation) that causes a defect in cell wall synthesis.
  • an auxotrophic mutation e.g., a dapA attenuation mutation
  • the genetically engineered microorganism can also include one or more lysis genes encoding one or more lysis elements (e.g., Listeriolysin O), as described herein.
  • the lysis element(s) can be released upon lysis of the microorganism and/or can be naturally exported by the microorganism.
  • the lysis element(s) can lyse the phagosome of the diseased cell to allow the contents of the lysed microorganism to escape into the cytoplasmic space of the diseased cell.
  • the genetically engineered microorganism carries a DNA payload (e.g., plasmid DNA or other DNA molecule) that is transferred into the cytoplasmic space of the diseased cell upon lysis of the microorganism and the phagosome.
  • the DNA can undergo nuclear localization, which can occur naturally or be guided by binding of a protein that includes a nuclear localization signal. Subsequently, the DNA can be transcribed to RNA, and the RNA can be translated to produce protein expression.
  • the protein can remain within the intracellular space of the diseased cell, can be transported to and incorporated into the cellular membrane, and/or can be secreted.
  • the protein can be a therapeutic agent, a detection marker, or a combination thereof, as described in further detail below.
  • the genetically engineered microorganism can alternatively or additionally carry an RNA payload (e.g., messenger RNA or other RNA molecule) that is transferred into the cytoplasmic space of the diseased cell upon lysis of the microorganism and the phagosome.
  • the RNA can be translated to produce protein expression.
  • the protein can remain within the intracellular space of the diseased cell, can be transported to and incorporated into the cellular membrane, and/or can be secreted.
  • the protein can be a therapeutic agent, a detection marker, or a combination thereof, as described in further detail below. Referring to FIG.
  • the genetically engineered microorganism can alternatively or additionally carry a protein payload.
  • the protein can be a therapeutic agent, a detection marker, or a combination thereof, as described in further detail below.
  • the protein can be expressed within the genetically engineered microorganism and subsequently delivered to the cell.
  • the protein is transferred into the cytoplasmic space of the diseased cell upon lysis of the microorganism and the phagosome.
  • the protein can remain within the intracellular space of the diseased cell, can be transported to and incorporated into the cellular membrane and/or other cellular compartments, and/or can be secreted.
  • the protein may not be transferred into the cytoplasmic space, e.g., the protein may remain within the phagosome, such that the genetically engineered microorganism may not include a lysis gene encoding a lysis element (e.g., listeriolysin O) that facilitates lyses of the endocytic vacuole.
  • a lysis element e.g., listeriolysin O
  • the protein may remain within the microorganism and/or can be present on the surface of the microorganism, such that the microorganism may not include an auxotrophic mutation that facilitates lysis of the microorganism (e.g., a dapA auxotrophic mutation).
  • the protein is capable of exerting a desired effect without being transported Attorney Docket No. MMS.003WO into the cytoplasmic space (e.g., if the protein is an enzyme that acts upon a small molecule that is capable of crossing the cell membrane and bacterial cell wall, as discussed further below).
  • the genetically engineered microorganism may not need to be internalized by the target cell, and may instead remain external to the target cell (e.g., attached to an external surface of the target cell).
  • the genetically engineered microorganisms of the present disclosure include one or more payload genes encoding at least one gene product that can be used to treat a target cell in a subject (e.g., a diseased and/or abnormal cell that is indicative of a disease).
  • the gene product(s) can be or include at least one therapeutic agent, such as a protein peptide, or nucleic acid molecule (e.g., an RNA molecule such as a catalytic RNA, a non-coding RNA, etc.).
  • the therapeutic agent can be any agent that exerts a therapeutic effect in the subject, such as obtaining a beneficial or intended clinical result including alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of the disease, steadying the disease in a non-advanced state, delaying the progress of the disease, and/or improvement or alleviation of disease symptoms.
  • the therapeutic agent exhibits therapeutic activity in the target cell, such as by directly killing the target cell, mediating killing of the target cell by another component, or inhibiting or otherwise interfering with cellular processes in the target cell.
  • the therapeutic agent may be a prodrug converting enzyme, a direct cytotoxic agent, or a combination thereof.
  • the therapeutic agent is or includes an enzyme that acts upon a substrate to produce a therapeutic effect.
  • the enzyme can be a prodrug converting enzyme.
  • a prodrug converting enzyme can be a protein having a function of converting an inactive prodrug into an active drug (e.g., an active metabolite) through an enzymatic reaction.
  • the prodrug converting enzyme enzymatically converts an inactive prodrug into an active drug capable of directly or indirectly having therapeutic effect.
  • the active drug can be an anti-cancer drug, a cytotoxic drug, Attorney Docket No. MMS.003WO anti-bacterial drug, an anti-parasite drug, a hormone, an immunosuppressive drug, or a combination thereof.
  • the prodrug converting enzyme activates a compound with little or no cytotoxicity into a therapeutically active product.
  • the prodrug converting enzyme activates a compound with little or no cytotoxicity into a toxic product.
  • the prodrug converting enzyme sensitizes the target cell therapy.
  • the prodrug converting enzyme can increase cell permeability to another therapeutic agent, restore hormonal responsiveness, and/or render the cell more sensitive to radiotherapy and/or chemotherapeutics.
  • the prodrug converting enzyme converts a precursor of 5-fluoruracil (5-FU) into 5-FU.
  • 5-FU is a chemotherapeutic agent that acts as a thymidylate synthase inhibitor, thus blocking DNA replication.
  • the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5-FU, such as a cytosine deaminase or a derivative (e.g., fusion protein) thereof.
  • FIG.2 illustrates the conversion of 5-FC into 5-FU by cytosine deaminase (CD).
  • the prodrug converting enzyme is selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450 (e.g., that acts on the prodrug as a substrate), an uracil phosphoribosyltransferase (UPRT), or a combination thereof, or a fusion protein comprising two or more thereof.
  • the enzyme is a fusion protein of a cytosine deaminase and a UPRT (CD:UPRT).
  • the cytosine deaminase is a yeast cytosine deaminase (yCD) and/or the UPRT is a yeast or bacterial URPT (yURPT or UPRT).
  • the prodrug converting enzyme is an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5- fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU.
  • the enzyme is a carboxylesterase.
  • the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU. In some embodiments, the prodrug converting enzyme is a ⁇ -glucuronidase. In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 6-methylpurine-deoxyriboside (MePdR) into 6-methylpurine. In some embodiments, the enzyme is a purine nucleoside phosphorylase (PNP). Attorney Docket No.
  • the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate (fludarabine monophosphate) into fluoroadenine.
  • the enzyme is a purine nucleoside phosphorylase (PNP).
  • the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil into 1-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl) 5-methyluracil monophosphate (FMAUMP).
  • the enzyme is a thymidine kinase (TK) and/or a thymidylate synthase (TS).
  • the prodrug is 5-fluorocytosine (5-FC), pentyl N-[1- [(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4- yl]carbamate (capecitabine), 5-FU glucuronide, 6-methylpurine-deoxyriboside (MePdR), fludarabine monophosphate, 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, or a combination of any two or more thereof.
  • 5-FC 5-fluorocytosine
  • the prodrug converting enzyme is a carboxypeptidase (e.g., a Pseudomonas sp. Carboxypeptidase G2 (CPG2)).
  • the prodrug converting enzyme is a ⁇ -lactamase (e.g., an Enterobacter cloacae ⁇ -lactamase).
  • the prodrug is ganciclovir, and the prodrug converting enzyme is a thymidine kinase (e.g., a viral thymidine kinase such as herpes simplex thymidine kinase).
  • the prodrug and active drug are small molecules that are capable of crossing the cell membrane of the target cell and the cell wall of the genetically engineered microorganism.
  • a prodrug/active drug combination that is capable of crossing cell membranes and cell walls is 5-FC/5-FU.
  • the prodrug converting enzyme does not need to be present in the cytoplasmic space of the target cell to convert the prodrug into the active drug.
  • the genetically engineered microorganism may not include a lysis gene encoding a lysis element that facilitates lysis of the endocytic vacuole of the target cell and/or may not include an auxotrophic mutation that facilitates lysis of the microorganism.
  • prodrug converting enzymes are provided in International Publication No. WO 1997/038087, U.S. Patent Application Publication No. 2020/0268794, U.S. Patent Application Publication No. 2020/0215111, U.S. Patent Application Publication No. 2020/0157518, U.S. Patent Application Publication No. Attorney Docket No. MMS.003WO 2019/0038727, U.S. Patent Application Publication No. 2017/0173092, the disclosures of which are incorporated by reference herein in their entirety.
  • prodrug converting enzymes are also provided in Aghi et al., Prodrug activation enzymes in cancer gene therapy, J Gene Med 2: 148-64 (2000); and Malekshah et al., Enzyme/Prodrug Systems for Cancer Gene Therapy, Curr Pharmacol Rep.2016; 2(6): 299–308, the disclosures of which are incorporated by reference in their entirety.
  • the use of a prodrug converting enzyme as a therapeutic agent may provide various advantages, such as reducing off-target cytotoxicity (e.g., killing of nontarget cells).
  • a payload gene encoding a prodrug converting enzyme e.g., a cytosine deaminase can be selectively delivered to target cells (e.g., diseased and/or abnormal cells of the GI tract) using a genetically engineered microorganism, as described herein.
  • target cells e.g., diseased and/or abnormal cells of the GI tract
  • the prodrug converting enzyme may be selectively expressed in the target cells, while nontarget cells may exhibit little or no expression of the prodrug converting enzyme.
  • the prodrug converting enzyme can be expressed by a genetically engineered microorganism that is selectively internalized by and/or selectively binds to the surface of target cells, as described herein.
  • a prodrug e.g., 5-FC
  • the prodrug converting enzyme present in and/or proximate to the target cells can convert the inactive prodrug into an active form (e.g., 5-FU) that results in killing of the target cells.
  • an active form e.g., 5-FU
  • the prodrug remains in its inactive form in nontarget cells lacking the prodrug converting enzyme, such that the nontarget cells are substantially unaffected.
  • Representative examples of prodrug converting enzymes and their associated polynucleotide sequences are provided in Table 4 below. Table 4: Prodrug Converting Enzymes Attorney Docket No. MMS.003WO Attorney Docket No.
  • the genetically engineered microorganism produces a therapeutic agent having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 6.
  • the genetically engineered microorganism includes a therapeutic agent of SEQ ID NO: 6.
  • the genetically engineered microorganism includes a therapeutic agent that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO: 8 or the coding region thereof.
  • the genetically engineered microorganism includes a therapeutic agent that is encoded by the sequence of any one of SEQ ID NO: 7 or SEQ ID NO: 8 or the coding region thereof. 2.
  • the therapeutic agent is a cytotoxic agent (e.g., a cytotoxic protein or peptide) that is capable of directly killing the target cell, also referred to herein as a “direct cytotoxic agent.”
  • a direct cytotoxic agent can kill the target cell by interfering with cellular processes, causing damage to cellular components, or suitable combinations thereof.
  • Examples of direct cytotoxic agents that may be used include microbial toxins, such as a clostridial toxin (e.g., botulinum neurotoxin, tetanus toxin, perfringolysin, ⁇ toxin, ⁇ toxin), a staphylococcal toxin, a Shiga toxin, an anthrax toxin, a diphtheria toxin, a pertussis toxin, a cholera toxin; caspases (e.g., constitutively active caspase-3); and suitable derivatives and combinations thereof.
  • a clostridial toxin e.g., botulinum neurotoxin, tetanus toxin, perfringolysin, ⁇ toxin, ⁇ toxin
  • staphylococcal toxin e.g., an anthrax toxin, a diphtheria toxin, a pertussi
  • the direct cytotoxic agent exhibits enhanced activity in target cells compared to the nontarget cells, e.g., direct cytotoxic agents that interfere with DNA replication may have an enhanced cytotoxic effect in rapidly dividing tumor cells compared to normal cells.
  • the direct cytotoxic agent may exhibit the same or similar activity in target cells versus nontarget cells. In either case, off-target effects (e.g., killing of nontarget cells) may be reduced by using a genetically engineered microorganism to selectively deliver a payload gene encoding a direct cytotoxic agent to target cells, or to selectively deliver the direct cytotoxic agent to target cells, as described herein.
  • the direct cytotoxic agent may be selectively expressed in and/or delivered to the target cells, Attorney Docket No. MMS.003WO thus resulting in selective killing of the target cells.
  • nontarget cells may exhibit little or no expression and/or receive little or no delivery of the direct cytotoxic agent and thus may remain substantially unaffected.
  • the therapeutic agent e.g., prodrug converting enzyme or direct cytotoxic agent
  • the therapeutic agent remains in the cytoplasm of the target cell.
  • the therapeutic agent is displayed on the surface of the target cell, e.g., the therapeutic agent can be attached to the surface of the target cell (e.g., by a GPI anchor).
  • the therapeutic agent is secreted by the target cell.
  • the nucleic acid molecule encoding the therapeutic agent can include a leader sequence for secretion of the therapeutic agent.
  • the therapeutic agent e.g., prodrug converting enzyme or direct cytotoxic agent
  • the mammalian promoter directs GI tract epithelial cell-specific expression.
  • a nucleic acid from the genetically engineered microorganism enters the nucleus of at least one target cell (e.g., a diseased and/or abnormal cell).
  • the nucleic acid is a DNA molecule (e.g., a plasmid).
  • the therapeutic agent e.g., prodrug converting enzyme or direct cytotoxic agent
  • a microbial promoter e.g., an RNA molecule encoding the therapeutic agent is translated in the target cell (e.g., a diseased and/or abnormal cell).
  • the genetically engineered microorganism has a deletion of an RNase gene to increase stability of the RNA molecule.
  • the RNase gene is RNase III (rnc).
  • the payload gene(s) encoding the therapeutic agent is present in a cassette that directs RNA self-amplification of the cassette in the target cells.
  • the cassette includes an RNA viral genome replication apparatus.
  • the RNA viral genome replication apparatus encodes an RNA- dependent RNA polymerase (RdRP).
  • RdRP is an alphavirus RdRP.
  • the alphavirus RdRP is nsp1, nsp2, nsp3, and/or nsp4.
  • the RNA molecule with the payload gene(s) encoding the therapeutic agent further includes an internal ribosome entry site (IRES).
  • the payload gene(s) are operably linked to a sequence element that causes the therapeutic agent (e.g., prodrug converting enzyme or direct cytotoxic Attorney Docket No. MMS.003WO agent) to be selectively expressed in the target cell, while producing little or no expression of the therapeutic agent in nontarget cells, thus reducing off-target effects of the therapeutic agent.
  • the sequence element can be a promoter and/or a cis-regulatory element (e.g., an enhancer) that is activated in the target cells due to the presence of transcription factors that are specifically present in the target cells, but is not activated in nontarget cells due to the absence of such transcription factors.
  • the transcription factors can be selectively expressed and/or overexpressed in cancerous and/or precancerous cells.
  • the payload gene(s) encoding the therapeutic agent includes one or more introns (e.g., one or more spliceosomal introns) such that the therapeutic agent is expressed in a first, active form (e.g., a catalytically active form) only in the target cells (e.g., diseased and/or abnormal cells), and is expressed in a second, nonactive form in nontarget cells (e.g., the genetically engineered microorganism and/or healthy cells).
  • a first, active form e.g., a catalytically active form
  • nontarget cells e.g., the genetically engineered microorganism and/or healthy cells.
  • the first form can be a protein or peptide produced from a spliced RNA molecule
  • the second form can be a protein or peptide produced from the unspliced RNA molecule
  • the first form can be a protein or peptide produced from a first splice variant of an RNA molecule
  • the second form can be a protein or peptide produced from a second splice variant of the RNA molecule (e.g., a variant produced by alternative splicing of the same RNA molecule).
  • introns in the payload gene can reduce off-target effects by ensuring that the therapeutic agent only exhibits significant activity when expressed in diseased and/or abnormal cells, and exhibits little or no activity when expressed by the genetically engineered microorganism (e.g., due to leaky expression, background activity of mammalian promoters in the microorganism) and/or when expressed by other nontarget cells (e.g., due to nonspecific binding of the microorganism to nontarget cells).
  • the prodrug converting enzyme can be capable of converting the inactive prodrug into the active drug only when the prodrug converting enzyme is expressed in a spliced form or as a splice variant produced specifically by the target cell.
  • the therapeutic agent is a direct cytotoxic agent
  • the direct cytotoxic agent may exhibit therapeutic activity only when expressed in a spliced form or as a splice variant produced specifically by the target cell.
  • the payload gene may not include any introns, e.g., if the payload gene is to be expressed in the microorganism for delivery of a protein payload to the target cell.
  • the genetically engineered microorganisms of the present disclosure include one or more payload genes encoding at least one gene product that can be used to detect a target cell in a subject (e.g., a diseased and/or abnormal cell that is indicative of a disease).
  • the gene product(s) can be or include at least one detection marker, such as a protein, peptide, and/or nucleic acid.
  • the detection marker can be an intracellular biomarker that is present within the target cell, a surface biomarker that is located on the surface of the target cell, a secretable biomarker that is exported by the target cell, or suitable combinations thereof.
  • the presence or absence of the detection marker can be correlated to presence or absence of the target cell in the patient, which in turn can be correlated to a positive or negative diagnosis, respectively.
  • the detection marker can be measured using any suitable technique. The appropriate technique can be selected based on the type of detection marker (e.g., protein, peptide, RNA, or DNA), whether the detection marker exhibits functional activity and/or functional properties (e.g., enzymatic activity, fluorescence, luminescence), and/or other relevant considerations.
  • the detection marker can be measured using one or more of the following: agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, circular dichroism (CD), chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, flow cytometry, fluorimetry, electrophoretic assay, enzymatic assay, enzyme linked immunosorbent assay (ELISA), gas chromatography, gravimetry, high pressure liquid chromatography (HPLC), immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, reverse transcription-polymerase chain reaction (RT-PCR), multiplex RT-PCR, quantitative PCR (qPCR), reverse transcription-qPCR (RT- qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), semi-quantitative PCR, semi- quantitative RT-PCR, gel electrophore
  • the detection marker is present in a biological sample collected from the subject, such as feces, blood, serum, plasma, mucus, urine, saliva, and/or a biopsy sample. Accordingly, the subject can be diagnosed by collecting the biological sample Attorney Docket No. MMS.003WO and analyzing the biological sample for the presence of the detection marker. In some embodiments, the detection marker is collected and measured in a noninvasive manner (e.g., without performing colonoscopy or endoscopy on the subject).
  • the detection marker can be measured without the use of a clinical laboratory instrument selected from a colonoscope, an endoscope, a radiography instrument (e.g., an X-ray machine), a computed tomography (CT) scanner, a magnetic resonance imaging (MRI) machine, a blood gas analyzer, and/or a urine chemistry analyzer.
  • a clinical laboratory instrument selected from a colonoscope, an endoscope, a radiography instrument (e.g., an X-ray machine), a computed tomography (CT) scanner, a magnetic resonance imaging (MRI) machine, a blood gas analyzer, and/or a urine chemistry analyzer.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • the detection marker can be measured using invasive techniques (e.g., via colonoscopy or endoscopy) and/or using one or more of the clinical laboratory instruments provided above.
  • the detection marker is or includes at least one protein or peptide.
  • the protein or peptide can be an intracellular protein or peptide that is present within the target cell, a surface protein or peptide that is located on the surface of the target cell, a secretable protein or peptide that is exported by the target cell, or suitable combinations thereof.
  • the protein or peptide can be any protein or peptide that is not endogenously produced by the cells of the subject (e.g., by target cells and/or by non-target cells within the subject).
  • the protein or peptide can be a naturally occurring protein or peptide that is endogenously produced by a different type of organism than the subject (e.g., a non- mammalian organism), or can be a derivative of a naturally occurring protein or peptide.
  • the protein or peptide can be a synthetic protein or peptide.
  • Synthetic proteins and peptides can be generated in various ways, such as by modifying the sequence of a naturally occurring protein or peptide (e.g., to increase stability, modify localization) or via de novo design.
  • a library of synthetic proteins or peptides are generated and screened for the desired activity.
  • the protein or peptide serves as the sole detection marker for determining the presence or absence of target cells in the subject. In other embodiments, however, the protein or peptide can be used in combination with another detection marker for determining the presence or absence of target cells in the subject, such as the RNA molecule encoding the protein or peptide, or a different RNA molecule.
  • the protein or peptide that serves as the detection marker is selected from a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT Attorney Docket No. MMS.003WO contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination of any two or more these.
  • a fluorescent marker e.g., a bioluminescent marker, an MRI contrast agent, a CT Attorney Docket No. MMS.003WO contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated
  • the detection marker is a protein or peptide that is easily detectable, e.g., based on binding to an antibody or other binding protein, via a noninvasive or invasive imaging technique, via an assay performed on a biological sample collected from the subject, etc.
  • the detection marker is a fluorescent protein.
  • the fluorescent protein can be selected from GFP, RFP, YFP, Sirius, Sandercyanin, shBFP- N158S/L173I, Azurite, EBFP2, mKalama1, mTagBFP2, TagBFP, shBFP, ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquoise, mTurquoise2, TagCFP, mTFP1, monomeric Midoriishi-Cyan, Aquamarine, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, Monomeric Azami Green, mWasabi, Clover, mNeonGreen, NowGFP, mClover3, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, lanRFP- ⁇ S83, mPapaya1, mCyRFP1, Monomeric Kusabira-Orange, mOrange,
  • the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713, iRFP720, or iSplit.
  • the detection marker is a fluorescent protein that becomes fluorescent upon contact with a metabolite found only in the mammalian cytoplasm.
  • the detection marker can be a near-infrared fluorescent protein that utilizes biliverdin as a cofactor to fluoresce, such as iRFP or iRFP670.
  • the detection marker can be Japanese freshwater eel (Anguilla japonica) UnaG protein, which fluoresces only upon binding to bilirubin.
  • the genetically engineered microorganism delivers one or more payload genes encoding at least one fluorescent protein to the target cells (e.g., diseased epithelial cells), the target cells express the fluorescent protein, and the target cells are detected based on fluorescence of the fluorescent protein.
  • the payload gene(s) can include one or more introns such that the fluorescent protein is expressed in a detectable form (e.g., a fluorescent form) only in the target cells, as described herein. Attorney Docket No.
  • the fluorescent protein is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay.
  • the fluorescent protein can be detected in situ in the subject using an endoscopic procedure or colonoscopic procedure (e.g., a white light endoscopic procedure or Laser-Induced Fluorescence Endoscopy (LIFE)).
  • the detection marker is a bioluminescent protein.
  • the bioluminescent protein can be selected from a Ca +2 regulated photoprotein (e.g., aequorin, symplectin, Mitrocoma photoprotein, Clytia photoprotein, and Obelia photoprotein), North American firefly luciferase, Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina luciferase, Metridina luciferase, Metrida luciferase, OLuc protein, red firefly luciferase, bacterial luciferase, or active variants thereof.
  • a Ca +2 regulated photoprotein e.g., aequorin, symplectin, Mit
  • the genetically engineered microorganism delivers one or more payload genes encoding at least one bioluminescent protein to the target cells (e.g., diseased epithelial cells), the target cells express the bioluminescent protein, and the target cells are detected based on luminescence of the bioluminescent protein.
  • the payload gene(s) can include one or more introns such that the bioluminescent protein is expressed in a detectable form (e.g., a luminescent form or a form that acts on a luminescent substrate) only in the target cells, as described herein.
  • the bioluminescent protein is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay.
  • a biological sample collected from the subject e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva
  • the bioluminescent protein can be detected in situ within the subject using an endoscopic procedure or colonoscopic procedure.
  • a substrate of the bioluminescent protein is administered during and/or before the endoscopic procedure or colonoscopic procedure.
  • Illustrative substrates include luciferin, or a pharmaceutically acceptable analog, derivative, or salt thereof.
  • the administration of the substrate of the bioluminescent protein is started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection.
  • the detection marker is an enzyme reporter.
  • the enzyme reporter catalyzes a reaction, which may be detected on the basis of Attorney Docket No. MMS.003WO change in, e.g., color, fluorescence, or luminescence.
  • Such reactions may use chromogenic, fluorogenic, or luminogenic substrates, which may be used in an in vitro assay to detect the target cells in a biological sample collected from the subject, and/or may be provided locally or systemically to the subject at the time of detection of the target cells.
  • the enzyme substrate is colorigenic, luminogenic, and/or fluorogenic.
  • the enzyme reporter can be selected from beta-galactosidase, chloramphenicol acetyltransferase, horseradish peroxidase, alkaline phosphatase, acetylcholinesterase, or catalase.
  • the enzyme reporter is beta-galactosidase
  • the substrate is selected from resorufin ⁇ -D-galactopyranoside, 5-dodecanoylaminofluorescein di- ⁇ -D- galactopyranoside, 5-bromo-4-chloro-3-indolyl ⁇ -D-galactopyranoside (X-Gal), or GALACTO-LIGHT PLUS.
  • the enzyme reporter is horseradish peroxidase
  • the substrate is selected from 3,3′,5,5′-Tetramethylbenzidine (TMB), 3,3’- Diaminobenzidine (DAB), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), or 5-Amino-2,3-dihydrophthalazine-1,4-dione (luminol).
  • the enzyme reporter is chloramphenicol acetyltransferase
  • the substrate is BODIPY FL-1- deoxychloramphenicol.
  • the genetically engineered microorganism delivers one or more payload genes encoding at least one enzyme reporter to the target cells (e.g., diseased epithelial cells), the target cells express the enzyme reporter, and the target cells are detected based on enzymatic activity of the enzyme reporter.
  • the payload gene(s) can include one or more introns such that the enzyme reporter is expressed in a detectable form (e.g., an enzymatically active form) only in the target cells, as described herein.
  • the enzymatic reporter is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay.
  • the enzymatic reporter can be detected in situ within the subject using an endoscopic procedure or colonoscopic procedure.
  • the substrate of the enzymatic reporter is administered during and/or before the endoscopic procedure or colonoscopic procedure.
  • the administration of the substrate is started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days prior to marker detection.
  • the detection marker is a secretable biomarker that is excreted in a biological sample collected from the subject.
  • the biological sample can be a biological fluid selected from blood, serum, plasma, mucus, urine, and/or saliva.
  • the biological sample can be a biopsy sample collected from the subject.
  • the secretable biomarker is an enzyme, a peptide hormone, or a protein or peptide antigen.
  • the secretable biomarker can be a secreted protein (or a subunit of peptide therefrom) selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2 (CCSP-2), Cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), or an artificial protein capable of tight binding with high specificity to a detection antibody or protein, or a subunit or fragment thereof, or a combination of any two or more thereof.
  • the secretable biomarker is a human chorionic gonadotropin (hCG).
  • hCG is a hormone composed of two subunits: the ⁇ and ⁇ subunits.
  • the hCG is an ⁇ subunit of human chorionic gonadotropin ( ⁇ hCG).
  • the hCG is a ⁇ subunit of human chorionic gonadotropin ( ⁇ hCG).
  • hCG is primarily catabolized by the liver, although about 20% is excreted in the urine. The ⁇ subunit is degraded in the kidney to make a core fragment which is measured by urine hCG tests. The pattern of excretion of hCG in urine throughout pregnancy is well established.
  • the hCG is measured using blood, serum, plasma, urine, and/or saliva.
  • the hCG e.g., ⁇ hCG
  • the hCG is optimized for distribution in body fluids (e.g., decreased or increased excretion, and increased stability).
  • the hCG is optimized for distribution in the blood.
  • the hCG is optimized for distribution in the urine.
  • the hCG is optimized for increased excretion (e.g., in urine or feces).
  • the hCG (e.g., ⁇ hCG) is optimized for decreased excretion, leading to accumulation of the hCG in the blood.
  • the hCG is optimized for increased stability (e.g., improved protein folding and/or stability against proteolysis) leading to increased accumulation.
  • the hCG (e.g., ⁇ hCG) is optimized for increased stability and decreased excretion, leading to accumulation in the blood.
  • the hCG (e.g., ⁇ hCG) has increased stability and increased excretion, leading to accumulation in the urine.
  • Attorney Docket No. MMS.003WO the hCG is measured using one anti-hCG antibody, or a fragment thereof.
  • the hCG is measured using two anti-hCG antibodies, or fragment(s) thereof. In some embodiments, the two anti-hCG antibodies, or fragment(s) thereof bind different epitopes. In some embodiments, the hCG is measured using an immunometric assay such as an ELISA. In some embodiments, the hCG is measured using an ELISA selected from direct ELISA, indirect ELISA, sandwich ELISA, or competitive ELISA. In some embodiments, a commercially available hCG detection kit is used.
  • a method for detecting diseased epithelial tissue includes: (i) administering to the target epithelial tissue (such as target tissue of the GI tract) of a subject in need thereof, a genetically engineered microorganism of any of the embodiments disclosed herein including an exogenous gene encoding an hCG or a fragment thereof; (ii) obtaining a biological sample from the subject selected from blood, serum, plasma, urine, or saliva; and (iii) measuring the hCG or a fragment thereof in the biological sample obtained from the subject.
  • the detection is performed using an agglutination assay, chemiluminescence, colorimetry, fluorimetry, a quantitative immunoassay, a dipstick assay, lateral flow immunoassay, ELISA, or a combination of any two or more thereof.
  • additional testing e.g., colonoscopy
  • the expression of hCG or a fragment thereof is detected in a biopsy sample using a technique selected from RT- PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH.
  • the expression of hCG is used for localizing diseased epithelial or GI tissue and/or epithelium of the ducts that are connected with GI (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct).
  • the secretable biomarker is an alkaline phosphatase.
  • the alkaline phosphatase is secreted alkaline phosphatase (SEAP). Alkaline phosphatases are normally membrane-bound, thus not secreted.
  • the alkaline phosphatase e.g., SEAP
  • the secreted alkaline phosphatase is measured using an enzymatic assay.
  • a chromogenic, fluorogenic, luminogenic and/or substrate is used in the assay.
  • Illustrative chromogenic alkaline phosphatase substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate Attorney Docket No. MMS.003WO (pNPP)), and 4-Chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphthoic acid 2,4- dimethylanilide phosphate.
  • BCIP 5-bromo-4-chloro-3-indolyl phosphate
  • pNPP p-nitrophenyl phosphate
  • 4-Chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphthoic acid 2,4- dimethylanilide phosphate 4-Chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphthoic acid 2,4- dimethylanilide phosphate.
  • Illustrative luminogenic alkaline phosphatase substrates include, but are not limited to, dioxetane phosphates (e.g., adamantyl dioxetane phenyl phosphate and chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD)).
  • Illustrative fluorogenic alkaline phosphatase substrates include, but are not limited to, 4-Methylumbelliferyl phosphate disodium salt (MUP), and 6,8-difluoro-7-hydroxy-4-methylcoumarin phosphate.
  • a substrate selected from p-nitrophenyl phosphate (pNPP), 4-Methylumbelliferyl phosphate disodium salt (MUP), or chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) is used to detect the alkaline phosphatase (e.g., SEAP).
  • the enzymatic assay uses a colorimetric, fluorometric and/or chemiluminescent readout.
  • pNPP is used as the substrate and the alkaline phosphatase (e.g., SEAP) is assayed from a biological sample, using a colorimetric readout.
  • MUP 4-methylumbelliferyl phosphate disodium salt
  • SEAP alkaline phosphatase
  • CSPD chloro-5-substituted adamantyl-1,2- dioxetane phosphate
  • SEAP alkaline phosphatase
  • a method for detecting diseased epithelial or GI tissue and and/or diseased epithelium of the ducts that are connected with gastrointestinal tissue includes: (i) administering to target epithelial cells (such as target cells of the GI tract) of a subject in need thereof, a genetically engineered microorganism of any of the embodiments disclosed herein including an exogenous gene encoding an alkaline phosphatase; (ii) obtaining a biological sample from the subject selected from blood, serum, or plasma; and (iii) measuring the alkaline phosphatase in the biological sample obtained from the subject.
  • the detection is performed using chemiluminescence, colorimetry, fluorimetry, a quantitative immunoassay, lateral flow immunoassay, ELISA, or a combination of any two or more thereof.
  • additional testing e.g., colonoscopy
  • the expression of the alkaline phosphatase is detected in a biopsy sample using a technique selected from RT-PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH.
  • the expression of the alkaline phosphatase is used for localizing diseased epithelial or GI tissue Attorney Docket No.
  • the secretable biomarker is marker is a luciferase.
  • the luciferase is Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof.
  • Gluc is naturally secreted from mammalian cells in an active form. Moreover, Gluc is excreted at least in urine.
  • the Gluc may be measured using blood, serum, plasma, urine, and/or saliva.
  • the luciferase is Gaussia luciferase (Gluc) or a derivative thereof.
  • the luciferase is measured using an enzymatic assay.
  • the luciferase is measured using a luciferin as a substrate.
  • the luciferin may be firefly luciferin, Latia luciferin, bacterial luciferin, dinoflagellate luciferin, vargulin, or foxfire.
  • ATP may be a cofactor.
  • the luciferin is coelenterazine. In some embodiments, the luciferase is measured using a luminescence readout.
  • the biological sample is selected from blood, urine, or saliva.
  • Gaussia luciferase (Gluc) is a small luciferase with numerous disulfide bonds. Gluc is stable in serum, however, it is not active in E. coli cytoplasm because the bacterial environment does not allow the disulfide bonds to form. Accordingly, in some embodiments, expression of Gluc is indicative of production and secretion of Gluc by diseased human cells.
  • the gene encoding Gluc comprises an intron to ensure production and secretion of Gluc by diseased human cells.
  • the Gluc does not require any cofactors for activity (e.g., ATP) and catalyzes the oxidation of the substrate coelenterazine in a reaction that leads to emission of blue light (480 nm). Accordingly, in some embodiments, the Gluc may be measured using luminescence. In other embodiments, the Gluc may be measured using an immunoassay such as a quantitative immunoassay, a lateral flow immunoassay, or an ELISA. In some embodiments, the Gluc is optimized for distribution in body fluids (e.g., decreased or increased excretion, and increased stability). In some embodiments, the Gluc is optimized for distribution in the blood. In some embodiments, the Gluc is optimized for distribution in the urine.
  • the Gluc is optimized for increased excretion (e.g., in urine or feces). In some embodiments, the Gluc is optimized for decreased excretion, leading to accumulation of the Gluc in the blood. In some embodiments, Attorney Docket No. MMS.003WO the Gluc is optimized for increased stability (e.g., improved protein folding and/or stability against proteolysis) leading to increased accumulation. In some embodiments, the Gluc is optimized for increased stability and decreased excretion, leading to accumulation in the blood. In some embodiments, the Gluc has increased stability and increased excretion, leading to accumulation in the urine.
  • the Gluc has increased stability and increased excretion, leading to accumulation in the urine.
  • a method for detecting diseased epithelial or GI tissue and/or diseased epithelium of the ducts that are connected with gastrointestinal tissue includes: (i) administering to target epithelial cells (such as target cells of the GI tract) of a subject in need thereof, a genetically engineered microorganism of any of the embodiments disclosed herein comprising an exogenous gene encoding an Gluc or a fragment thereof; (ii) obtaining a biological sample from the subject selected from blood, serum, plasma, urine, or saliva; and (iii) measuring the Gluc or a fragment thereof in the biological sample obtained from the subject.
  • target epithelial cells such as target cells of the GI tract
  • the detection is performed using a luminescence assay.
  • additional testing e.g., colonoscopy
  • the expression of Gluc or a fragment thereof is detected based on luminescence, during colonoscopy.
  • coelenterazine may be administered to the subject before or during colonoscopy.
  • the expression of Gluc or a fragment thereof may be detected in a biopsy sample using a technique selected from RT-PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH.
  • the expression of Gluc is used for localizing diseased GI tissue and/or diseased epithelium of the ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct).
  • the secretable biomarker is human carcinoembryonic antigen (CEA). CEA is naturally expressed in some normal adult tissues, and in tumors expressing the same self-antigen.
  • the secretable biomarker is colon cancer secreted protein 2 (CCSP2).
  • CCSP2 expression is generally absent in normal body tissues, but is induced in colon adenomas and colon cancers.
  • the secretable biomarker is cathepsin B.
  • Cathepsin B belongs to a family of lysosomal cysteine proteases and plays an important role in intracellular proteolysis. It is found both in serum and urine. Attorney Docket No. MMS.003WO
  • the secretable biomarker is secretable by epithelial cells of GI tissue. Accordingly, in such embodiments, the secretable biomarker can be synthesized and sorted in the secretory pathway in mammalian cells that express the secretable biomarker.
  • the secretable biomarker includes an ER signal sequence, optionally located at or near the N-terminus.
  • the ER signal directs the ribosomes that are synthesizing the secretable biomarker to the rough ER.
  • the secretable biomarker includes a native signal peptide. Additionally or alternatively, in some embodiments, the secretable biomarker includes a heterologous signal sequence. In some embodiments, the heterologous signal sequence is selected from the signal sequence of interleukin-2, CD5, the Immunoglobulin Kappa light chain, trypsinogen, serum albumin, or prolactin.
  • the Gaussia luciferase (Gluc) comprises a native signal sequence. Additionally or alternatively, the Gaussia luciferase (Gluc) comprises a heterologous signal sequence.
  • the ⁇ hCG comprises a native signal sequence. Additionally or alternatively, the ⁇ hCG comprises a heterologous signal sequence.
  • the genetically engineered microorganism delivers one or more payload genes encoding at least one secretable biomarker to the target cells (e.g., diseased epithelial cells), the target cells express the secretable biomarker, and the target cells are detected based on presence of the secretable biomarker.
  • the payload gene(s) can include one or more introns such that the secretable biomarker is expressed in a detectable form only in the target cells, as described herein.
  • the secretable biomarker is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay.
  • the secretable biomarker is measured by using an enzymatic assay (e.g., where the secretable biomarker has enzymatic activity) or an immunoassay.
  • the secretable biomarker can be measured in the biological sample using an assay selected from agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, CD, chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, fluorimetry, flow cytometry, electrophoretic assay, enzymatic assay, ELISA, gas chromatography, gravimetry, HPLC, immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, or a combination of any two or more thereof.
  • an assay selected from agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, CD, chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, fluorimetry, flow cytometry, electrophoretic assay, enzymatic as
  • the expression of the secretable biomarker is detected in a biopsy sample using a technique selected from RT-PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH.
  • the expression of the secretable biomarker is used for localizing diseased epithelial or GI tissue and/or epithelium of the ducts that are connected with GI tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct).
  • the detection marker is an MRI contrast agent.
  • the MRI contrast agent can be a protein or peptide that causes the accumulation of a magnetic responsive atom, such as transition metal ions (e.g., Cu 2+ , Fe 2+ /Fe 3+ , Co 2+ , and Mn 2+ ), or lanthanide metal ions (e.g., Eu 3+ , Gd 3+ , Ho 3+ , and Dy 3+ ).
  • transition metal ions e.g., Cu 2+ , Fe 2+ /Fe 3+ , Co 2+ , and Mn 2+
  • lanthanide metal ions e.g., Eu 3+ , Gd 3+ , Ho 3+ , and Dy 3+
  • the MRI contrast agent can cause sequestration or chelation of metal ions (e.g., Fe 3+ ) or can catalyze a biochemical reaction that leads to change in accumulation of ions (e.g., cleavage of a caged synthetic Gd 3+ compound), and thereby allow the detection of the target cells via MRI.
  • metal ions e.g., Fe 3+
  • the MRI contrast agent can cause sequestration or chelation of metal ions (e.g., Fe 3+ ) or can catalyze a biochemical reaction that leads to change in accumulation of ions (e.g., cleavage of a caged synthetic Gd 3+ compound), and thereby allow the detection of the target cells via MRI.
  • the MRI contrast agent can be selected from ferritin, transferrin receptor-1 (TfR1), Tyrosinase (TYR), beta-galactosidase, manganese-binding protein MntR, creatine kinase (CK), Magnetospirillum magnetotacticum magA, divalent metal transporter DMT1, protamine-1 (hPRM1), urea transporter (UT-B), and ferritin receptor Timd2 (T-cell immunoglobulin and mucin domain containing protein 2), sodium iodide symporter, E. coli dihydrofolate reductase, or norepinephrine transporter, or active variants thereof.
  • TfR1 transferrin receptor-1
  • TYR Tyrosinase
  • beta-galactosidase manganese-binding protein MntR
  • CK creatine kinase
  • Magnetospirillum magnetotacticum magA divalent metal transport
  • the genetically engineered microorganism delivers one or more payload genes encoding at least one MRI contrast agent to the target cells (e.g., diseased epithelial cells), the target cells express the MRI contrast agent, and the target cells are detected based on presence of the MRI contrast agent.
  • the payload gene(s) can include one or more introns such that the MRI contrast agent is expressed in a detectable form (e.g., a form that causes the accumulation of magnetic responsive atoms) only in the target cells, as described herein.
  • the detection of the target cells is performed using an MRI procedure.
  • the MRI procedure is noninvasive.
  • a substrate of the MRI contrast agent is administered during and/or before the MRI procedure.
  • the substrate of the MRI contrast agent is a source of magnetic responsive atoms, which can be accumulated by the MRI contrast agent within or on the surface of the target cells.
  • a caged synthetic Gd 3+ compound comprising a galactoside may be administered when the MRI contrast agent is beta-galactosidase.
  • the administration of the substrate of the MRI contrast agent may be started prior Attorney Docket No. MMS.003WO to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection.
  • the detection marker is a PET reporter.
  • the PET reporter can be a protein or peptide that causes the accumulation of a PET probe (e.g., a positron emitting radioisotope) in or on the surface of diseased epithelial cells.
  • the PET probe causes the accumulation of the PET probe through binding to a receptor, antibody, an enzyme, or a cellular transport mechanism.
  • Illustrative PET probes include [ 18 F]FHBG, [ 18 F]FEAU, [ 124 I]FIAU, [ 18 F or 11 C]BCNA, [ 11 C] ⁇ -galactosyl triazoles, [ 18 F]L- FMAU, [ 18 F]FESP, [ 11 C]Raclopride, [ 11 C]N-methylspiperone, [ 18 F]FES, 68 Ga-DOTATOC, [ 18 F]fluoropropyl-trimethoprim, Na 124 I, or a 225 Ac-DOTA chelate.
  • the PET reporter is selected from thymidine kinase, deoxycytidine kinase, Dopamine 2 Receptor, estrogen receptor ⁇ surface protein binding domain, somatostatin receptor subtype 2, carcinoembryonic antigen, a sodium iodide symporter, a single-chain antibody specific to 1,4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (DOTA), or E. coli dihydrofolate reductase, or a variant thereof.
  • DOTA 1,4,7,10-tetraazacyclododecane-1
  • DOTA 4,7,10-tetraacetic acid
  • E. coli dihydrofolate reductase E. coli dihydrofolate reductase
  • the genetically engineered microorganism delivers one or more payload genes encoding at least one PET reporter to the target cells (e.g., diseased epithelial cells), the target cells express the PET reporter, and the target cells are detected based on presence of the PET reporter.
  • the payload gene(s) can include one or more introns such that the PET reporter is expressed in a detectable form (e.g., a form that causes the accumulation of a PET probe) only in the target cells, as described herein.
  • the detection of the target cells is performed using a PET imaging procedure.
  • one or more PET probes are administered during and/or before the PET imaging procedure.
  • the administration of the PET probe may be started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection.
  • the detection marker is a SPECT reporter.
  • the SPECT reporter can be a protein or peptide that causes the accumulation of a SPECT probe (e.g., a gamma-ray emitting radioisotope) in or on the surface of the target cells.
  • the SPECT reporter causes the accumulation of the SPECT probe through binding to a receptor, antibody, an enzyme, or a cellular transport mechanism.
  • MMS.003WO SPECT probes include Sodium pertechnetate ( [ 99 mTc]NaTcO4), Na 123 I, Na 125 I, Na 131 I, [ 123 I]- NKJ64, [125I]-NKJ64, [ 131 I]-(R)-N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine, [ 123 I]- NKJ64, [125I]-(R)-N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine, [ 131 I]-(R)-N-methyl-3- (2-iodophenoxy)-3-phenylpropanamine, [ 123 I] ⁇ -CIT (2 ⁇ -carbomethoxy-3 ⁇ -(4- iodophenyl)tropane), [ 125 I] ⁇ -CIT (2 ⁇ -carbomethoxy-3 ⁇ -(4-iodophenyl)tropane), [ 131 I] ⁇ -
  • the SPECT reporter is selected from sodium ion symporter, norepinephrine transporter, sodium iodide symporter, dopamine receptor, or dopamine transporter. Accordingly, in some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one SPECT reporter to the target cells (e.g., diseased epithelial cells), the target cells express the SPECT reporter, and the target cells are detected based on presence of the SPECT reporter.
  • the target cells e.g., diseased epithelial cells
  • the payload gene(s) can include one or more introns such that the SPECT reporter is expressed in a detectable form (e.g., a form that causes the accumulation of a SPECT probe) only in the target cells, as described herein.
  • the detection of the target cells is performed using a SPECT imaging procedure.
  • one or more SPECT probes are administered during and/or before the SPECT imaging procedure.
  • the administration of the SPECT probe may be started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection.
  • the detection marker is a photoacoustic reporter.
  • the genetically engineered microorganism delivers a one or more nucleic acid(s) encoding at least one photoacoustic reporter to target cells (e.g., diseased epithelial cells).
  • target cells e.g., diseased epithelial cells
  • the detection of the target cells is performed using an endoscopic procedure, or colonoscopic procedure.
  • Illustrative photoacoustic reporters include any fluorescent proteins disclosed herein. Attorney Docket No. MMS.003WO Representative examples of detection markers and their associated polynucleotide sequences are provided in Table 5 below.
  • the genetically engineered microorganism produces a detection marker having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 9.
  • the genetically engineered microorganism includes a detection marker of SEQ ID NO: 9. Additional details and examples of detection markers are disclosed in International Publication Nos. WO 2021/247480 and WO 2022/261290, the disclosures of which are incorporated by reference herein in their entirety. 2.
  • the detection marker is or includes at least one RNA molecule.
  • the at least one RNA molecule can include a messenger RNA (mRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a catalytic RNA (e.g., a ribozyme), a non- coding RNA, a riboswitch, or a combination thereof.
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • catalytic RNA e.g., a ribozyme
  • non- coding RNA e.g., a riboswitch, or a combination thereof.
  • the RNA molecule is a naturally occurring RNA molecule or a derivative thereof.
  • the naturally occurring RNA molecule can be any RNA molecule that is not endogenously produced by the cells of the subject (e.g., by target cells and/or non-target cells), such as an RNA molecule that is endogenously produced by a different type of organism than the subject (e.g., a non- mammalian organism).
  • the RNA molecule can include a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to a naturally occurring RNA molecule.
  • the RNA molecule can be an artificial RNA molecule (also known as a synthetic RNA molecule).
  • RNA molecules can be generated in various ways, such as by modifying the sequence of a naturally occurring Attorney Docket No. MMS.003WO RNA molecule (e.g., to increase stability, spliceability) or via de novo design.
  • a library of artificial RNA molecules is generated and screened for the desired activity.
  • the RNA molecule serves as the sole detection marker for determining the presence or absence of target cells in the subject. In other embodiments, however, the RNA molecule can be used in combination with another detection marker for determining the presence or absence of target cells in the subject, such as a protein or peptide encoded by the RNA molecule, or a different protein or peptide.
  • the RNA molecule that serves as the detection marker is a mRNA encoding a protein or peptide.
  • the protein or peptide can be any protein or peptide that is not endogenously produced by the cells of the subject (e.g., by target cells and/or by non-target cells within the subject).
  • the protein or peptide can be a naturally occurring protein or peptide that is endogenously produced by a different type of organism than the subject (e.g., a non-mammalian organism), or can be a derivative of a naturally occurring protein or peptide.
  • the protein or peptide can be a synthetic protein or peptide.
  • the protein or peptide produced by the mRNA can optionally also serve as a detection marker, in addition or alternatively to the mRNA itself.
  • the protein or peptide can be an enzyme, a hormone, an antigen, a fluorescent molecule, a bioluminescent molecule, etc., that is capable of being detected via any of the detection techniques provided herein.
  • the mRNA encodes for one or more of the following: a fluorescent marker, a bioluminescent marker, a CT contrast agent, an MRI contrast agent, a Positron Emission Tomography (PET) reporter, a Single Photon Emission Computed Tomography (SPECT) reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination thereof.
  • PET Positron Emission Tomography
  • SPECT Single Photon Emission Computed Tomography
  • an enzyme reporter e.g., a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination thereof.
  • the mRNA encodes for a fluorescent protein selected from GFP, RFP, YFP, Sirius, Sandercyanin, shBFP-N158S/L173I, Azurite, EBFP2, mKalama1, mTagBFP2, TagBFP, shBFP, ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquoise, mTurquoise2, TagCFP, mTFP1, monomeric Midoriishi-Cyan, Aquamarine, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, Monomeric Azami Green, mWasabi, Clover, mNeonGreen, NowGFP, mClover3, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, lanRFP- ⁇ S83, mPapaya1, mCyRFP1, Monomeric Kusabir
  • the mRNA encodes for a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713, iRFP720, or iSplit.
  • the near-infrared fluorescent protein is iRFP670, which requires biliverdin to fluoresce. In embodiments where the genetically engineered microorganism does not make biliverdin, iRFP670 fluorescence provides evidence that the iRFP670 was expressed in mammalian cells.
  • the mRNA encodes for a bioluminescent protein selected from a Ca +2 regulated photoprotein (e.g., aequorin, symplectin, Mitrocoma photoprotein, Clytia photoprotein, and Obelia photoprotein), North American firefly luciferase, Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina luciferase, Metridina luciferase, Metrida luciferase, OLuc protein, red firefly luciferase, bacterial luciferase, or active variants thereof.
  • a Ca +2 regulated photoprotein e.g., aequorin, s
  • the RNA molecule is a catalytic RNA molecule that catalyzes a specific biochemical reaction, such as a ribozyme.
  • ribozymes include, but are not limited to, hammerhead ribozyme, hepatitis delta virus (HDV) ribozyme, HDV-like ribozyme, hairpin ribozyme, Varkud satellite (VS) ribozyme, glmS riboswitch, twister ribozyme, twister sister ribozyme, hatchet ribozyme, pistol ribozyme, Hovlinc ribozyme, Long Interpersed Nuclear Element-1 (LINE-1) ribozyme, beta-globin co-transcriptional cleavage ribozyme (CoTC ribozyme), Vg1 ribozyme, and protein-responsive ribozymes.
  • HDV hepatitis delta virus
  • VS Varkud satellite
  • ribozyme glmS riboswitch
  • twister ribozyme twister sister ribozyme
  • the RNA molecule includes one or more features to improve in vivo stability.
  • the RNA molecule can be a circular RNA, which may be more stable than linear RNAs.
  • the RNA molecule can have a secondary Attorney Docket No. MMS.003WO structure that is associated with enhanced longevity in vivo, such as one or more hairpins, long range interactions, G-quadruplexes, or pseudoknots, or combinations thereof. Hairpins, also known as stem-loop structures, can include a hybridized stem and a single stranded loop, and can optionally contain mismatches and/or bulges.
  • Pseudoknots can include nested stem-loop structures, with half of one stem intercalated between the two halves of another stem.
  • G- quadruplexes can be composed of guanine-rich sequences and can be formed from G-quartets composed of four guanine nucleotides that interact with each other through Hoogsteen hydrogen bonding.
  • the RNA molecule can include a 5’ hairpin structure and/or 3’ poly A tail to increase stability.
  • the RNA molecule can bind to a stabilization factor, which can be a protein, peptide, nucleic acid, or combination thereof.
  • the RNA molecule is capable of self-replicating and/or self-amplifying.
  • the payload gene encoding the RNA molecule can be present in a cassette that directs RNA self-amplification of the cassette in target cells.
  • the cassette includes an RNA viral genome replication apparatus, such an RNA- dependent RNA polymerase (RdRP).
  • RdRP is an alphavirus RdRP.
  • the alphavirus RdRP includes nsp1, nsp2, nsp3, and/or nsp4.
  • RNA molecules described herein can have any suitable length, such as a length within a range from 100 nt to 20 knt, 100 nt to 10 knt, 100 nt to 5 knt, 100 nt to 1 knt, 100 nt to 500 nt, 100 nt to 200 nt, 200 nt to 20 knt, 200 nt to 10 knt, 200 nt to 5 knt, 200 nt to 1 knt, 200 nt to 500 nt, 500 nt to 20 knt, 500 nt to 10 knt, 500 nt to 5 knt, 500 nt to 1 knt, 1 knt to 20 knt, 1 knt to 10 knt, 1 knt to 5 knt, 5 knt to 10 knt, or 10 knt to 20 k
  • an RNA molecule can have a length of at least 50 nt, 100 nt, 500 nt, 1 knt, 2 knt, 5 knt, or 10 knt; and/or a length of no more than 20 knt, 15 knt, 10 knt, 5 knt, 2 knt, 1 knt, or 500 nt.
  • the RNA molecule has at least two forms, including a first form when expressed by a target cell and a second, different from when expressed by a non-target cell (e.g., the genetically engineered microorganism).
  • the RNA molecule can be encoded by a DNA sequence including one or more introns (e.g., spliceosomal introns) that are spliced out in the RNA molecule to produce spliced RNA and/or different RNA splice variants, as described herein.
  • introns e.g., spliceosomal introns
  • the first form of the RNA molecule that is produced by the target cell e.g., a spliced RNA or first splice variant
  • the second form of the RNA molecule that is produced by the non-target cell e.g., an unspliced RNA or second splice Attorney Docket No. MMS.003WO variant
  • the first form of the RNA molecule that is produced by the target cell is a functional form of a catalytic RNA
  • the second form of the RNA molecule that is produced by the nontarget cell is a nonfunctional form of the catalytic RNA
  • the first form of the RNA molecule that is produced by the target cell can have improved stability compared to the second form of the RNA molecule that is produced by the nontarget cell.
  • the RNA molecule is detected using RT-PCR, multiplex RT-PCR, qPCR, RT-qPCR, dPCR, ddPCR, semi-quantitative PCR, semi-quantitative RT- PCR, gel electrophoresis, RNA sequencing, FISH (e.g., RNA-FISH), CISH, or suitable combinations thereof.
  • the RNA molecule can be detected in a biological sample from the subject using a whole RNA extraction and qPCR sampling technique.
  • the biological sample can include, for example, feces, blood, serum, plasma, mucus, urine, and/or saliva.
  • the RNA molecule is present within cells in the biological sample.
  • the total RNA can be extracted from the cells in the biological sample by disrupting the biological sample (e.g., via vortexing, centrifugation, buffers, surfactants) and/or lysing the cells (e.g., via a lysis buffer).
  • the RNA can then be converted to cDNA using suitable primers, such as random hexamers to reverse transcribe all RNA, an oligo dT primer to selectively reverse transcribe mammalian RNA only, or with primers specific to the gene encoded by the RNA (e.g., the payload gene).
  • an enrichment step can be performed before cDNA conversion using techniques such as bead-based pull down for either mammalian RNA (e.g., using oligo dT probes) or specific RNA (e.g., using gene-specific probes).
  • mammalian RNA e.g., using oligo dT probes
  • specific RNA e.g., using gene-specific probes.
  • the cDNA can then be quantified using qPCR.
  • qPCR is performed using primers that are capable of hybridizing and amplifying cDNA produced from a first form of the RNA molecule produced by target cells (e.g., a spliced RNA molecule, a first splice variant), but are not capable of hybridizing and amplifying cDNA produced from a second form of the RNA molecule produced by non-target cells (e.g., an unspliced RNA molecule, a second splice variant).
  • the first form of the RNA can include a first exon directly ligated to a second exon
  • the second form of the RNA molecule can include an intron interposed between the first and second exons.
  • the qPCR primer can be designed to hybridize to the portion of the corresponding cDNA at the interface between the first and second exons, e.g., the 5’ region of the primer hybridizes to the first exon in the cDNA and the 3’ region of the primer hybridizes to the second exon, or vice-versa.
  • the number of nucleotides in the 5’ region Attorney Docket No. MMS.003WO and the 3’ region can be selected so that if the intron is present in the cDNA, either the 5’ region or the 3’ region of the primer will fail to hybridize to the cDNA.
  • the 5’ region of the primer includes at least one, two, three, four, five, six, seven, eight, nine, or 10 nucleotides that hybridize to the first exon; and/or the 3’ region of the primer includes at least one, two, three, four, five, six, seven, eight, nine, or 10 nucleotides that hybridize to the second exon.
  • the RNA molecule is a catalytic RNA (e.g., a ribozyme)
  • the catalytic RNA can be detected using an enzymatic assay.
  • the catalytic RNA can be extracted from a biological sample and/or enriched as described above.
  • the presence of the catalytic RNA can then be measured, e.g., by combining the catalytic RNA with a substrate and quantifying the amount of RNA based on the amount of converted substrate.
  • the in vivo activity of the catalytic RNA molecule can create products (e.g., nucleic acid fragments) that can be detected in a biological sample from the subject using RT-PCR, multiplex RT-PCR, qPCR, RT-qPCR, dPCR, ddPCR, semi- quantitative PCR, semi-quantitative RT-PCR, gel electrophoresis, RNA sequencing, and/or any of the other techniques described herein.
  • the genetically engineered microorganisms of the present disclosure deliver a nucleic acid to target cells (e.g., diseased and/or abnormal cells).
  • the nucleic acid can be a DNA molecule (e.g., a plasmid DNA, also referred to herein as a payload plasmid).
  • the payload plasmid is present in multiple copies (ranging from about 1 to about 300 copies, from about 20 to about 50 copies, from about 2 to about 10 copies, or from about 5 to about 10 copies) per cell, or is a single copy plasmid. Copy number may depend on the particular genetic characteristics of the plasmid.
  • the DNA molecule (e.g., payload plasmid) harbors one or more payload genes encoding the gene products described herein (e.g., therapeutic agents, such as prodrug converting enzymes, and/or detection markers).
  • the DNA molecule can also include one or more sequence elements operably linked to the payload genes that control the expression of the gene products.
  • the sequence element(s) may control and regulate the transcription, transcript stability, translation, protein stability, cellular localization, and/or secretion of the gene products.
  • the sequence element(s) may prevent expression of the gene product by the genetically engineered microorganism.
  • MMS.003WO sequence element(s) may allow expression (e.g., transcription and/or translation) of the gene product by the genetically engineered microorganism.
  • the gene products are expressed in one form by the target cell and in another form by the genetically engineered microorganism, as described herein.
  • the one or more payload genes are operably linked to a mammalian promoter.
  • the one or more payload genes include at least one microbial repressor binding site to inhibit bacterial transcription.
  • the one or more payload genes include microbial transcription terminators.
  • the one or more payload genes include at least one intron, as described herein.
  • the one or more payload genes include codon usage optimized for mammalian expression.
  • certain optional sequence elements present in the payload gene e.g., mammalian promoters, microbial repressor binding sites (e.g., operators), internal ribosome entry sites
  • the use of introns that are not spliced by genetically engineered microorganism allows for different forms of the gene product to differentiate between expression by the mammalian cells versus background expression by the genetically engineered microorganism (e.g., due to leaky expression, background activity of mammalian promoters in the microorganism). Therefore, in some embodiments, the presence of a particular form of the gene product provides a true readout of the presence of the target cells (e.g., diseased epithelial cells), with little or no background expression in the genetically engineered microorganism. Accordingly, in some embodiments, the one or more payload genes may be operably linked to a mammalian promoter.
  • the mammalian promoter directs expression in the specific target cell type, such as GI tract epithelial cell-specific expression.
  • suitable mammalian promoters that direct GI tract epithelial cell-specific expression are MUC2 gene promoter, T3 b gene promoter, intestinal fatty acid binding protein gene promoter, lysozyme gene promoter, and villin gene promoter.
  • the mammalian promoter directs an inducible GI tract epithelial cell- specific expression.
  • an illustrative example of a suitable inducible mammalian promoter includes a cytochrome P450 promoter element that is transcriptionally up-regulated in response to a lipophilic xenobiotic such as ⁇ -napthoflavone.
  • the inducible mammalian promoter may be regulated by tetracycline, cumate, or an estrogen.
  • the inducible mammalian promoter may be a Tet-On or Tet-Off promoter.
  • the one or more payload genes may be inducible and/or repressible, and optionally controlled by delivering the inducer or repressor to the subject.
  • the microbial repressor binding site(s), which are optionally present in the one or more payload genes, may repress the expression of the payload genes in bacteria, while exerting little or no repressive effect in mammalian cells.
  • the repressor sequence may be selected from one or more lac operator(s), one or more ara operator(s), one or more trp operator(s), one or more SOS operator(s), one or more integration host factor (IHF) binding sites, one or more histone-like protein HU binding sites, or a combination of two or more thereof.
  • the microbial transcription termination site(s) can cause premature termination of the transcription of the one or more payload genes in the genetically engineered microorganism, without causing premature termination of the transcription of the one or more payload genes in mammalian cells.
  • the one or more payload genes include a rho-independent microbial transcription termination site.
  • the one or more payload genes include a 5’ untranslated region, and the 5’ untranslated region includes a rho-independent microbial transcription termination site.
  • the rho-independent microbial transcription termination site includes a short hairpin followed by a run of 4–8 Ts (e.g., TTTTTT and TTTTT).
  • Illustrative rho-independent microbial transcription termination sites are T7 terminator, rrnB terminator, and T0 terminator.
  • the DNA molecule e.g., payload plasmid
  • the DNA molecule includes at least one binding site for a DNA binding protein.
  • the at least one binding site for a DNA binding protein forms an array of multiple adjacent binding sites for the DNA binding protein.
  • the DNA binding protein includes one or more nuclear localization signals (NLS).
  • the DNA binding protein binds the DNA molecule (e.g., a plasmid) and promotes the nuclear translocation of the DNA molecule (e.g., a plasmid) via the one or more NLS.
  • the NLS is SV40 T antigen NLS sequence (KKKRKV).
  • the DNA binding protein is NF ⁇ B.
  • the genetically engineered microorganism includes a gene encoding the DNA binding protein having the one or more NLS. Without being bound by theory, it is believed that the DNA binding protein comprising one or more NLS binds the at least one binding site for the DNA binding protein on the plasmid and promotes nuclear translocation of the plasmid via the action of the one or more NLS. Thus, in these embodiments, the target cells express the Attorney Docket No. MMS.003WO payload genes from the DNA molecule delivered by the microorganism.
  • the gene encoding the DNA binding protein is genomically integrated, is present on a payload plasmid harboring one or more payload genes, or is present on another plasmid.
  • the genetically engineered microorganisms of the present disclosure deliver an RNA molecule (e.g., an mRNA molecule) encoding the gene products described herein (e.g., therapeutic agents, such as prodrug converting enzymes, and/or detection markers).
  • the payload gene(s) encoding the gene product may be operably linked to a microbial promoter (e.g., a proD promoter).
  • the genetically engineered microorganism delivers an mRNA molecule encoding the gene products to the cytoplasm of the target cells, which are capable of translating the mRNA to produce the encoded gene products.
  • the payload gene(s) encoding the gene products includes an internal ribosome entry site(s) (IRES).
  • the IRES can promote translation of the mRNA molecule delivered by the genetically engineered microorganism in the target cell.
  • the mRNA sequence that is delivered includes an element that imparts stability on the mRNA molecule.
  • Non-limiting examples of the elements that impart stability on the mRNA molecule include 5’ hairpin structures and 3’poly A tails.
  • a genetic change in the genetically engineered microorganism can improve stability of the mRNA molecule.
  • the genetic change is a deletion of an RNase gene.
  • the RNase gene is RNase III (rnc).
  • the genetically engineered microorganism delivers one or more mRNA molecules encoding the therapeutic agent and/or detection marker to the target cell (e.g., diseased epithelial cells)
  • the one or more payload gene(s) encoding the RNAs are integrated in the genome of the genetically engineered microorganism.
  • the one or more payload gene(s) are present on a plasmid.
  • the payload gene(s) encoding the therapeutic agent (e.g., prodrug converting enzyme) and/or detection marker may be operably linked to a microbial promoter.
  • suitable microbial promoters include a natural promoter of any chromosomal gene, plasmid gene, or bacteriophage gene that functions in the genetically engineered microorganism (e.g., E. coli).
  • the microbial promoter is a synthetic promoter derived from a promoter consensus sequence.
  • the microbial promoter is a constitutive promoter.
  • the microbial promoter is an inducible promoter.
  • suitable inducible Attorney Docket No. MMS.003WO microbial promoter include the araBAD promoter and the lac promoter. Accordingly, in some embodiments, the payload gene(s) encoding the therapeutic agent and/or detection marker may be inducible and/or repressible, and optionally controlled by delivering the inducer and/or repressor to the subject.
  • the promoter is a T7 or T3 promoter, and the genetically engineered microorganism expresses a T7 or T3 RNA polymerase gene to support expression of the payload gene(s).
  • the genetically engineered microorganism expresses a T7 RNA polymerase (T7RNAP) encoded by a T7RNAP gene, and harbors one or more payload genes encoding the gene products described herein (e.g., therapeutic agents and/or detection markers) under the control of a T7 promoter.
  • T7RNAP is integrated on the bacterial chromosome.
  • the T7RNAP is present on a plasmid.
  • the T7RNAP is controlled by an inducible promoter (e.g., an araBAD or lacUV5 promoter).
  • the bacteria can express mRNA encoding the therapeutic agent and/or the detection marker.
  • the genetically engineered microorganism delivers mRNA encoding the therapeutic agent and/or the detection marker to the target cell (e.g., diseased epithelial cells).
  • the mRNA encoding the therapeutic agent and/or the detection marker that is delivered to the target cell can include an internal ribosome entry site.
  • the genetically engineered microorganism delivers the therapeutic agent and/or the detection marker to the target cell.
  • An internal ribosome entry site is an RNA element that allows for translation initiation in a cap-independent manner.
  • the IRES may be selected from an IRES from an encephalomyocarditis virus (EMCV), an IRES from a hepatitis C virus (HCV), or an IRES from a cricket paralysis virus (CrPV).
  • EMCV encephalomyocarditis virus
  • HCV hepatitis C virus
  • CrPV cricket paralysis virus
  • the IRES(s) present in the payload gene(s) encoding the therapeutic agent and/or detection marker allow for the production of the therapeutic agent and/or detection marker in mammalian cells using an mRNA produced in the genetically engineered microorganism.
  • One or more intron(s), which are optionally present in the payload gene(s) encoding the therapeutic agent and/or detection marker, can prevent the functional expression of the therapeutic agent and/or detection marker in bacteria, while allowing expression of payload gene(s) encoding the therapeutic agent and/or detection marker in mammalian cells, irrespective of whether the mRNA encoding the therapeutic agent and/or detection marker may be transcribed in the genetically engineered microorganism or a mammalian cell.
  • the intron may be a spliceosomal intron.
  • the intron creates a frameshift or a premature stop codon in an unspliced mRNA encoding the therapeutic agent and/or detection marker. Therefore, in some embodiments, the genetically engineered microorganism provides a true readout of the presence of target cells (e.g., diseased cells), without background expression of the detection marker in the genetically engineered microorganism. In some embodiments, the genetically engineered microorganism delivers to the target cells an RNA molecule that is capable of self-replicating and/or self-amplifying. In some embodiments, the payload gene(s) encoding the therapeutic agent and/or the detection marker are present in a cassette that directs RNA self-amplification of the cassette in the target cells.
  • the cassette comprises an RNA viral genome replication apparatus.
  • the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP).
  • the RdRP is an alphavirus RdRP.
  • the alphavirus RdRP includes nsp1, nsp2, nsp3, and/or nsp4.
  • the payload gene(s) encoding the therapeutic agent and/or the detection marker includes codon usage optimized for mammalian expression.
  • the one or more payload genes optionally further include a sequence element selected from Kozak sequences, 2A peptide sequences, mammalian transcription termination sequences, polyadenylation sequences (pA), leader sequences for protein secretion, or a combination of any two or more thereof.
  • the Kozak sequence is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts.
  • the Kozak sequence present in the one or more payload genes can improve correct translation initiation.
  • the Kozak sequence has the following nucleotide sequence: 5’-(GCC)GCCRCCAUGG-3’.
  • the 2A peptides when present, can function by preventing the synthesis of a peptide bond between the glycine and proline residues found at the end of the 2A peptides, and that the 2A peptides allow production of equimolar levels of multiple proteins from the same mRNA.
  • the 2A peptides become attached to C-terminus upstream protein, while the downstream protein starts with a proline.
  • the 2A peptide is selected from E2A ((GSG)QCTNYALLKLAGDVESNPGP), F2A ((GSG)VKQTLNFDLLKLAGDVESNP GP), P2A ((GSG)ATNFSLLKQAGDVEENPGP), or Attorney Docket No.
  • the GSG sequence (which is included in the parentheses) may be optionally present.
  • the polyadenylation sequences (pA) cause addition of a polyA tail to mRNA, which can be important for the nuclear export, translation, and stability of mRNA.
  • the mammalian transcription termination sequences can terminate transcription and promote the addition of polyA tail.
  • the one or more payload genes include a sequence element that is both a mammalian transcription termination sequence and a polyadenylation sequence, such as a SV40 terminator, hGH terminator, BGH terminator, or rbGlob terminator.
  • the one or more payload genes optionally include leader sequences for protein secretion. In some embodiments, the one or more payload genes optionally include upstream sequences for display of the corresponding protein or peptide on a mammalian cell surface.
  • the genetically engineered microorganisms of the present disclosure deliver a protein or peptide that is a therapeutic agent (e.g., a prodrug converting enzyme) and/or a detection marker.
  • the payload gene(s) encoding the therapeutic agent (e.g., prodrug converting enzyme) and/or detection marker may be operably linked to a microbial promoter (e.g., a proD promoter).
  • suitable microbial promoters include a natural promoter of any chromosomal gene, plasmid gene, or bacteriophage gene that functions in the genetically engineered microorganism (e.g., E. coli).
  • the microbial promoter is a synthetic promoter derived from a promoter consensus sequence.
  • the microbial promoter is a constitutive promoter.
  • the microbial promoter is an inducible promoter.
  • suitable inducible microbial promoters include the araBAD promoter and the lac promoter.
  • the payload gene(s) encoding the therapeutic agent and/or detection marker may be inducible and/or repressible, and optionally controlled by delivering the inducer and/or repressor to the subject.
  • the payload gene(s) can be present on a plasmid, can be integrated into a genomic site, or suitable combinations thereof. Representative examples of DNA constructs including a therapeutic agent, detection marker, and additional sequence elements are provided in Table 6 below. Attorney Docket No. MMS.003WO Table 6: DNA Constructs Attorney Docket No.
  • the genetically engineered microorganism includes a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 10.
  • the genetically engineered microorganism includes the sequence of SEQ ID NO: 10. IV. Compositions and Methods of Use
  • about 10 3 to about 10 11 viable genetically engineered microorganisms are administered to a subject, depending on the species of the subject (e.g., a human or non-human subject), as well as the disease or condition that is being diagnosed and/or treated.
  • about 10 5 to about 10 9 viable genetically engineered microorganisms of the present disclosure are administered to a subject.
  • the genetically engineered microorganisms of the present disclosure may be administered between 1 and about 50 times, e.g., prior to detection of an expressed marker.
  • the genetically engineered microorganisms may be administered from 1 to about 21 times, or from 1 to about 14 times, or from 1 to about 7 times, e.g., prior to the marker detection.
  • the genetically engineered microorganisms may be administered starting between about 1 hour to about 2 months prior to marker detection.
  • the administration of the genetically engineered microorganisms may be started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days.
  • the genetically engineered microorganisms of the present disclosure may be administered by any route as long as they are capable of invading their target cells upon Attorney Docket No. MMS.003WO administration and capable of delivery of their payload.
  • the payload that the genetically engineered microorganisms of the present disclosure deliver can include a nucleic acid molecule (e.g., plasmid DNA harboring one or more payload genes, introns, and/or additional sequence elements; or an mRNA harboring one or more payload genes, introns, and/or additional sequence elements), and/or can include a protein or peptide, as already described.
  • the genetically engineered microorganisms of the present technology are administered by an oral and/or rectal route.
  • the genetically engineered microorganisms of the present disclosure can be administered along with a pharmaceutically acceptable carrier and/or diluent.
  • “pharmaceutically acceptable” may refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject.
  • the particular pharmaceutically acceptable carrier and/or diluent employed can be varied as desired.
  • diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame.
  • carriers examples include proteins (e.g., as found in skim milk), sugars (e.g., sucrose), or polyvinylpyrrolidone.
  • the carriers can be used at any suitable concentration, such as a concentration within a range from 0.1% to 30% (w/v), or within a range from 1% to 10% (w/v).
  • the pharmaceutically acceptable carriers and/or diluents which may be used for delivery may depend on specific routes of administration. Any such carrier or diluent can be used for administration of the genetically engineered microorganisms of the present disclosure, so long as the genetically engineered microorganisms of the present disclosure are still capable of invading a target cell and delivering the payload that they carry to the target cells.
  • a pharmaceutical composition may be any combination of an active ingredient (e.g., the genetically engineered microorganisms) with a carrier, inert or active, that makes the composition suitable for diagnostic and/or therapeutic use.
  • the pharmaceutical compositions of the present disclosure can be formulated for oral and/or rectal administration. Lyophilized forms are also included, so long as the genetically engineered microorganisms are invasive and capable of delivering their payload upon contact Attorney Docket No. MMS.003WO with a target cell or upon administration to the subject.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients (e.g., carriers and/or diluents), such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate), and/or wetting agents (e.g., sodium lauryl sulphate).
  • pharmaceutically acceptable excipients e.g., carriers and/or diluents
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and/or preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily est
  • the preparations may also contain buffer salts, flavoring, coloring agents, and/or sweetening agents as appropriate.
  • the pharmaceutical compositions provided herein may be administered rectally in the forms of suppositories, pessaries, pastes, powders, creams, ointments, solutions, emulsions, suspensions, gels, foams, sprays, or enemas. These dosage forms can be manufactured using conventional processes as described in Remington: The Science and Practice of Pharmacy, supra. Rectal suppositories are solid bodies for insertion into rectum, which are solid at ordinary temperatures but melt or soften at body temperature to release the genetically engineered microorganisms of the present disclosure inside the rectum.
  • Pharmaceutically acceptable carriers utilized in rectal suppositories can include bases or vehicles, such as stiffening agents, which produce a melting point in the proximity of body temperature, when formulated with the pharmaceutical compositions provided herein; and antioxidants, including bisulfite and sodium metabisulfite.
  • Suitable vehicles include, but are not limited to, cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol), spermaceti, paraffin, white and yellow wax, appropriate mixtures of mono-, di- and triglycerides of fatty acids, or hydrogels (such as polyvinyl alcohol, hydroxyethyl methacrylate, polyacrylic acid, Attorney Docket No.
  • the genetically engineered microorganisms of the present disclosure are administered as a single composition, or are administered individually at the same or different times and via the same or different route (e.g., oral and rectal) of administration.
  • the genetically engineered microorganisms of the present disclosure are provided in a mixture or solution suitable for rectal instillation and that includes sodium thiosulfate, bismuth subgallate, vitamin E, and/or sodium cromolyn.
  • a pharmaceutical composition of the present disclosure includes, in a suppository form, butyrate, and glutathione monoester, glutathione diethylester or other glutathione ester derivatives.
  • the suppository can optionally include sodium thiosulfate and/or vitamin E.
  • the pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the genetically engineered microorganisms of the present disclosure are formulated as an enema formulation.
  • the enema formulation can include a reducing agent (or any other agent having a similar mode of action).
  • the enema formulation can optionally comprise polysorbate-80 (or any other suitable emulsifying agent), and/or any short chain fatty acid (e.g., a five, four, three, or two carbon fatty acid) as a colonic epithelial energy source, such as sodium butyrate (4 carbons), proprionate (3 carbons), acetate (2 carbons), etc., and/or any mast cell stabilizer, such as cromolyn sodium (GASTROCROM) or Nedocromil sodium (ALOCRIL).
  • the pharmaceutical composition includes from about 10 5 to about 10 9 viable genetically engineered microorganisms of the present disclosure.
  • the composition includes cromolyn sodium
  • the cromolyn sodium can be present in an amount from about 10 mg to about 200 mg, or from about 20 mg to about 100 mg, or from about 30 mg to about 70 mg.
  • the composition includes polysorbate-80, the polysorbate-80 can be provided at a concentration from about 1% (v/v) to about 10% (v/v).
  • the composition includes sodium butyrate, the sodium buyrate can be present in an amount of about 500 to about 1500 mg.
  • the composition suitable for administration as an enema is formulated to include genetically engineered microorganisms of the present disclosure, Attorney Docket No. MMS.003WO cromolyn sodium, and polysorbate-80.
  • the composition further includes alpha-lipoic acid and/or L-glutamine and/or N-acetyl cysteine and/or sodium butyrate (1.1 gm).
  • the compositions may, if desired, be presented in a pack or dispenser device and/or a kit that may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the present technology provides compositions that are useful for treating and/or detecting a disease, such as a disease of the GI tract and/or associated tissue.
  • a pharmaceutical composition for treating and/or detecting a target cell indicative of a disease can include a genetically engineered microorganism as disclosed herein, and, optionally, a pharmaceutically acceptable carrier.
  • the genetically engineered microorganism can be a non-pathogenic and/or auxotrophic microorganism.
  • the genetically engineered microorganism can deliver a payload gene specifically to the target cell.
  • the payload gene can be part of a larger nucleic acid molecule, such as a DNA molecule (e.g., plasmid DNA).
  • the payload gene can encode at least one gene product, such as a therapeutic agent, a detection marker, or a combination thereof.
  • the payload gene can be operably linked to a mammalian promoter for expression by the target cell, e.g., in embodiments where the genetically engineered microorganism is configured to deliver a DNA molecule including the payload gene to the target cell.
  • the mammalian promoter can be active and/or specific for epithelial expression (e.g., GI tract epithelial cell-specific expression).
  • the payload gene can be operably linked to a microbial promoter for expression by the genetically engineered microorganism, e.g., in embodiments where the genetically engineered microorganism is configured to deliver an RNA molecule including the payload gene to the target cell, or in embodiment where the genetically engineered microorganism is configured to deliver a protein encoded by the payload gene to the target cell.
  • the present disclosure provides genetically engineered microorganisms that are administered for the purpose of treating and/or identifying target cells (e.g., diseased and/or abnormal cells) in a subject.
  • the genetically engineered microorganisms have been genetically altered to invade diseased and/or abnormal cells and deliver a nucleic acid encoding a prodrug converting enzyme, enabling the abnormal cells to convert a non-toxic prodrug into a pharmacologically active drug, and thereby provide a targeted treatment to the diseased and/or abnormal cells.
  • the engineered microorganisms delivers the prodrug converting enzyme directly.
  • the engineered microorganisms additionally produce a detection marker or deliver a nucleic acid encoding the detection marker that allows detection of diseased and/or abnormal cells.
  • the genetically engineered microorganism includes a targeting gene encoding a targeting element that facilitates binding to and/or entry into the target cell.
  • the targeting element can be a surface protein that specifically interacts with a surface marker on the target cell.
  • the target cell is a diseased epithelial cell
  • the surface protein specifically interacts with one or more cell membrane receptors which are not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, common bile duct, etc., but are exposed to the luminal side of diseased epithelial cells of the GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, common bile duct, etc., in a subject suffering from a disease.
  • the present technology provides a genetically engineered microorganism useful in the treating of cells having mislocalized and/or aberrantly expressed cell surface molecules in the GI tract, and optionally, for diagnosing, prognosing, and/or evaluating a disease or condition associated with cells having mislocalized and/or aberrantly expressed cell surface molecules in the GI tract.
  • the genetically engineered microorganism includes a lysis gene encoding a lysis element that allows the genetically engineered microorganism and/or a payload plasmid including the payload gene to enter the cytoplasm of the target cell.
  • the lysis element can lyse an endocytic vacuole to allow the genetically engineered microorganism and/or payload plasmid to escape the phagosome.
  • the payload plasmid can subsequently enter the nucleus of the target cell.
  • the genetically engineered microorganism can also include an auxotrophic mutation in an essential gene that causes lysis of the genetically engineered microorganism within the target cell to release the payload plasmid into the cytoplasm of the target cell.
  • the genetically engineered microorganism can also include a functional copy of the essential gene to serve as a selection mechanism.
  • the targeting gene and lysis gene are integrated into the genome of the genetically engineered microorganism, while the payload gene and functional copy of the essential gene are located on an episomal plasmid of the genetically engineered microorganism.
  • the genetically engineered microorganism can also include a payload gene encoding at least one therapeutic agent.
  • the therapeutic agent can be a prodrug converting enzyme that converts an inactive precursor to a therapeutically active product that kills the target cell.
  • the prodrug converting enzyme is an enzyme that converts a precursor of 5-FU (e.g., 5-FC) into 5-FU, such as a cytosine deaminase or a derivative thereof (e.g., a CD:UPRT fusion protein).
  • the therapeutic agent can be a direct cytotoxic agent that directly kills the target cell.
  • the genetically engineered microorganism can also include a payload gene encoding at least one detection marker.
  • the detection marker can be a protein, peptide, or nucleic acid molecule (e.g., an RNA molecule).
  • the detection marker is a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination of any two or more these.
  • a fluorescent marker e.g., a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination of any two or more these.
  • an ultrasound reporter e.g., a bacterial gas
  • the detection marker may be detectable in situ in the subject (e.g., using an endoscopic or colonoscopic procedure) and/or may be detectable in a biological sample from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva; and/or from a biopsy sample).
  • a single genetically engineered microorganism includes a first payload gene encoding at least one therapeutic agent, and a second payload gene encoding at least one detection marker.
  • a first genetically engineered microorganism includes a first payload gene encoding at least one therapeutic agent
  • a second genetically engineered microorganism includes a second payload gene encoding at least one detection marker.
  • the first genetically engineered microorganism can be administered to the subject before, concurrently with, or after the second genetically engineered microorganism.
  • the genetically engineered microorganism is non- pathogenic, auxotrophic, and includes an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s).
  • the cell membrane receptor is not exposed on the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but is exposed on the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc. in a subject suffering from a disease.
  • the surface protein can promote binding and invasion of the microorganism in the Attorney Docket No. MMS.003WO diseased epithelial cells.
  • the microorganism can also include one or more payload gene(s) encoding at least one detection marker and/or therapeutic agent (e.g., prodrug converting enzyme) operably linked to a promoter to drive mammalian or bacterial RNA expression.
  • the promoter is a mammalian promoter.
  • the mammalian promoter directs epithelial-specific expression, such as GI tract epithelial cell- specific expression.
  • the promoter is a bacterial promoter (or a bacteriophage promoter that functions in the genetically engineered microorganism), and the resulting mRNA is translatable by the genetically engineered microorganism and/or a mammalian cell.
  • the genetically engineered microorganism of the present disclosure delivers a protein to target cells (e.g., diseased epithelial cells).
  • the one or more payload genes encoding the therapeutic agent and/or detection marker is operably linked to a microbial promoter.
  • the one or more payload genes encoding the therapeutic agent and/or detection marker includes one or more microbial transcription terminators.
  • the one or more payload genes encoding the therapeutic agent and/or detection marker include one or more bacterial ribosome binding sites (e.g., Shine-Dalgarno sequences).
  • the microbial promoter is inducible and/or repressible.
  • the microbial promoter is constitutive.
  • the one or more payload genes encoding the therapeutic agent and/or detection marker are inserted on a plasmid.
  • the one or more payload genes encoding the therapeutic agent and/or detection marker are stably integrated on the chromosome.
  • the present technology provides a genetically engineered microorganism useful in the detection of mislocalized and/or aberrantly expressed cell surface molecules in the GI tract, and optionally, for prognosing, evaluating, and/or treating a disease or condition, where the genetically engineered microorganism is capable of delivering to the diseased cells an RNA molecule that is capable of self-replicating or self-amplifying.
  • the RNA molecule that is capable of self-replicating or self-amplifying produces an mRNA encoding one or more therapeutic agents and/or detection markers.
  • the RNA molecule that is capable of self-replicating or self-amplifying is produced by expression of a cassette that comprises an RNA viral genome replication apparatus and genes encoding one or more detection markers.
  • the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP).
  • the RdRP is an alphavirus RdRP.
  • the alphavirus RdRP includes nsp1, nsp2, nsp3, and/or nsp4.
  • the one or more payload genes encoding the therapeutic agent and/or detection marker have codon usage optimized for mammalian expression.
  • the present technology provides methods for treating and/or detecting a target cell in a subject, such as a diseased and/or abnormal cell.
  • the target cell can be a diseased epithelial cell indicative of a disease of the GI tract and/or associated tissues, such as a precancerous lesion, a GI tract cancer, IBD (e.g., ulcerative colitis, Crohn’s disease), IBS, or Barrett’s esophagus.
  • Illustrative precancerous lesions and GI tract cancers include squamous cell carcinoma of anus, low-grade squamous intraepithelial lesions (LSIL) of anus, high-grade squamous intraepithelial lesions (HSIL) of anus, colorectal cancer (e.g., colorectal adenocarcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer), colorectal polyposis (e.g., Peutz-Jeghers syndrome, juvenile polyposis syndrome, MUTYH-associated polyposis, familial adenomatous polyposis/Gardner’s syndrome, Cronkhite-Canada syndrome), carcinoid tumor, pseudomyxoma peritonei, duodenal adenocarcinoma, premalignant adenoma of small bowel, distal bile duct carcinoma, BilIN (e.g., BilIN-1, BilIN
  • the precancerous lesion is an adenoma.
  • the precancerous lesion includes a polyp such as a sessile polyp, a serrated polyp (e.g., hyperplastic polyps, sessile serrated adenomas/polyps, traditional serrated adenoma), a sessile serrated polyp, a flat polyp, a sub-pedunculated polyp, pedunculated polyp, and a combination thereof.
  • the polyp is a diminutive polyp.
  • the precancerous lesion is a BilIN selected from BilIN-1, BilIN-2, BilIN-3, or cholangiocarcinoma.
  • the precancerous lesion is a PanIN selected from PanIN-1, PanIN-2, PanIN-3, or pancreatic ductal adenocarcinoma. In some embodiments, the precancerous lesion has a size within a range from about 0.05 mm to about 30 mm. In some embodiments, the precancerous lesion has a size less than about 0.1 mm, less than about 0.25 mm, less than about 0.5 mm, less than about 1 mm, less than about 2 mm, less than about 5 mm, less than about 8 mm, less than about 10 mm, less than about 15 mm, less than about 20 mm, less than about 25 mm, or less than about 30 mm.
  • the cancer is a polyp, an adenoma, or a frank cancer.
  • the cancer is CRC, Lynch syndrome, familial adenomatous polyposis, hereditary non-polyposis colon cancer (HNPCC), or a sporadic cancer.
  • the cancer is a BilIN (e.g., BilIN-1, BilIN-2, BilIN-3, cholangiocarcinoma) or a PanIN (e.g., PanIN-1, PanIN-2, PanIN-3, pancreatic ductal adenocarcinoma).
  • the present technology provides a method for treating a subject, where the method includes administering a genetically engineered microorganism to the subject (e.g., to a GI tract of the subject).
  • the genetically engineered microorganism can include a payload gene encoding a therapeutic agent, such as a prodrug converting enzyme or direct cytotoxic agent.
  • the genetically engineered microorganism can also include a targeting gene encoding a targeting element (e.g., invasin) that binds to a surface marker on a target cell (e.g., a diseased and/or abnormal cell of the GI tract) and/or promotes internalization of the genetically engineered microorganism by the target cell.
  • a targeting element e.g., invasin
  • the surface marker can be a cell surface marker that is mislocalized in the target cell, such as an integrin.
  • the genetically engineered microorganism can optionally include a lysis gene encoding a lysis element (e.g., a lysin) that lyses an endocytic vacuole of the target cell.
  • the genetically engineered microorganism can optionally include an auxotrophic mutation, e.g., to facilitate lysis of the genetically engineered microorganism in the target cell.
  • the lysis gene and/or the auxotrophic mutation to facilitate lysis may be omitted.
  • the genetically engineered microorganism is administered multiple times during a course of treatment, such as at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more.
  • the genetically engineered microorganism may be administered once every 12 hours, once every 24 hours, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 week, once every month, once every 2 months, once every 3 months, once every 6 months, or once every 12 months.
  • the genetically engineered microorganism may be administered only a single time during the course of treatment.
  • the method can further include administering a prodrug to the subject.
  • the prodrug can be 5-FC
  • the active drug can be 5-FU
  • the prodrug converting enzyme can be or include a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof (e.g., a Attorney Docket No. MMS.003WO CD:UPRT fusion protein).
  • the prodrug can be administered via any suitable route, such as an oral route, a rectal route, a sublingual route, an intravenous route, an intramuscular route, a subcutaneous route, a transdermal route, an intranasal route, or a vaginal route.
  • the prodrug may be administered via the same route as the genetically engineered microorganism (e.g., an oral route, such as part of a single oral capsule or as two different oral capsules), or may be administered via a different route than the genetically engineered microorganism.
  • the timing of the administration of the genetically engineered microorganism and the prodrug may be varied as desired.
  • the genetically engineered microorganism is administered concurrently with the prodrug.
  • the genetically engineered microorganism is administered before the prodrug, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the genetically engineered microorganism is administered to the subject.
  • the prodrug is administered before the genetically engineered microorganism, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the prodrug is administered to the subject.
  • the prodrug is administered multiple times during a course of treatment, such as at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more.
  • the prodrug may be administered once every 12 hours, once every 24 hours, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 week, once every month, once every 2 months, once every 3 months, once every 6 months, or once every 12 months.
  • the genetically engineered microorganism may be administered only a single time during the course of treatment.
  • the administration frequency of the prodrug may be the same as the administration frequency of the genetically engineered microorganism, or the administration frequency of the prodrug may be different than the administration frequency of the genetically engineered microorganism.
  • the genetically engineered microorganism includes a second payload gene encoding a detection marker.
  • the detection marker can be a protein, peptide, or a nucleic acid molecule, as described herein.
  • the detection marker can include one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI Attorney Docket No. MMS.003WO contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter.
  • the method can include administering a second genetically engineered microorganism including the second payload gene encoding the detection marker.
  • the second genetically engineered microorganism can include any of the other sequence elements described herein, such as a targeting gene encoding a targeting element, a lysis gene encoding a lysis element, an auxotrophic mutation, and/or additional sequence elements.
  • the second genetically engineered microorganism is administered concurrently with the genetically engineered microorganism.
  • the second genetically engineered microorganism is administered before the genetically engineered microorganism, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the genetically engineered microorganism is administered to the subject.
  • the genetically engineered microorganism is administered before the second genetically engineered microorganism, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the second genetically engineered microorganism is administered to the subject.
  • the method further includes detecting the detection marker (e.g., encoded by the second payload gene of the genetically engineered microorganism or the second genetically engineered microorganism).
  • the method can include collecting a biological sample from the subject after administration of the genetically engineered microorganism and/or the second genetically engineered microorganism.
  • the biological sample can include feces, blood, serum, plasma, mucus, urine, saliva, or a combination thereof.
  • the biological sample is collected using a noninvasive technique (e.g., without performing colonoscopy or endoscopy).
  • the biological sample can be collected at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days after administration of the genetically engineered microorganism and/or the second genetically engineered microorganism to the subject.
  • multiple biological samples are collected at multiple time points, such as at least two, three, four, five, 10, 20, or more different time points. For example, Attorney Docket No.
  • MMS.003WO biological samples can be collected once every hour, once every two hours, once every 12 hours, once every 24 hours, once every 48 hours, or once every week after administration of the genetically engineered microorganism and/or the second genetically engineered microorganism to the subject.
  • the method can further include detecting whether at least one detection marker is present in the biological sample.
  • the detection marker can be or include a protein, a peptide, or a nucleic acid.
  • the detection marker is a gene product encoded by a payload gene that is transferred to the target cell by the genetically engineered microorganism and/or the second genetically engineered microorganism.
  • the biological sample includes a plurality of cells, including the target cell (e.g., diseased and/or abnormal cells) and non-target cells (e.g., the genetically engineered microorganism, healthy cells).
  • the method can include processing the biological sample to extract any detection marker present on the target cells, within the target cells, and/or secreted by the target cells.
  • the method can further include performing an assay on the extracted detection marker to determine the presence and/or amount of the detection marker.
  • the assay can include agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, CD, chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, flow cytometry, fluorimetry, electrophoretic assay, enzymatic assay, ELISA, gas chromatography, gravimetry, HPLC, immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, RT-PCR, multiplex RT-PCR, qPCR, RT-qPCR, dPCR, ddPCR, semi-quantitative PCR, semi-quantitative RT-PCR, gel electrophoresis, RNA sequencing, FISH (e.g., RNA-FISH), CISH, or suitable combinations thereof.
  • FISH e.g., RNA-FISH
  • CISH e.g.,
  • the fecal sample can contain diseased and healthy epithelial cells sloughed off from the GI tract, as well as the genetically engineered microorganism and/or the second genetically engineered microorganism.
  • the fecal sample can be collected by defecation into a collection device, or can be collected during a procedure, such as an enema, a fecal swab, or an endoscopy.
  • the fecal sample can be tested immediately, or can be stored in a buffer prior to testing, such as an aqueous buffer, a glycerol-based buffer, a polar solvent based-buffer, an osmotic balance buffer, or other buffer sufficient for preserving the fecal sample.
  • the fecal sample can optionally be refrigerated, for example, at 4 °C, 0 °C, -20 °C, -80 °C, or lower.
  • Attorney Docket No. MMS.003WO The fecal sample can be disrupted in the presence of a buffer and/or a surfactant to form a suspension.
  • the buffer can be a biologically compatible buffer, such as Hanks’ balanced salt solution, Alsever’s solution, Earle’s balanced salt solution, Gey’s balanced salt solution, phosphate buffered saline, Puck’s balanced salt solution, Ringer’s balanced salt solution, Simm’s balanced salt solution, TRIS-buffered saline, or Tyrode’s balanced salt solution.
  • the surfactant can be an ionic or non-ionic surfactant, such as Tween-20 or Triton- X-100.
  • the fecal sample can be disrupted in the presence of a ribonuclease inhibitor.
  • the ribonuclease inhibitor can be solvent based, protein based, or another type of method to prevent RNA destruction, including, for example, Protector RNase Inhibitor (Roche), RNasin® (Promega), SUPERase- InTM (Thermo Fisher Scientific), RNaseOUTTM (Thermo Fisher Scientific), ANTI-RNase, Recombinant RNase Inhibitor, or a cloned RNase Inhibitor.
  • the fecal sample is disrupted by vortexing, shaking, stirring, rotating, or otherwise mechanically agitating the fecal sample with the buffer, surfactant, and/or ribonuclease inhibitor to form the suspension.
  • the detection marker can be extracted from the suspension using techniques such as centrifugation, filtration, targeted probes that specifically bind the detection marker, antibodies, column-based techniques, bead-based techniques, solvent-based techniques, chromatographic techniques, or suitable combinations thereof.
  • the detection marker is an intracellular marker
  • the cells present in the fecal sample can be lysed using a lysis buffer and/or mechanical lysis techniques, and the lysate can be processed via centrifugation, filtration, column-based techniques, bead-based techniques, solvent-based techniques, chromatographic techniques, etc., to extract the detection marker from the lysate.
  • the detection marker is or includes an RNA molecule
  • the fecal sample can be treated with DNase to degrade DNA.
  • the detection marker extracted from the biological sample can then be measured or otherwise analyzed using one or more of the techniques described herein. Presence of the detection marker in the biological sample can indicate that the target cell is present in the subject, which can be indicative of the subject having the disease or being at risk of developing the disease.
  • the method can include other techniques for detecting the presence of the detection marker.
  • the detection marker can be detected in situ within the subject using techniques such as endoscopy, colonoscopy, MRI, CT Attorney Docket No. MMS.003WO imaging, PET imaging, or a combination thereof. enzyme.
  • the method can optionally include administering the substrate the subject, such as a substrate of a bioluminescent protein, a substrate of an MRI contrast agent, a PET probe, a substrate of an enzyme reporter, a SPECT probe, or a combination of any two or more thereof.
  • the substrate such as a substrate of a bioluminescent protein, a substrate of an MRI contrast agent, a PET probe, a substrate of an enzyme reporter, a SPECT probe, or a combination of any two or more thereof.
  • the substrate can be administered prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection.
  • the substrate may be administered after the administration of the genetically engineered microorganism and/or the second genetically engineered microorganism.
  • the method can further include selecting the subject for a treatment if the detection marker is determined to be present, thus indicating that the subject has the disease or is at risk of developing the disease, and/or indicating that previous treatments (e.g., treatment with a genetically engineered microorganism including a payload gene encoding a therapeutic agent) did not sufficiently treat or otherwise mitigate the disease.
  • the treatment is or includes surgery, e.g., to remove diseased tissue.
  • the treatment is or includes administration of a therapeutic agent.
  • the therapeutic agent can be selected from a chemotherapeutic agent, a cytotoxic agent, an immune checkpoint inhibitor, an immunosuppressive agent, a sulfa drug, a corticosteroid, an antibiotic, or a combination of any two or more thereof.
  • the treatment is or includes administering a third genetically engineered microorganism to the subject.
  • the third genetically engineered microorganism can include a payload gene encoding a therapeutic agent, which may the same payload gene and therapeutic agent as the genetically engineered microorganism, or can be a different payload gene and therapeutic agent.
  • the third genetically engineered microorganism can include any of the other sequence elements described herein, such as a targeting gene encoding a targeting element, a lysis gene encoding a lysis element, an auxotrophic mutation, and/or additional sequence elements.
  • the methods described herein are performed on a subject that is at low risk for cancer (e.g., CRC) and/or colorectal polyps. In some embodiments, the subject has no previous incidence of cancer and/or colorectal polyps.
  • the method is performed on a subject that is high or medium risk for cancer and/or colorectal Attorney Docket No. MMS.003WO polyps.
  • the subject has a prior incidence of cancer and/or colorectal polyps, and/or has a family history of cancer and/or colorectal polyps.
  • the subject has a genetic predisposition to cancer.
  • the subject has FAP, attenuated FAP, Lynch syndrome, oligopolyposis, Peutz-Jeghers syndrome, MUTYH- associated polyposis, juvenile polyposis syndrome, and/or Cowden syndrome.
  • the subject is at least 45 years of age, at least 50 years of age, at least 55 years of age, or at least 60 years of age.
  • the present technology provides compositions and methods that are useful for treating diseased GI tissue.
  • the present technology relates to a method for treating diseased epithelial tissue, where the method includes administering to the GI tract of a subject in need thereof, a genetically engineered microorganism engineered to direct expression of a prodrug converting enzyme specifically in diseased epithelial cells of the GI tract.
  • the diseased epithelial tissue is selected from GI tract epithelium and/or bile duct epithelium.
  • the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 5-FC into 5-FU.
  • the enzyme is selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450 (e.g., that acts on the prodrug as a substrate), a uracil phosphoribosyltransferase (yUPRT), and a combination thereof or a fusion protein comprising two or more thereof.
  • the enzyme is a fusion protein of a cytosine deaminase and a UPRT.
  • the cytosine deaminase and/or the UPRT is derived from yeast.
  • the present technology relates to a method for treating a disease characterized by the presence of diseased GI tissue in a subject in need thereof.
  • the method includes administering to the epithelial tissue of a subject, an engineered microorganism that specifically invades diseased cells of the epithelium, and directs expression of a prodrug converting enzyme in the diseased cells.
  • the method further includes administering a prodrug.
  • the genetically engineered microorganism includes an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue.
  • the surface protein promotes binding and/or invasion of the microorganism in the diseased epithelial cells.
  • the genetically engineered microorganism disclosed herein can include one or more gene(s) encoding a surface protein, where the surface protein specifically interacts with Attorney Docket No.
  • the surface protein promotes the binding and invasion specifically of epithelial cells of diseased GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., by the genetically engineered microorganism.
  • the genetically engineered microorganism disclosed herein includes a second exogenous gene encoding a lysin that lyses the endocytotic vacuole, and thereby contributes to pore formation, breakage, or degradation of the phagosome.
  • the lysin is a cholesterol-dependent cytolysin.
  • the lysin is selected from the group consisting of listeriolysin O, ivanolysin O, streptolysin, perfringolysin, botulinolysin, leucocidin, and a mutant derivative thereof.
  • the lysin is listeriolysin O (SEQ ID NO: 3), or a mutant derivative thereof (without limitation, e.g., SEQ ID NO: 4).
  • the present technology relates to a method for treating a disease characterized by the presence of diseased GI tissue in a subject in need thereof.
  • the diseases that may be treated using the genetically engineered microorganisms and/or using the methods disclosed herein include precancerous lesions, GI tract cancers, ulcerative colitis, Crohn’s disease, Barrett ’s esophagus, irritable bowel syndrome and irritable bowel disease.
  • GI tract cancers and precancerous syndromes include squamous cell carcinoma of anus, colorectal cancer (CRC) (including colorectal adenocarcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer), colorectal polyposis (including Peutz-Jeghers syndrome, juvenile polyposis syndrome, MUTYH-associated polyposis, familial adenomatous polyposis/Gardner's syndrome, and Cronkhite-Canada syndrome), carcinoid, pseudomyxoma peritonei, duodenal adenocarcinoma, distal bile duct carcinomas, pancreatic ductal adenocarcinomas, gastric carcinoma, signet ring cell carcinoma (SRCC), gastric lymphoma (MALT lymphoma), linitis plastic (Brinton ’s disease), and squamous cell carcinoma of esophagus and adenocarcino
  • the diseased epithelial cells from subjects suffering from one or more of these indications may be treated using the genetically engineered microorganisms of the present technology.
  • the genetically engineered microorganisms can specifically bind to diseased epithelial cells by specifically interacting with one or more cell membrane receptor(s) Attorney Docket No. MMS.003WO that are exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc.
  • the genetically engineered microorganisms may not bind to normal (non-diseased) epithelial cells because the one or more cell membrane receptor(s) are not exposed to the luminal side of the normal epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc.
  • the genetically engineered microorganism delivers one or more nucleic acid(s) encoding at least one detection marker and/or prodrug converting enzyme to the diseased epithelial cells (target cells).
  • the diseased epithelial cells can express the at least one detection marker and/or prodrug converting enzyme, allowing their detection or targeted delivery of the prodrug converting enzyme to them.
  • the diseased epithelial cells can be identified as the cells that accumulate the at least one detection marker and/or prodrug converting enzyme inside them or on their surface, while the detection marker and/or prodrug converting enzyme is not present in or on the surface of the surrounding healthy cells (normal epithelial cells).
  • Detection of the diseased epithelial cells may be carried out using a suitable technique such as colonoscopy, endoscopy, magnetic resonance imaging, CT scan, PET scan, SPECT scan, etc.
  • a method for detecting and/or treating a target cell in a subject can include administering at least one genetically engineered microorganism of the present disclosure to the subject.
  • the genetically engineered microorganism is administered via an oral and/or rectal route.
  • the genetically engineered microorganism can be administered into a lumen of the GI tract of the subject.
  • the genetically engineered microorganism can be administered as part of a pharmaceutical composition including a pharmaceutically acceptable excipient, as described herein.
  • the method optionally includes administration of a colon cleansing agent, such as a laxative.
  • the colon cleansing agent is administered prior to and/or after the administration of the microorganism.
  • the present technology relates to a method for diagnosing and treating a disease characterized by the presence of diseased GI tissue in subject in need thereof.
  • the method comprises administering to the epithelial tissue of a subject, an engineered microorganism that specifically invades diseased cells of the epithelium, and directs expression of a prodrug converting enzyme and a detection marker in Attorney Docket No. MMS.003WO the diseased cells.
  • the method further includes administering a prodrug.
  • the prodrug is administered where diseased tissue is detected.
  • the methods include administering to the GI tract of a subject in need thereof, a genetically engineered microorganism that directs expression of a prodrug converting enzyme and a detection marker specifically in diseased cells.
  • the method further includes administering a prodrug.
  • the prodrug is administered to diseased tissue that is detected.
  • the method can further involve detecting the expression of the detection marker to thereby detect the diseased epithelial cells.
  • the genetically engineered microorganism is non-pathogenic, auxotrophic, and includes an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s).
  • the cell membrane receptor may not be exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but may be exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., in the subject suffering from a disease.
  • the expression and/or localization of the one or more cell membrane receptor(s) can confer the specificity for diseased or abnormal cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., on the microorganism.
  • the surface protein can thus promote binding and invasion of the microorganism in the diseased epithelial cells.
  • the microorganism can also include one or more gene(s) encoding a prodrug converting enzyme and a detection marker operably linked to a promoter (e.g., a mammalian or bacterial promoter).
  • the microorganism delivers a nucleic acid (e.g., a DNA or an mRNA molecule) for expression of the detection marker in diseased epithelial cells.
  • the diseased epithelial cells express a prodrug converting enzyme and a detection marker, thereby allowing their detection.
  • the method further includes administering a prodrug.
  • the prodrug is administered when diseased tissue is detected (e.g., when potential cancerous or malignant tissue is detected).
  • the promoter may be a mammalian promoter. In some embodiments, the mammalian promoter directs GI tract epithelial cell-specific expression.
  • the promoter is a bacterial promoter
  • the resulting mRNA is translatable in the bacterial or mammalian cell.
  • the present technology provides a method for diagnosis and/or prognosis of a disease or disorder in a subject, the method comprising (i) administering Attorney Docket No. MMS.003WO to the GI tract of a subject in need thereof, a genetically engineered microorganism disclosed herein; and (ii) detecting the expression of the detection marker, thereby detecting the diseased epithelial cells.
  • the microorganism can include an exogenous gene encoding a surface protein, where the surface protein specifically interacts with one or more cell membrane receptor(s), which are not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but is exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., in the subject suffering from a disease.
  • the surface protein promotes binding and invasion of the microorganism in the diseased epithelial cells.
  • the microorganism can also include one or more gene(s) encoding the detection marker operably linked to a promoter.
  • the promoter is a mammalian promoter.
  • the mammalian promoter that is active or specific for epithelial expression or GI tract epithelial cell-specific expression.
  • the mammalian promoter directs GI tract epithelial cell-specific expression.
  • the microorganism delivers a DNA molecule (e.g., a plasmid) to diseased epithelial cells.
  • the genetically engineered microorganism is administered via an oral or rectal route.
  • the method further includes administration of a colon cleansing agent comprising a laxative.
  • the colon cleansing agent including the laxative is administered prior to the administration of the microorganism.
  • the genetically engineered microorganism is non-pathogenic.
  • the genetically engineered microorganism is auxotrophic.
  • the genetically engineered microorganism is non-pathogenic and auxotrophic.
  • the present technology provides a genetically engineered microorganism for use in a method of diagnosis and/or prognosis of a disease or disorder in a subject, the method comprising (i) administering to the gastrointestinal tract of a subject in need thereof, disclosed herein; and (ii) detecting the expression of the detection marker, thereby detecting the diseased epithelial cells.
  • the microorganism can include an exogenous gene encoding a surface protein, where the surface protein specifically interacts with one or more cell membrane receptor(s), which are not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but is exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, Attorney Docket No. MMS.003WO etc., in the subject suffering from a disease.
  • the surface protein promotes binding and invasion of the microorganism in the diseased epithelial cells.
  • the microorganism can also include one or more gene(s) encoding the detection marker operably linked to a promoter.
  • the promoter is a mammalian promoter.
  • the mammalian promoter directs GI tract epithelial cell-specific expression.
  • the genetically engineered microorganism is administered via an oral or rectal route.
  • the method further includes administration of a colon cleansing agent comprising a laxative.
  • the colon cleansing agent including the laxative is administered prior to the administration of the microorganism.
  • the genetically engineered microorganism is non-pathogenic. In some embodiments, the genetically engineered microorganism is auxotrophic. In some embodiments, the genetically engineered microorganism is non-pathogenic and auxotrophic.
  • methods of selecting a subject suffering from or suspected to be suffering from a disease for a treatment comprising: (i) administering to the gastrointestinal tract of the subject a genetically engineered microorganism of any one of the embodiments disclosed herein; (ii) detecting elevated expression of the detection marker compared to surrounding normal epithelial cells; and (iii) selecting the subject for treatment if expression of the detection marker is observed compared to surrounding normal epithelial cells.
  • the disease is selected from a precancerous lesion, cancer, ulcerative colitis, Crohn’s disease, Barrett’s esophagus, irritable bowel syndrome, and irritable bowel disease.
  • the treatment is surgery or administration of a therapeutic agent.
  • the surgery removes diseased tissue.
  • the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, an immune checkpoint inhibitor, an immunosuppressive agent, a sulfa drug, a corticosteroid, an antibiotic, and a combination of any two or more thereof. Also disclosed herein, in various aspects, are methods of treating a cancer in a patient.
  • these methods comprise: (i) administering to the GI tract of the subject a genetically engineered microorganism of the present technology; (ii) detecting the expression of the detection marker, thereby detecting the diseased epithelial cells; and (iii) administering a treatment if the expression of the detection marker is observed.
  • the treatment is surgery or administration of a therapeutic agent.
  • the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic Attorney Docket No. MMS.003WO agent, an immune checkpoint inhibitor, an immunosuppressive agent, a sulfa drug, a corticosteroid, an antibiotic, and a combination of any two or more thereof.
  • Example 1 Delivery of DNA Encoding a Chemotherapeutic to Diseased Cells by Genetically Engineered Bacterial Strains
  • Bacteria of the current disclosure can specifically detect diseased cells and deliver a prodrug converting enzyme to diseased cells, thereby enabling targeted drug delivery to diseased cells. Without being bound by theory, it is hypothesized that payload delivery to diseased cells proceeds through four distinct steps. As shown in FIG. 1C and FIG. 10A, the genetically engineered microorganisms of the current disclosure can bind to diseased epithelial cells through mislocalized receptors, and undergo internalization (FIG. 1C).
  • the bacteria may undergo lysis, inter alia, due to the dapA attenuation mutation, which causes a defect in cell wall synthesis (FIG.1C).
  • Listeriolysin O LLO, or Hly; SEQ ID NO: 3
  • LLO listeriolysin O
  • the plasmid carrying the prodrug converting enzyme can undergo nuclear localization, which can occur naturally or be guided by binding of a protein that includes a nuclear localization signal.
  • the plasmid carrying the prodrug converting enzyme can drive the expression of the prodrug converting enzyme in the diseased epithelial cells of the GI tract and/or epithelium of the ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct).
  • the diseased epithelial cells may exhibit mislocalized receptors and/or expression of novel mammalian membrane receptors.
  • the novel receptors may be receptors that are normally not found in the cells and/or receptor formed by translocations and other genomic rearrangement.
  • FIG.3 shows a schematic of a strain for DNA transfer.
  • the strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG. 3 (top panel)).
  • invasin inv
  • LLO listeriolysin O
  • FIG. 3 top panel
  • MMS.003WO plasmid containing a payload gene under a eukaryotic promoter and selection based on a wild- type alr gene (FIG.3 (bottom panel)).
  • the above strain was further modified as shown in FIG.4 for a targeted delivery of a chemotherapeutic to diseased epithelial cells.
  • the strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv) and listeriolysin O (hlyA) (FIG. 4 (top panel)).
  • the strain carries a plasmid having an intron- containing gene encoding a chimeric bifunctional yeast cytosine deaminase (yCD) and the yeast uracil phosphoribosyltransferase (yUPRT, fusion protein is yCD:UPRT) gene under a eukaryotic promoter.
  • the plasmid also carries a promoter-less iRFP670 gene following an internal ribosome entry site (IRES) from an encephalomyocarditis virus (EMCV) (SEQ ID NO: 10, FIG.4 (bottom panel)).
  • the yCD:UPRT gene which contains an intron, was placed under the control of a eukaryotic promoter, to prevent the expression of yCD:UPRT enzyme in bacteria.
  • the bacteria from this strain represented in FIG. 4 were coincubated with HEK293T cells (an immortalized cancer cell line) for two hours followed by washing away of extracellular bacteria. Bacteria lacking invasin or listeriolysin O were used as controls. To determine the efficiency of gene transfer, the HEK293T cells were analyzed by flow cytometry. As shown in FIG.5A, iRFP670 expression could be detected when the strain comprising invasin and listeriolysin O was used.
  • the bacteria lacking invasin or listeriolysin O showed only background levels of iRFP670 expression (FIG.5A).
  • the HEK293T cells were further incubated in the presence of increasing concentrations of 5-fluorocytosine (5-FC) and their viability was measured.
  • yCD:UPRT deaminates the nontoxic prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic compound 5-fluorouracil (5-FU), which is used in the treatment of malignant tumors.
  • HEK293T cells incubated with the strain comprising invasin and listeriolysin O shown in FIG.4) were killed.
  • FIG.5B shows a schematic of a strain for RNA transfer.
  • the strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG.6 (top panel)).
  • This strain carries a plasmid containing a payload (illustrated herein using the iRFP670 gene) under a prokaryotic promoter and selection based on a wild-type alr gene (FIG.6 (bottom panel)).
  • the iRFP670 gene followed an internal ribosome entry site (IRES) from an encephalomyocarditis virus (EMCV) (SEQ ID NO: 10, FIG.6 (bottom panel)). DNA transfer and RNA transfer to diseased cells were studied. Without wishing to be bound by theory, the mechanism of DNA transfer is shown in FIG.1C.
  • the bacteria of the strain represented in FIG.3 (with the iRFP670 gene as the payload gene) were coincubated with HEK293T cells (an immortalized cancer cell line) for one hour followed by washing away of extracellular bacteria. Bacteria lacking invasin or listeriolysin O were used as controls. To determine the efficiency of gene transfer, the HEK293T cells were analyzed by flow cytometry.
  • FIG.7A iRFP670 expression could be detected when the strain comprising invasin and listeriolysin O was used.
  • the bacteria lacking invasin or listeriolysin O showed only background level of iRFP670 expression (FIG.7A).
  • FIG. 1D the mechanism of RNA transfer is shown in FIG. 1D.
  • the genetically engineered microorganisms of the current disclosure can bind to diseased epithelial cells through mislocalized receptors and undergo internalization (FIG.1D). Upon internalization, the bacteria may undergo lysis, inter alia, due to the dapA attenuation mutation, which causes a defect in cell wall synthesis (FIG. 1D).
  • Listeriolysin O (LLO, or Hly; SEQ ID NO: 3) can then be released and lyses the phagosome or is naturally exported from the E. coli strain.
  • the mRNA transcribed in the bacteria can then be transferred to the diseased cells, as shown in FIG. 1D, and translated in diseased cells under the control of the EMCV IRES, which thereby drives the expression of the prodrug converting enzyme in the diseased epithelial cells of GI tract and/or epithelium of the Attorney Docket No. MMS.003WO ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and common bile duct).
  • gastrointestinal tissue e.g., epithelial tissue lining the bile duct, pancreatic duct, and common bile duct.
  • HEK293T cells an immortalized cancer cell line
  • Bacteria lacking invasin or listeriolysin O were used as controls.
  • the HEK293T cells were analyzed by flow cytometry.
  • iRFP670 expression could be detected when the strain comprising invasin and listeriolysin O was used.
  • the bacteria lacking invasin or listeriolysin O showed only background level of iRFP670 expression (FIG.7B).
  • Invasin + and listeriolysin O + dapA ⁇ , alr ⁇ , dadX ⁇ rnc + bacteria, or invasin + and listeriolysin O + wild type bacteria harboring the plasmid shown were used as controls.
  • Each strain carried the wild-type alr gene-selected plasmid harboring the iRFP670 gene under a prokaryotic promoter and an EMCV IRES (FIG.6 (bottom panel)).
  • the HEK293T cells were analyzed by flow cytometry to identify the portion of the HEK293T cells that expressed iRFP670 under the control of EMCV IRES.
  • RNA delivery by the strains disclosed herein may be improved by mutations that reduce activity of an RNase gene such as RNase III.
  • Example 3 In Vivo Bacterial Targeting of Diseased Cells The data above show that genetic transfer depends on invasin. The following experiment was carried out to confirm that the initial binding to diseased tissue depends on presence of a receptor for bacteria on the cells. Briefly, ⁇ 1 integrin in HCT116 cells was knocked out.
  • invasin + and dapA ⁇ , alr ⁇ , dadX ⁇ bacteria were coincubated with the cells for one hour followed by washing away of extracellular bacteria.
  • dapA ⁇ , alr ⁇ , dadX ⁇ bacteria lacking invasin were used as a control.
  • Each strain carried the wild-type alr gene-selected plasmid expressing mNeonGreen gene under a prokaryotic promoter.
  • the HCT116 cells were analyzed by flow cytometry.
  • FIG.10B wild type HCT116 cells were positive for the presence of mNeonGreen only when bacteria expressing wild-type invasin (“invasin + ”) were used. Only background levels of mNeonGreen positive cells were observed when bacteria expressing a truncated invasin incapable of binding to ⁇ 1 integrin were used (“invasin-”) (FIG. 10B). As expected, as shown in FIG. 10C, HCT116 cells lacking ⁇ 1 integrin showed background levels of mNeonGreen positive cells even when invasin + bacteria were used. Wild type HCT116 cells were identified as mNeonGreen positive when invasin + bacteria were used (FIG.10C).
  • FIG. 9A shows a schematic representation of the model. Upon injection of (Z) 4-hydroxytamoxifen, these mice develop tumors in the distal colon but not in the proximal colon (FIG. 9A (right panel)).
  • FIG. 9B shows a representative confocal fluorescence microscopy image of colon tissue section showing healthy tissue and diseased tissue.
  • the development of tumors in this model was studied by analyzing the ⁇ 1 integrin localization at 3, 5, 7, 11, 13, and 21 days after the injection of 4-hydroxytamoxifen.
  • progressive induction of colorectal cancer in Apcfl/fl,VilCre mice was seen as demonstrated by confocal fluorescence microscopy images of colon tissue sections taken 3 days (FIG.9C), 5 days (FIG.
  • FIG.12A Three hours later, the colon was extracted from the mice and analyzed using epifluorescence microscopy (FIG.12A). As shown in FIG.12B, GFP expressing bacteria were observed binding to diseased tissue as early as 5 days after induction of tumor induction by Apc deletion. GFP expressing bacteria were not observed in normal tissue. There was no detectable RFP expressing bacteria in diseased or normal tissue. The experiment was re-performed after swapping fluorescent markers.
  • tumors were induced in the Apcfl/fl,VilCre mice and 3 to7 days later, bacteria of the following strains were co-administered to the mice by enema: • Experimental bacteria: invasin + and alr ⁇ , dadX ⁇ bacteria harboring a wild-type alr gene-selected plasmid expressing an RFP gene under a prokaryotic promoter, and • Control bacteria: invasin- and alr ⁇ , dadX ⁇ bacteria harboring a wild-type alr gene- selected plasmid expressing a GFP gene under a prokaryotic promoter. Three hours later, colon was extracted from the mice and analyzed using epifluorescence microscopy (FIG.13A).
  • RFP expressing bacteria were observed binding to diseased tissue as early as 5 days after induction of tumor induction by Attorney Docket No. MMS.003WO Apc deletion. RFP expressing bacteria were not observed in normal tissue. There was no detectable GFP expressing bacteria in diseased or normal tissue.
  • FIG. 14A shows the number of bacteria in tumor and non-tumor samples: Table 7: Number of Bacteria Detected The number of bacteria was plotted. As shown in FIG. 14B, no bacteria were associated with normal tissue. In contrast, diseased tissue showed the presence of hundreds of bacteria (FIG. 14B).
  • FIG. 14C shows fluorescence microscopy from different mice showing bacteria that were associated with tumor tissue (right images) but not in non-tumor tissue (left images).
  • Gentamicin was added to the cells to kill any non-invaded bacteria. After incubation for 1 hour, the HCT116 cells were lysed using a detergent, and serial dilutions of the lysate was plated (FIG.15A). As shown in FIG. 15B, thousands of invasin + bacteria could be detected in the lysate. In contrast, no bacteria lacking invasin were detected in the lysate. As shown in FIG. 15B, the number of invaded bacteria decreased with time, which could be due to their lysis. These results demonstrate, inter alia, invasin-dependent invasion of the bacteria disclosed herein in diseased cells.
  • tumors were induced in Apcfl/fl,VilCre mice. Two weeks later, the surgery noted above to seal the colon was performed on the mice, followed by administration of an enema with invasin + alr ⁇ , dadX ⁇ bacteria harboring a wild-type alr gene-selected plasmid expressing GFP and a kanamycin resistance marker gene under a prokaryotic promoter. After 3 hours, the proximal colon tissue (non-tumor tissue) and distal colon tissue (having tumors) specimens were extracted and viewed by fluorescence and phase contrast microscopy. As shown in FIG.16A, the bacteria were seen associated with tumor tissue but not non-tumor tissue.
  • the colon tissue was added into a 1.5 mL tube with 200 ⁇ L PBS and vortexed twice for 10 seconds each (sample # 1). Serial dilutions of the supernatants were spotted on an LB plate supplemented with diaminopimelic acid. As shown in FIG. 16B, bacteria associated with but not internalized within tumor tissue could grow on LB plates supplemented with diaminopimelic acid and kanamycin. The washed colon tissue was incubated with gentamycin to kill any remaining extracellular bacteria, followed by washing away of the gentamycin and a treatment with 0.3% Triton-X (#2), then centrifuged at 400g for 1 min to remove tissue fragments. The supernatant was sample #3.
  • sample #4 The tissue pellet was resuspended in PBS (sample #4). The rest of the supernatant was centrifuged 21000g for 5 minutes, and the pellet was resuspended in PBS (sample #5). Serial dilutions of the samples #2–#5 were spotted on a LB plate supplemented with diaminopimelic acid and kanamycin. As shown in FIG.16C, each of sample # 2 to sample # 5 could grow on LB plates supplemented with diaminopimelic acid and kanamycin. Each of Attorney Docket No. MMS.003WO sample # 1 to sample # 5 and the input bacteria were serially diluted and plated on LB plates supplemented with diaminopimelic acid.
  • Example 5 Self-Amplifying RNA for Amplification of the Gene Encoding a Prodrug Converting Enzyme in the Diseased Cells
  • FIG. 17 A schematic representation of amplification of a payload in target cells using self-amplifying RNA is shown in FIG. 17.
  • FIG.18 A schematic representation of the improvement of the signal from RNA transfer of payload using self-amplifying RNA is illustrated in FIG.18. The cell on the left side in FIG.18 shows RNA transfer without amplification.
  • the cell on the right side in FIG.18 shows the amplification of RNA in target cells using self-amplifying RNA.
  • This strategy will first be tested using an iRFP670 reporter.
  • a construct for amplification of mRNA encoding iRFP670 or GFP will be made.
  • the construct will include, under the control of a prokaryotic promoter, an IRES, the functional components of an RNA- dependent RNA polymerase (RdRP) or viral genome replication apparatus, which includes nsp1, nsp2, nsp3 and nsp4, an iRFP670 or GFP sequence (indicated in FIG.17 as a “payload sequence”), and a poly(A) tail.
  • RdRP RNA- dependent RNA polymerase
  • an RdRP will be produced upon translation of the nsp1-4 genes present on the (+) strand of the construct.
  • the RdRP is expected to produce a (-) strand using the (+) strand as a template (FIG. 17).
  • the RdRP is then expected to produce another copy (+) strand using the (-) strand as a template (FIG.17). It is expected that the RdRP will catalyze multiple rounds of production of the (-) strand and (+) strand. Transcription of the (-) strand will produce an mRNA encoding the iRFP670 or GFP, which is translated to the Attorney Docket No. MMS.003WO iRFP670 or GFP.
  • a construct for amplification of mRNA encoding yCD:UPRT or other potential payloads to aid in treatment or detection of diseased lesions will be made.
  • the construct will include, under the control of a prokaryotic promoter, the functional components of an RNA- dependent RNA polymerase (RdRP) or viral genome replication apparatus, which includes nsp1, nsp2, nsp3 and nsp4, a yCD:UPRT sequence (indicated in FIG.
  • an RdRP will be produced upon translation of the nsp1-4 genes present on the (+) strand of the construct.
  • the RdRP is expected to produce a (-) strand using the (+) strand as a template (FIG. 17).
  • the RdRP is then expected to produce another copy (+) strand using the (-) strand as a template (FIG.17). It is expected that the RdRP will catalyze multiple rounds of production of the (-) strand and (+) strand. Transcription of the (-) strand will produce an mRNA encoding the yCD:UPRT, which is translated to the yCD:UPRT.
  • mislocalized receptors are attractive targets for differentiating normal and dysplastic cells in the gastrointestinal tract as they can freely interact with bacteria resident in the intestinal lumen.
  • studies to investigate receptor mislocalization were performed using a mouse model of adenoma formation.
  • Apc L/L ;Villin-CreER mice were used as a mouse model of adenoma formation, as described in Example 3 above. Briefly, 100 ⁇ M 4-hydroxytamoxifen was freshly prepared in 1x PBS and 2x 50 ⁇ L injections were placed into the distal colon of mice. Colon tissue was harvested at various time points after the injection of 4-hydroxytamoxifen.
  • Images were acquired using either a 20x (Nikon Plan Apo), 40x objective (Nikon Plan Apo), or 60x oil immersion objective (Nikon Plan Apc VC) using a Nikon 90i upright microscope equipped with a Hamamatsu Orca-ER CCD camera and an APC line 1200 light source.
  • a mouse model of adenoma formation that allows for specific temporal and special staging of adenomas in the mouse colon, dysplasia was induced and stained for the localization of the cell surface receptor ⁇ 1 integrin, in addition to ⁇ -catenin, which becomes mislocalized when Apc is knocked out.
  • EcN coli Nissle 1917
  • the bacterial protein invasin from Yersina pseudotuberculosis is a binding partner of ⁇ 1 integrin.
  • EcN was isolated from a capsule of Mutaflor® and confirmed by established PCR methods and by whole genome sequencing.
  • the cryptic plasmids of Mutaflor, pMUT1 and pMUT2 were cured from the strain by counterselection. Briefly, each plasmid was isolated and engineered to contain an ampicillin resistance marker and the sucrose counter selection marker sacB. Engineered plasmids were transformed into the wild-type EcN strain and selected by ampicillin. Strains were passaged in ampicillin for multiple days followed by selection on LB-agar plates containing 10% sucrose. Isolated colonies were selected and loss of the plasmid was confirmed by miniprep and PCR.
  • the cryptic plasmids pMUT1 and pMUT2 were removed from EcN and the alanine racemase genes alr and dadX were knocked out to render the strain auxotrophic for the essential cell wall component, D- alanine.
  • Gene knockouts of alr and dadX were carried out using established lambda red recombineering methods. All strains were cured of antibiotic markers to generate fully antibiotic sensitive strains. Strains lacking episomes were cultured in lysogeny broth (LB) containing 1 mM D-alanine, whereas strains containing episomes expressing the alr gene were cultured in LB lacking D-alanine.
  • the inv gene encoding for invasin was then integrated at the lambda phage attachment site.
  • an episomal plasmid containing the alr gene and encoding the fluorescent protein mNeonGreen was transformed into the strain to generate a fluorescent EcN strain lacking any antibiotic resistance markers.
  • the invasin protein is not optimally active in E. coli grown at 37 °C, strains were grown at 30 °C unless otherwise indicated.
  • a control strain of EcN expressing a truncated invasin incapable of binding ⁇ 1 integrin (invasin trunc ) and the flurorescent protein mScarlet was also prepared.
  • HCT116 cells ATCC® CCL-247 or HCT116 cells with the gene ITGB1 encoding ⁇ 1 integrin knocked out (Abcam ab273724) were used for in vitro invasion assays.
  • Attorney Docket No. MMS.003WO Cells were cultured in DMEM media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were seeded on 96-well tissue culture treated plates at densities ranging from 50–90% confluence depending on the experiment.
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • HCT116 cells at approximately 80% confluence were washed once with PBS, followed by addition of 100 ⁇ L of media containing bacteria at the starting multiplicity of infection (MOI) noted. Plates were incubated for two hours at 37 °C with 5% CO2 and then washed in prewarmed PBS to remove unbound bacteria, followed by the addition of DMEM with 10% FBS and 40 ⁇ g/mL gentamycin to kill extracellular bacteria. Following 1 hour of incubation, cells were detached from plates using TrypLE (Thermo-Fischer) and then analyzed by flow cytometry.
  • mice received orthotopic injections for tumor induction as described in Example 6 above 5 or 7 days prior to bacterial enema.
  • Bacterial cultures invasin (mNeongreen); invasin trunc (mScarlet)) were grown in LB overnight. Bacteria were diluted 1:33 and grown to mind-log phase for 2 hours with shaking at 30 °C. The OD600 was measured, adjusted to 0.5 in SMEM (Thermo Fisher) and combined. Mice were anesthetized with isoflurane, colon flushed to remove any fecal material, and given the bacterial enema. Anuses were then sealed using Vetbond tissue adhesive (3M) for 3.5 hours.
  • Vetbond tissue adhesive (3M)
  • mice were then sacrificed and the colons were flushed 4x 20 mL with 1x PBS.
  • the colons were lateralized and placed luminal side down on microscope slides. Images were acquired using a 20x objective (Nikon) on a Nikon Eclipse Ti-S/L inverted microscope affixed with an Andor Zyla scMOS camera and a Lumencor Light Engine light source.
  • the OD600 of all cultures were taken and adjusted to 0.2 in DMEM and then plated for CFU on LB-agar with appropriate additive to determine MOI.
  • Serial dilutions on the adjusted bacterial cultures were performed at 1:2 in DMEM for a total of 6 dilutions total.
  • the media from the tissue culture plates was aspirated and the plates washed once with pre-warmed PBS. 100 ⁇ L of pre-warmed DMEM without antibiotic was added to each well, and then 100 ⁇ L of corresponding bacterial dilution in DMEM was subsequently added to that, with the two plates being inoculated identically.
  • the plates were statically incubated for 1 hour at 37 °C and 5% CO2.
  • mice with adenomas induced in the distal colon for either one week or two weeks were given enemas containing EcN strains either expressing invasin or invasin trunc .
  • strains harbored an episome containing a kanamycin resistance gene to Attorney Docket No. MMS.003WO differentiate the introduced EcN strains from native gut microbiota. The anus was sealed by z- suture techniques after enema injection. After 2 hours of enema injection, the mice were sacrificed followed by removal of the colon and washing with PBS.
  • the colon was divided into proximal (non-tumor lesion) and distal sections (tumor lesion) and incubated in the presence of collagenase (2 mg/mL) and gentamycin (100 ⁇ g/mL) for 30 minutes at 37 oC After washing out collagenase and gentamycin with PBS, tissue was dissociated with TrypLE TM Express for 30 minutes at 37 oC on the shaker, then broken down into single cells using 16 G and 18 G needles. Samples were filtrated with 100 ⁇ M filter and then stained with an antibody to the epithelial marker Epcam for 20 minutes on ice. 7-AAD dye was used to exclude dead cells prior to the sample sorting.
  • Epcam+ fraction epidermal cells
  • the supernatant was removed after the centrifuge and the palette was incubated with Triton X-100 (0.25%) for lysing cells at 37 oC for 15 minutes. Lysate was plated on media containing kanamycin to select for the introduced EcN strain and incubated overnight at 37 oC prior to counting the colony forming units. Gentamycin protection assays were performed to determine if invasin expressing EcN was endocytosed after binding to ⁇ 1 integrin.
  • EcN expressing invasin showed a clear, MOI dependent survival rate as compared to EcN expressing invasin trunc (FIG. 20).
  • This technique was then adapted to an in vivo system. As before, mice with adenomas induced in the distal colon for either one week or two weeks were given enemas containing EcN strains either expressing invasin or invasin trunc . In addition, strains harbored an episome containing a kanamycin resistance gene to differentiate the introduced EcN strains from native gut microbiota.
  • mice After 2 hours, the mice were sacrificed and the colons were removed and washed. The colons were then divided into proximal and distal sections and dissociated in the presence of gentamycin. The cells from the dissociated tissue were then stained with an antibody to the epithelial cell marker Epcam and sorted by flow cytometry to generate a population of only epithelial cells. These sorted cells were then lysed and the lysate was plated on media containing kanamycin to select for the introduced EcN strain. The data indicated that EcN expressing invasin were consistently endocytosed into dysplastic cells as opposed to normal cells (FIG. 21). These results indicate that EcN expressing invasin are selectively endocytosed into dysplastic tissue. Attorney Docket No.
  • Example 9 In Vitro Delivery of a Prodrug Converting Enzyme Using Genetically Engineered Microorganisms
  • This example describes the construction of an engineered EcN strain capable of identifying mislocalized receptors along the gut epithelium and delivering a translational fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase (yCD:UPRT).
  • yCD:UPRT has high activity for the conversion of the antifungal 5-FC to cytotoxic 5-FU. This fusion was added to a high copy episome expressed under the ProD constitutive prokaryotic promoter.
  • FIG.22 shows a schematic of a strain for protein transfer.
  • the strain has dapA ⁇ , alr ⁇ , dadX ⁇ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG. 22 (top panel)).
  • This strain carries a plasmid containing a payload gene (e.g., yCD:UPRT, SEQ ID Nos: 6 and 7) under a prokaryotic promoter and selection based on a wild-type alr gene (FIG. 22 (bottom panel)).
  • a payload gene e.g., yCD:UPRT, SEQ ID Nos: 6 and 7
  • the studies in this example used an engineered EcN strain as depicted in FIG.22, except that the dapA ⁇ auxotrophic mutation and listeriolysin O gene were omitted.
  • HCT116 cells either containing or lacking ⁇ 1 integrin at approximately 50% confluence were washed once with prewarmed PBS, followed by addition of 100 ⁇ L of media containing bacteria and allowed to incubate 2 hours at 37 °C and 5% CO2.
  • gentamycin was added to a final concentration of 40 ⁇ g/mL and the plates were washed in prewarmed PBS to remove unbound bacteria. After washing, the cells received 200 ⁇ L of DMEM with 10% FBS and 40 ⁇ g/mL gentamycin and were incubated overnight at 37 °C and 5% CO2.
  • yCD:UPRT conversion of 5-FC to 5-FU may lead to not only killing of cell expressing the enzyme, but also nearby cells. This bystander effect may be desirable for treatments where the enzyme may not be delivered to every diseased cell.
  • a mixture of 1:1 WT HCT116: ⁇ 1 integrin knockout HCT116 was seeded onto plates and the 5-FC dose response experiment was performed as before. In addition to staining for living cells, the presence of ⁇ 1 integrin on cells was also stained, allowing the levels of each population to be measured by flow cytometry.
  • Attorney Docket No. MMS.003WO These results indicate that invasin-expressing EcN can successfully deliver the prodrug converting enzyme yCD:UPRT to ⁇ 1 integrin-expressing mammalian cells, and that the enzyme successfully converted 5-FC to 5-FU resulting in cell killing.
  • Example 10 In Vivo Delivery of a Prodrug Converting Enzyme Using Genetically Engineered Microorganisms This example describes studies investigating in vivo activity of an engineered EcN strain capable of identifying mislocalized receptors along the gut epithelium and delivering yCD:UPRT.
  • the protein fusion When combined with administration of the antifungal 5-FC, the protein fusion produced 5-FU at the pre-cancerous lesion, leading to a reduction in adenoma burden.
  • Mice were injected as described in Example 6 above to initiate tumor formation, and treatment began on day 3 post injection.
  • bacteria were grown overnight at 30 °C and diluted 1:30 in LB and grown for 2 hours to mid-log phase. The OD600 was measured and the effective OD600 of 4.5 was calculated.
  • Bacteria were pelleted and resuspended in 30 ⁇ L of either 155 mM 5-FC (+/- yCD:UPRT) or 1x PBS (+/- invasin).
  • mice were anesthetized and colons were flushed to remove feces. After 15 minutes, mice were given bacterial enemas and the anuses were sutured shut. After 1 hour, the sutures were removed. Mice received 2x 375 ⁇ L intraperitoneal (IP) injections of 250 mg/kg 5-FC. Mice in the 5-FU treatment group received IP injections of 25 mg/kg 5-FU every other day over the course of the experiment. Mice were treated for 13 days. At day 11, tumor sizes were monitored via colonoscopy. On day 14, mice were euthanized and the colons were removed, flushed, and lateralized. Tumors were imaged on a Nikon SMZ18 dissection scope at 0.75x magnification.
  • IP intraperitoneal
  • Colon tissue was preserved in 10% formalin for histology. Harvested tissue was fixed in 10% formalin, paraffin embedded, and sectioned into 4-5 ⁇ m. Antigen retrieval was performed using sodium citrate buffer and a pressurized Decloaking Chamber (Biocare Medical, NxGen). Antibodies and dilutions for immunohistochemistry (IHC) are as follows: rabbit monoclonal anti-Cleaved Caspase-3 (Asp175)(1:400, Cell Signaling Technologies (CST), 9661) and mouse monoclonal anti ⁇ -catenin (1:100, BD Biosciences, 610164). Biotin- conjugated secondary donkey anti-mouse or anti-rabbit antibodies were used (1:500, Jackson ImmunoResearch).
  • IHC immunohistochemistry
  • Vectastain Elite ABC immunoperoxidase detection kit (Vector Laboratories, PK6100) followed by Signalstain DAB substrate kit (CST, 8049) were used for visualization. All IHC antibody dilutions were carried out in Signalstain Antibody Diluent (CST, 8112). For cleaved caspase-3 analysis, positive regions were counted in the base of 45 intact crypts in the proximal colon. The tissue was mounted using Invitrogen Prolong Gold Attorney Docket No. MMS.003WO (P36930) mounting medium.
  • Images were acquired using either a 20x (Nikon Plan Apo), 40x objective (Nikon Plan Apo), or 60x oil immersion objective (Nikon Plan Apc VC) using a Nikon 90i upright microscope equipped with a Hamamatsu Orca-ER CCD camera and an APC line 1200 light source.
  • Dyplasia was induced in mice and the adenoma was allowed to develop for three days.
  • Mice were given twice daily enemas of engineered bacteria resuspended in 5-FC, as well as twice daily IP injections of 250 mg/kg 5-FC (FIG.26). Control groups either received no treatment or IP injections of 25 mg/kg 5-FU every two days.
  • mice On day 14 post injection, mice were sacrificed and the surface area of dysplastic tissue was measured. Mice receiving both the engineered EcN and 5-FC showed similar reduced lesion size both visually (FIG. 27) and quantitatively (FIG.28) as compared to 5-FU injection. Mice administered bacteria lacking the yCD:UPRT enzyme and mice administered 5-FC with no bacteria showed similar levels of dysplastic tissue to the untreated control. To test the advantage of targeting dysplastic tissue using the ⁇ 1 integrin-invasin interaction, a similar experiment was performed with engineered bacteria expressing the yCD:UPRT enzyme and either invasin or invasin trunc .
  • mice were given bacterial enemas prepared in PBS in addition to twice daily IP injections of 250 mg/kg 5-FC.
  • Mice treated with invasin expressing bacteria showed lower levels of dysplastic tissue when compared to mice treated with invasintrunc expressing bacteria, suggesting that specific targeting to dysplastic tissue by the ⁇ 1 integrin-invasin interaction had a therapeutic advantage (FIG.29).
  • normal tissue was harvested from the mouse proximal colon and stained for the cleavage of caspase 3 as a marker for apoptosis.
  • the system uses targeting to dysplastic tissue based on bacterial access to ⁇ 1 integrin, in accordance with results in the previous examples showing that ⁇ 1 integrin is mislocalized to the apical membrane of intestinal epithelial cells early in the dysplasia development process.
  • results of the present example show that invasin- expressing bacteria bind and are internalized into cells in vivo, and that this effect can be used to convert 5-FC to 5-FU, leading to localized cell killing.
  • This approach may be a viable therapy to slow the development of adenomas in genetically predisposed populations, such as those suffered from familial adenomatous polyposis or Lynch syndrome.
  • Example 11 Delivery of a Direct Cytotoxic Agent Using Genetically Engineered Microorganisms
  • An engineered EcN strain is designed to deliver a mammalian cytotoxic protein as the payload, such as constitutively active caspase-3, which causes cell death.
  • the strain can be generally similar to the strain design shown in FIG.22, except that the payload gene is a gene for the mammalian cytotoxic protein.
  • Mice are injected as described in Example 6 above to initiate tumor formation, and treatment begins on day 3 post injection. For each enema (twice daily), bacteria are grown overnight at 30 °C and diluted 1:30 in LB and grown for 2 hours to mid-log phase.
  • the OD600 is measured and the effective OD600 of 4.5 is calculated.
  • Bacteria are pelleted and resuspended in 30 ⁇ L of 1x PBS. Mice are anesthetized and colons are flushed to remove feces. After 15 Attorney Docket No. MMS.003WO minutes, mice are given bacterial enemas and the anuses are sutured shut. After 1 hour, the sutures were removed. Mice are treated for 13 days. At day 11, tumor sizes are monitored via colonoscopy. On day 14, mice are euthanized and the colons were removed, flushed, and lateralized. Tumors were imaged on a Nikon SMZ18 dissection scope at 0.75x magnification.
  • mice treated with bacteria producing the cytotoxic protein are expected to have smaller tumors than mice treated with control bacteria that do not produce the cytotoxic protein and mice treated with control bacteria that express the cytotoxic protein and a truncated invasin. Additional examples of aspects of the present technology are described below as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples and do not limit the present technology. 1.
  • a method for treating a subject comprising: administering a genetically engineered microorganism to a gastrointestinal (GI) tract of the subject, wherein the genetically engineered microorganism comprises: a targeting gene encoding a targeting element that binds to a surface marker on a target cell in the GI tract, and a payload gene encoding a therapeutic agent.
  • the therapeutic agent is a prodrug converting enzyme that converts a prodrug into an active drug
  • the method further comprises administering the prodrug to the subject.
  • the prodrug comprises a precursor of 5- fluorouracil (5-FU) and the active drug comprises 5-FU. 4.
  • the method of Clause 3, wherein the precursor of 5-FU comprises 5- fluorocytosine (5-FC). 5.
  • the prodrug converting enzyme comprises a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof.
  • Attorney Docket No. MMS.003WO 6 The method of Clause 4 or 5, wherein the prodrug converting enzyme comprises a fusion protein of a cytosine deaminase and an uracil phosphoribosyltransferase (CD:UPRT). 7.
  • the CD:UPRT comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 6.
  • the CD:UPRT comprises a sequence of SEQ ID NO: 6.
  • the payload gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO: 8.
  • the payload gene comprises a sequence of any one of SEQ ID NO: 7 or SEQ ID NO: 8.
  • the precursor of 5-FU comprises pentyl N- [1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4- yl]carbamate (capecitabine).
  • the prodrug converting enzyme comprises a carboxylesterase.
  • the method of Clause 3, wherein the precursor of 5-FU comprises 5-FU glucuronide. 14.
  • the method of Clause 13, wherein the prodrug converting enzyme comprises a ⁇ -glucuronidase. 15. The method of Clause 2, wherein the prodrug converting enzyme comprises a purine nucleoside phosphorylase. Attorney Docket No. MMS.003WO 16. The method of Clause 15, wherein the prodrug comprises 6-methylpurine- deoxyriboside and the active drug comprises 6-methylpurine. 17. The method of Clause 15, wherein the prodrug comprises 9-beta-D- arabinofuranosyl-2-fluoroadenine monophosphate and the active drug comprises fluoroadenine. 18.
  • the prodrug comprises 1-(2-deoxy-2-fluoro- b-D-arabinofuranosyl)uracil
  • the active drug comprises 1-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl) 5-methyluracil monophosphate.
  • the prodrug converting enzyme comprises a thymidine kinase, a thymidine synthase, or a combination thereof.
  • the prodrug comprises ganciclovir
  • the prodrug converting enzyme comprises a viral thymidine kinase. 21.
  • the method of Clause 33 wherein the cancer is one or more of the following: colorectal cancer, Lynch syndrome cancer, familial adenomatous polyposis, hereditary non- polyposis colon cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, or a sporadic cancer.
  • Attorney Docket No. MMS.003WO 35 The method of any one of Clauses 1–34, wherein the payload gene comprises at least one intron. 36. The method of Clause 35, wherein the at least one intron comprises a spliceosomal intron. 37. The method of any one of Clauses 1–36, wherein the payload gene is operably linked to a promoter. 38.
  • the method of Clause 37, wherein the promoter is a mammalian promoter. 39. The method of Clause 37, wherein the promoter is a microbial promoter. 40. The method of any one of Clauses 1–39, wherein the targeting element comprises one or more of the following: an invasin, an intimin, an adhesin, a flagellin, a component of a type III secretion system, a leptin, or an antibody. 41. The method of any one of Clauses 1–40, wherein the surface marker is mislocalized on the target cell. 42.
  • the targeting element comprises an invasin and the surface marker comprises an integrin.
  • the invasin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1, or comprises a sequence of SEQ ID NO: 1. 45.
  • the genetically engineered microorganism further comprises a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell.
  • the lysis element comprises a lysin.
  • the lysin comprises listeriolysin O. 51.
  • the method of Clause 50 wherein the lysin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4, or comprises a sequence of any one of SEQ ID NO: 3 or SEQ ID NO.4.
  • 52. The method of any one of Clauses 1–51, wherein the genetically engineered microorganism comprises an auxotrophic mutation.
  • the auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell. 54.
  • the auxotrophic mutation comprises a mutation in one or more of the following: dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF.
  • Attorney Docket No. MMS.003WO 55 The method of any one of Clauses 52–54, wherein the auxotrophic mutation comprises a deletion of an essential gene, and the payload gene is located on a plasmid comprising a functional copy of the essential gene. 56.
  • the plasmid comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or a coding region thereof, or the plasmid comprises a sequence of SEQ ID NO: 5 or the coding region thereof.
  • the genetically engineered microorganism is configured to deliver a DNA molecule comprising the payload gene to the target cell.
  • the DNA molecule comprises a plasmid.
  • the method of Clause 57 or 58, wherein the DNA molecule comprises at least one binding site for a DNA binding protein that comprises one or more nuclear localization signals.
  • 60. The method of Clause 59, wherein the genetically engineered microorganism comprises a gene encoding the DNA binding protein.
  • 61. The method of any one of Clauses 1–56, wherein the genetically engineered microorganism is configured to deliver an RNA molecule comprising the payload gene to the target cell.
  • 62. The method of Clause 61, wherein the RNA molecule comprises an mRNA molecule.
  • MMS.003WO 64 The method of Clause 63, wherein the genetically engineered microorganism comprises a cassette that directs RNA self-amplification of the cassette in the target cell, and the payload gene is present in the cassette.
  • the cassette comprises an RNA viral genome replication apparatus.
  • the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP).
  • RdRP RNA-dependent RNA polymerase
  • 67 The method of Clause 66, wherein the RdRP comprises an alphavirus RdRP.
  • the alphavirus RdRP comprises one or more of nsp1, nsp2, nsp3, or nsp4. 69.
  • the method of any one of Clauses 61–68, wherein the genetically engineered microorganism comprises a deletion of an RNase gene. 70. The method of Clause 69, wherein the RNase gene comprises RNase III. 71. The method of any one of Clauses 1–56, wherein the genetically engineered microorganism is configured to deliver a protein comprising the therapeutic agent to the target cell. 72. The method of Clause 71, wherein the payload gene is operably linked to a microbial promoter. 73. The method of Clause 72, wherein the microbial promoter is a constitutive promoter. 74.
  • any one of Clauses 1–76 further comprising administering a second genetically engineered microorganism to the subject, wherein the second genetically engineered microorganism comprises a second payload gene encoding a detection marker.
  • the second genetically engineered microorganism is administered before, concurrently with, or after administration of the genetically engineered microorganism to the subject.
  • the detection marker comprises a protein, a peptide, or a nucleic acid molecule. 80.
  • the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter.
  • the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter.
  • the method of any one of Clauses 76–82 further comprising performing an endoscopic or colonoscopic procedure to detect the detection marker in the subject.
  • Attorney Docket No. MMS.003WO 84 The method of any one of Clauses 81–83, further comprising: upon detection of the detection marker, administering a third genetically engineered microorganism to the GI tract of the subject, wherein the third genetically engineered microorganism comprises the targeting gene and the payload gene.
  • the method of any one of Clauses 1–84 wherein the subject is low risk for colorectal cancer (CRC) or colorectal polyps.
  • CRC colorectal cancer
  • 86 The method of Clause 85, wherein the subject has no previous incidence of CRC or colorectal polyps. 87.
  • FAP familial adenomatous polyposis
  • FAP familial adenomatous polyposis
  • Lynch syndrome oligopolyposis
  • MAP MUTYH-associated polyposis
  • juvenile polyposis syndrome or Cowden syndrome.
  • a genetically engineered microorganism comprising: a targeting gene encoding a targeting element that binds to a surface marker on a target cell in a gastrointestinal (GI) tract; and a payload gene encoding a therapeutic agent.
  • the therapeutic agent is a prodrug converting enzyme that converts a prodrug into an active drug.
  • the genetically engineered microorganism of Clause 97 wherein the prodrug comprises a precursor of 5-fluorouracil (5-FU) and the active drug comprises 5-FU.
  • the genetically engineered microorganism of Clause 98 wherein the precursor of 5-FU comprises 5-fluorocytosine (5-FC).
  • the prodrug converting enzyme comprises a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof. 101.
  • CD:UPRT uracil phosphoribosyltransferase
  • the genetically engineered microorganism of Clause 98, wherein the precursor of 5-FU comprises pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2- yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine).
  • the prodrug converting enzyme comprises a carboxylesterase.
  • the genetically engineered microorganism of Clause 98, wherein the precursor of 5-FU comprises 5-FU glucuronide.
  • the genetically engineered microorganism of Clause 110, wherein the prodrug comprises 9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate and the active drug comprises fluoroadenine.
  • the genetically engineered microorganism of Clause 97 wherein the prodrug comprises 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, and the active drug comprises 1- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5-methyluracil monophosphate.
  • the prodrug converting enzyme comprises a thymidine kinase, a thymidine synthase, or a combination thereof.
  • the genetically engineered microorganism of Clause 97 wherein the prodrug comprises ganciclovir, and the prodrug converting enzyme comprises a viral thymidine kinase.
  • the therapeutic agent is a direct cytotoxic agent.
  • the direct cytotoxic agent comprises a microbial toxin or a caspase.
  • the direct cytotoxic agent comprises a cytotoxic protein or peptide.
  • the genetically engineered microorganism of Clause 123 wherein the cancer is one or more of the following: colorectal cancer, Lynch syndrome cancer, familial adenomatous polyposis, hereditary non-polyposis colon cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, or a sporadic cancer.
  • the targeting element comprises one or more of the following: an invasin, an intimin, an adhesin, a flagellin, a component of a type III secretion system, a leptin, or an antibody.
  • the targeting element comprises one or more of the following: an invasin, an intimin, an adhesin, a flagellin, a component of a type III secretion system, a leptin, or an antibody.
  • the surface marker is mislocalized on the target cell.
  • the surface marker is present on a luminal side of the target cell and is not present on a luminal side of a non-target cell.
  • the genetically engineered microorganism of Clause 133 or 134 wherein the targeting gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or a coding region thereof, or comprises a sequence of SEQ ID NO: 2 or the coding region thereof.
  • the targeting element promotes internalization of the genetically engineered microorganism by the target cell.
  • the genetically engineered microorganism of Clause 139, wherein the lysin comprises listeriolysin O. 141.
  • the genetically engineered microorganism of Clause 140 wherein the lysin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4, or comprises a sequence of any one of SEQ ID NO: 3 or SEQ ID NO.4.
  • the genetically engineered microorganism of Clause 142 wherein the auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell.
  • the plasmid comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, Attorney Docket No. MMS.003WO 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or a coding region thereof, or the plasmid comprises a sequence of SEQ ID NO: 5 or the coding region thereof. 147.
  • the genetically engineered microorganism of Clause 151 or 152, wherein the RNA molecule comprises a self-replicating or self-amplifying RNA molecule.
  • the genetically engineered microorganism of Clause 153 wherein the genetically engineered microorganism comprises a cassette that directs RNA self- amplification of the cassette in the target cell, and the payload gene is present in the cassette.
  • the genetically engineered microorganism of Clause 154 wherein the cassette comprises an RNA viral genome replication apparatus. Attorney Docket No. MMS.003WO 156.
  • RdRP RNA-dependent RNA polymerase
  • the genetically engineered microorganism of Clause 157 wherein the alphavirus RdRP comprises one or more of nsp1, nsp2, nsp3, or nsp4.
  • the detection marker comprises a protein, a peptide, or a nucleic acid molecule.
  • the genetically engineered microorganism of Clause 166 or 167 wherein the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter.
  • the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter.
  • the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter
  • a method for treating a disease characterized by the presence of diseased gastrointestinal (GI) tissue in a subject in need thereof comprising: (a) administering to the epithelial tissue of a subject, a genetically engineered microorganism that specifically invades diseased cells of the epithelium, and directs expression of a prodrug converting enzyme in the diseased cells, and (b) administering a prodrug. 171.
  • GI diseased gastrointestinal
  • the method of Clause 170 wherein the genetically engineered microorganism comprises an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue; and wherein the surface protein promotes binding and/or invasion of the microorganism in the diseased epithelial cells.
  • CRC colorectal cancer
  • MMS.003WO 173 Attorney Docket No. MMS.003WO 173.
  • the method of Clause 172, wherein the subject has no previous incidence of CRC or colorectal polyps.
  • Clause 170 or 171 wherein the subject has familial adenomatous polyposis (FAP), attenuated FAP, Lynch syndrome (hereditary non-polyposis colorectal cancer (HNPCC)), oligopolyposis, Peutz-Jeghers syndrome (PJS), MUTYH- associated polyposis (MAP), juvenile polyposis syndrome, or Cowden syndrome.
  • FAP familial adenomatous polyposis
  • HNPCC human non-polyposis colorectal cancer
  • JS oligopolyposis
  • MAP MUTYH- associated polyposis
  • juvenile polyposis syndrome or Cowden syndrome.
  • the prodrug converting enzyme is selected from: an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5- fluorouracil (5-FU), the enzyme optionally selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, a uracil phosphoribosyltransferase (UPRT), and a combination thereof or a fusion protein comprising two or more thereof; an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU, optionally the enzyme being a carboxylesterase; an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU,
  • MMS.003WO (MePdR) into 6-Methylpurine optionally the enzyme being a purine nucleoside phosphorylase (PNP); an enzyme capable of catalyzing conversion of 9-beta-D-Arabinofuranosyl-2- fluoroadenine monophosphate(fludarabine monophosphate) into fluoroadenine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); and/or an enzyme capable of catalyzing conversion of 1-(2-Deoxy-2-fluoro-b-D- arabinofuranosyl)uracil into 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5- methyluracil monophosphate (FMAUMP), optionally the enzyme being a thymidine kinase (TK) and/or a thymidylate synthase (TS).
  • PNP purine nucleoside phosphorylase
  • TK thymidine
  • the prodrug is 5-fluorocytosine (5-FC), pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin- 4-yl]carbamate (capecitabine), 5-FU glucuronide, 6-Methylpurine-deoxyriboside (MePdR), fludarabine monophosphate, 1-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, and a combination of any two or more thereof. 181.
  • the prodrug is 5-fluorocytosine (5-FC), pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin- 4-yl]carbamate (capecitabine), 5-FU glucuron
  • the method of Clause 185 wherein the nucleic acid is a plasmid molecule.
  • the method of Clause 186, wherein the plasmid molecule comprises a gene encoding the prodrug converting enzyme.
  • the method of Clause 186 or 187, wherein the plasmid molecule comprises at least one binding site for a DNA binding protein.
  • the method of Clause 188, wherein the at least one binding site for the DNA binding protein is selected from SV40 enhancer, a transcription factor binding site, and a bacterial operator.
  • the method of Clause 189, wherein the DNA binding protein comprises one or more nuclear localization signal(s) (NLS). 191.
  • the NLS is an SV40 T antigen NLS sequence (KKKRKV).
  • the microorganism comprises a gene encoding the DNA binding protein. 193.
  • the genetically engineered microorganism has a deletion of an RNase gene.
  • the genetically engineered microorganism further comprises a gene encoding a detection marker.
  • the detection marker is selected from a fluorescent protein, a bioluminescent protein, a contrast agent for magnetic resonance imaging (MRI), a Positron Emission Tomography (PET) reporter, an enzyme reporter, a contrast agent for use in computerized tomography (CT), a Single Photon Emission Computed Tomography (SPECT) reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), and ion channel reporters (e.g., cAMP activated cation channel), and a combination of any two or more thereof.
  • MRI magnetic resonance imaging
  • PET Positron Emission Tomography
  • CT computerized tomography
  • SPECT Single Photon Emission Computed Tomography
  • the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713 (iRFP), iRFP720 and iSplit. 213.
  • the detection marker is a secretable biomarker that is excreted in a biological fluid selected from mucus, saliva, and urine.
  • the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two or more thereof.
  • the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two
  • the method of Clause 214 or 215, wherein the detecting comprises: (c1) obtaining a biological sample of the subject; and (c2) evaluating the sample for the presence or amount of the secretable biomarker.
  • the method of Clause 216, wherein the biological sample is selected from blood, plasma, serum, urine, feces, saliva, and mucus, or a combination of any two or more thereof. 218.
  • the method of Clause 223, wherein the surface protein is an invasin, or a fragment thereof. Attorney Docket No. MMS.003WO 225.
  • the method of Clause 223, wherein the surface protein comprises a peptide or protein that specifically binds to the surface of cancerous cells and/or pre-cancerous cells. 227.
  • the method of Clause 226, wherein the peptide or protein is selected from a leptin, an antibody, or a fragment thereof.
  • the method of Clause 243 wherein the selection mechanism is a marker causing complementation of a mutation in an essential gene and/or a nutritional marker.
  • the method of Clause 246, wherein the essential gene and/or a nutritional marker is alr and/or dadX. 248.
  • the precancerous lesion comprises a polyp selected from sessile polyp, serrated polyp (e.g., hyperplastic polyps, sessile serrated adenomas/polyps, and traditional serrated adenoma), sessile serrated polyp, flat polyp, sub- pedunculated polyp, pedunculated polyp, and a combination thereof.
  • serrated polyp e.g., hyperplastic polyps, sessile serrated adenomas/polyps, and traditional serrated adenoma
  • the precancerous lesion comprises a biliary intraepithelial neoplasm (BilIN) selected from BilIN-1, BilIN-2, BilIN-3, and cholangiocarcinoma.
  • BilIN biliary intraepithelial neoplasm
  • the precancerous lesion comprises a pancreatic intraepithelial neoplasm (PanIN) selected from PanIN -1, PanIN -2, PanIN -3 and pancreatic ductal adenocarcinoma (PDAC).
  • PDAC pancreatic ductal adenocarcinoma
  • the method of Clause 252, wherein the cancer comprises a polyp, an adenoma, or a frank cancer. 260.
  • the cancer comprises Lynch syndrome cancer, familial adenomatous polyposis, hereditary non-polyposis colon cancer (HNPCC), pancreatic ductal adenocarcinoma (PDAC), cholangiocarcinoma, or a sporadic cancer.
  • HNPCC hereditary non-polyposis colon cancer
  • PDAC pancreatic ductal adenocarcinoma
  • cholangiocarcinoma or a sporadic cancer.
  • a genetically engineered microorganism comprising an exogenous gene encoding a surface protein, wherein the surface protein specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue, and which is optionally epithelial tissue of the gastrointestinal tract, wherein the surface protein promotes binding and invasion of epithelial cells of diseased tissue, Attorney Docket No.
  • microorganism comprises a gene encoding a prodrug converting enzyme that is capable of converting a non-toxic prodrug to a pharmacologically active compound inside a diseased cell, the gene encoding a prodrug converting enzyme being operably linked to a promoter.
  • the prodrug converting enzyme is selected from: an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5- fluorouracil (5-FU), the enzyme optionally selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, a uracil phosphoribosyltransferase (yUPRT), and a combination thereof or a fusion protein comprising two or more thereof that; an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU, optionally the enzyme being a carboxylesterase; an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU
  • 265. The genetically engineered microorganism of any one of Clauses 261–264, wherein the prodrug converting enzyme is expressed from a mammalian promoter.
  • the genetically engineered microorganism of Clause 270 wherein the at least one binding site for the DNA binding protein is selected from SV40 enhancer, a transcription factor binding site, and a bacterial operator. 272.
  • RdRP RNA-dependent RNA polymerase
  • the genetically engineered microorganism of Clause 284, wherein the at least one intron is a spliceosomal intron. 286.
  • the genetically engineered microorganism of any one of Clauses 261–285, wherein the engineered microorganism further comprises a gene encoding a detection marker. 287.
  • the detection marker is selected from a fluorescent protein, a bioluminescent protein, a contrast agent for magnetic resonance imaging (MRI), a Positron Emission Tomography (PET) reporter, an enzyme reporter, a contrast agent for use in computerized tomography (CT), a Single Photon Emission Computed Tomography (SPECT) reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), and ion channel reporters (e.g., cAMP activated cation channel), and a combination of any two or more thereof. 288.
  • MRI magnetic resonance imaging
  • PET Positron Emission Tomography
  • CT computerized tomography
  • SPECT Single Photon Emission Computed Tomography
  • the genetically engineered microorganism of Clause 287 wherein the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713 (iRFP), iRFP720 and iSplit. 289.
  • the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two or more thereof.
  • the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two or more thereof.
  • the genetically engineered microorganism of Clause 292 wherein the Escherichia coli Nissle 1917 or the derivative thereof harbors a plasmid pMUT1 and/or a plasmid pMUT2, and/or derivatives thereof. 294.
  • the genetically engineered microorganism of Clause 297, wherein the surface protein comprises a peptide or protein that specifically binds to the surface of cancerous cells and/or pre-cancerous cells. 299.
  • Attorney Docket No. MMS.003WO 300 The genetically engineered microorganism of Clause 299, wherein the antibody, or a fragment thereof is selected from a single-domain antibody (sdAb) and an scFv fragment.
  • sdAb single-domain antibody
  • scFv fragment 301.
  • the microbial surface protein is selected from invasin, intimin and adhesin.
  • the genetically engineered microorganism of Clause 304 or 305 wherein the gene encoding the surface protein and/or the gene encoding the lysin is integrated in the genome of the microorganism. 307.
  • the genetically engineered microorganism of Clause 310 wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a plasmid. 313.
  • the selection mechanism is an antibiotic resistance marker selected from kanamycin resistance gene and tetracycline resistance gene.
  • Attorney Docket No. MMS.003WO 317 The genetically engineered microorganism of Clause 315, wherein the selection mechanism is a marker causing complementation of a mutation in an essential gene and/or a nutritional marker.
  • the genetically engineered microorganism of Clause 316 wherein the essential gene and/or a nutritional marker is selected from dapA, dapD, murA, alr, dadX, murI, dapE, thyA and a combination of any two or more thereof.
  • the genetically engineered microorganism of Clause 318 wherein the essential gene and/or a nutritional marker is alr and/or dadX.
  • the genetically engineered microorganism of Clause 320 wherein the inactivation of alr and dadX genes is caused by a point mutation, a frameshift mutation or a nonsense mutation. 322.
  • a pharmaceutical composition comprising the genetically engineered microorganism of any one of Clauses 96–169 or 261–323, and a pharmaceutically acceptable carrier. 325.
  • a method for treating a subject comprising administering the genetically engineered microorganism of any one of Clauses 96–169 or 261–323 or the pharmaceutical composition of Clause 324 to the subject.
  • Attorney Docket No. MMS.003WO Conclusion Although many of the embodiments are described above with respect to compositions and methods for treating and/or detecting diseases of the GI tract and/or associated tissues, the technology is applicable to other applications and/or other approaches, such as diagnosing and/or treating other types of diseases. Moreover, other embodiments in addition to those described herein are within the scope of the technology.
  • the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, may refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
  • the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • percent identity and sequence similarity may be performed using the BLAST algorithm, which is described in Altschul et al. (J. Mol. Biol.215:403-410 (1990)).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • the term “subject” may broadly refer to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.). To the extent any materials incorporated herein by reference conflict with the present disclosure, the present disclosure controls. It will also be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the technology. Further, while advantages associated with certain embodiments of the technology have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the technology. Accordingly, the disclosure and Attorney Docket No. MMS.003WO associated technology can encompass other embodiments not expressly shown or described herein.

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Abstract

Genetically engineered microorganisms and associated methods are provided. In some embodiments, a method for treating a subject includes administering a genetically engineered microorganism to the subject. The genetically engineered microorganism can include a targeting gene encoding a targeting element that binds to a surface marker on a target cell. The genetically engineered microorganism can include a payload gene encoding a therapeutic agent, such as a prodrug converting enzyme.

Description

Attorney Docket No. MMS.003WO ENGINEERED MICROORGANISMS FOR DELIVERY OF THERAPEUTIC AGENTS CROSS-REFERENCE TO RELATED APPLICATION(S) The present application claims the benefit of priority to U.S. Provisional Application No.63/431,538, filed December 9, 2022, which is incorporated by reference herein in its entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING The present application contains an electronic Sequence Listing in XML file format named “MMS_003WO_SL,” created on November 28, 2023, and having a size of 21.2 kilobytes, the contents of which are incorporated by reference herein in their entirety. TECHNICAL FIELD The present technology generally relates to engineered microorganisms, and in particular, to the use of genetically engineered microorganisms for delivery of therapeutic agents. BACKGROUND The gastrointestinal (GI) tract takes in food, digests it to extract and absorb energy and nutrients, and expels the remaining waste as feces. GI diseases are the diseases involving the organs that form the GI tract, which include the mouth, esophagus, stomach, small intestine, large intestine, and rectum. GI diseases include Barrett’s esophagus, inflammatory bowel disease (IBD) (e.g., Crohn’s disease, ulcerative colitis), irritable bowel syndrome (IBS), precancerous syndromes, and cancer. The diagnosis and treatment of GI diseases is challenging. For example, despite strong evidence demonstrating that screening of individuals with average colorectal cancer risk reduces mortality, compliance amongst individuals is limited due to the invasiveness, discomfort, and fear associated with colonoscopy. Techniques like endoscopy, colonoscopy, and computed tomography (CT) scanning can aid diagnosis by facilitating viewing of the lumen of the GI tract. For example, focal, irregular, or asymmetrical gastrointestinal wall thickening on a CT scan can indicate a malignancy. Segmental or diffuse gastrointestinal wall thickening can indicate an ischemic, inflammatory, or infectious disease. However, endoscopy, colonoscopy, etc., are invasive procedures and uncomfortable for the patient, particularly for Attorney Docket No. MMS.003WO patients that are at high risk for cancers of the GI tract, who may need these procedures more frequently. Moreover, colonoscopy is not very efficient in diagnosis of certain types of precancerous lesions such as flat polyps, which are easily missed during colonoscopy. Moreover, there is no treatment for small polyps other than their surgical removal. Some polyps are cancerous and even malignant at the time of their removal. Accordingly, a less invasive technique that provides treatment and optionally monitoring of diseased gastrointestinal tissue is desirable. BRIEF DESCRIPTION OF THE DRAWINGS Many aspects of the present disclosure can be better understood with reference to the following drawings. The components in the drawings are not necessarily to scale. Instead, emphasis is placed on illustrating clearly the principles of the present disclosure. FIGS. 1A–1E are schematic illustrations of payload delivery to diseased GI tissue using genetically engineered microorganisms. FIG. 1A illustrates localization of cell membrane receptors in healthy cells versus diseased cells of the GI tract. FIG. 1B illustrates selective binding of genetically engineered microorganisms to diseased cells via mislocalized cell membrane receptors. FIG. 1C illustrates transfer of a DNA payload. FIG. 1D illustrates transfer of an RNA payload. FIG.1E illustrates transfer of a protein payload. FIG.2 illustrates the conversion of 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) by cytosine deaminase (CD). FIG.3 shows a schematic of a strain for DNA transfer. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3). The strain carries a plasmid containing a payload gene under a eukaryotic promoter and selection based on a wild-type alr gene. FIG.4 shows a schematic of a strain for DNA transfer. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv) and listeriolysin O (hlyA). The strain carries a plasmid having an intron-containing gene encoding a chimeric bifunctional yeast cytosine deaminase (yCD) and the yeast uracil phosphoribosyltransferase (yUPRT) gene under a eukaryotic promoter. The plasmid also carries an iRFP670 gene under the control of an internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) (SEQ ID NO: 10). Attorney Docket No. MMS.003WO FIGS. 5A and 5B demonstrate killing of cancer cells in the presence of 5- fluorocytosine (5-FC) upon delivery of yeast cytosine deaminase. FIG. 5A is a bar graph demonstrating invasin- and LLO-dependent genetic transfer as assayed by the expression of iRFP670. FIG.5B shows invasin- and LLO-dependent, 5-fluorocytosine-mediated cell killing. FIG.6 shows a schematic of a strain for RNA transfer. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv) and listeriolysin O (hlyA; LLO). Bottom panel shows a schematic representation of a plasmid that the strain carries, a plasmid containing an iRFP670 gene under a prokaryotic promoter and under the control of EMCV IRES. FIGS. 7A and 7B demonstrate delivery of DNA and RNA by the bacterial strains disclosed herein. FIG. 7A demonstrates the expression of an iRFP670 reporter under the control of a eukaryotic promoter. FIG. 7B demonstrates the expression of an iRFP670 reporter expressed under a prokaryotic promoter and translated under the control of EMCV IRES. FIGS.8A and 8B demonstrate that deletion of ribonuclease III (RNase III, rncΔ) improves RNA delivery as determined by flow cytometry. FIG. 8A is a flow cytometry plot and FIG.8B is a graph illustrating the flow cytometry data of FIG.8A. FIGS. 9A–9H show the inducible mislocalization of β1 integrin in the Apcfl/fl,VilCre mice used herein. FIG.9A (left panel) shows a schematic representation of the mice, which develop tumors in distal colon after injection of 4-hydroxytamoxifen (right panel). FIG. 9B shows a representative confocal fluorescence microscopy image of a colon tissue section showing healthy tissue and diseased tissue. Progressive induction of colorectal cancer in Apcfl/fl,VilCre mice is shown as demonstrated by confocal fluorescence microscopy images of colon tissue sections taken 3 days (FIG. 9C), 5 days (FIG.9D), 7 days (FIG. 9E), 11 days (FIG.9F), 13 days (FIG.9G), and 3 weeks (FIG.9H) after the injection of 4-hydroxytamoxifen. Images are at 20x and 60x magnification. 60x magnification images (larger panels in FIGS. 9C–9H) depict staining for β1 integrin and cell nuclei. FIGS.10A–10C show the bacteria disclosed herein are internalized in cells in a receptor and ligand-dependent matter. FIG. 10A is a schematic representation of binding of invasin+ bacteria to cells expressing β1 integrin. FIG. 10B shows that invasin is required for bacterial binding to cells. Bacteria lacking invasin or harboring an invasin gene were incubated with HCT116 colorectal cancer cells in vitro, and internalization was measured by gentamicin Attorney Docket No. MMS.003WO protection assay. FIG. 10C shows that β1 integrin is required for bacterial binding to cells. Bacteria harboring an invasin gene were incubated in vitro with HCT116 colorectal cancer cells or HCT116 cells having the chromosomal gene for production of β1 integrin knocked out, and internalization was measured by gentamicin protection assay. FIGS. 11A and 11B show the bacteria disclosed herein can discriminate between diseased versus non-diseased tissue at 3 weeks. FIG.11A is a schematic representation of the experiment. Briefly, tumorigenesis was induced in Apcfl/fl;Vil-Cre-ERT2 mice by injecting 4-hydroxytamoxifen. Three weeks later, bacteria expressing wild-type invasin (“invasin+”) and GFP, or bacteria expressing a truncated invasin incapable of binding β1 integrin (“invasin-”) and RFP were administered to the mice by enema.3 hours later, the mice were sacrificed, and the colon was extracted and colon tissue was subjected to epifluorescence microscopy. FIG. 11B shows internalization of invasin+ bacteria but not the invasin- bacteria in diseased cells as examined by epifluorescence microscopy. FIGS. 12A and 12B show the bacteria disclosed herein can discriminate between diseased versus non-diseased tissue early during tumorigenesis. FIG. 12A is a schematic representation of the experiment. Briefly, tumorigenesis was induced in Apcfl/fl;Vil- Cre-ERT2 mice by injecting 4-hydroxytamoxifen. 3 to 7 days later, invasin+ bacteria expressing GFP or invasin- bacteria expressing RFP were administered to the mice by enema. 3 hours later, the mice were sacrificed, and the colon was extracted and colon tissue was subjected to epifluorescence microscopy. FIG. 12B illustrates epifluorescence microcopy of dysplastic and normal tissue at 5 and 7 days post induction of adenoma formation. E. coli Nissle 1917 (EcN) expressing invasin are shown in green and EcN expressing a truncated invasin incapable of binding β1 integrin (invasintrunc) are shown in red. Images are at 20x magnification. Internalization of invasin+ bacteria but not the invasin- bacteria in diseased cells is observed as early as 5 to 7 days. FIGS. 13A and 13B show the bacteria disclosed herein can discriminate between diseased versus non-diseased tissue early during tumorigenesis. FIG. 13A is a schematic representation of the experiment. Briefly, tumorigenesis was induced in Apcfl/fl;Vil- Cre-ERT2 mice by injecting 4-hydroxytamoxifen. 3 to 7 days later, invasin+ bacteria expressing RFP or invasin- bacteria expressing GFP were administered to the mice by enema. 3 hours later, the mice were sacrificed, and colon was extracted and colon tissue was subjected to epifluorescence microscopy. FIG. 13B illustrates epifluorescence microcopy of dysplastic and normal tissue at 5 and 7 days post induction of adenoma formation. EcN expressing invasin Attorney Docket No. MMS.003WO are shown in red and EcN expressing invasintrunc are shown in green. Images are at 20x magnification. Internalization of invasin+ bacteria but not the invasin- bacteria in diseased cells is observed as early as 5 to 7 days post induction. FIGS.14A–14C show that the bacteria disclosed herein invade only in the tumor tissue. FIG.14A shows a surgical intervention to increase bacterial concentration in the colon. FIG.14B is a graph showing the number of bacteria that were present in non-tumor and tumor tissue. FIG. 14C shows fluorescence microscopy from different mice showing bacteria that were present in tumor tissue (right images) but not in non-tumor tissue (left images). FIGS. 15A and 15B show the endocytosis of bacteria disclosed herein in an invasin-dependent manner. FIG.15A shows a schematic representation of the experiment. FIG. 15B is a graph that plots the number of invasin+ and invasin- bacteria internalized in HCT116 colon cancer cells as a function of time. FIGS.16A–16C show the endocytosis of bacteria disclosed herein in an invasin- dependent manner. Briefly, tumor bearing Apcfl/fl,VilCre mice were administered invasin+ fluorescent bacteria by enema. After 2 hours, the proximal colon tissue (non-tumor tissue) and distal colon tissue (having tumors) were extracted and processed as in FIGS.16A to 16C. FIG. 16A shows fluorescence microscopy and phase contrast images of colon tissue showing bacteria associated with tumor tissue but not non-tumor tissue. FIG.16B shows the results of a semi-quantitative assay for bacteria that adhered to but did not invade colon tissue. The colon tissue was added into 1.5 mL tube with 200 µL PBS and vortexed twice for 10 seconds each. Serial dilutions of the supernatants were spotted on an LB plate. FIG.16C shows the results of a semi-quantitative assay for bacteria that invaded colon tissue. The washed colon tissue was incubated with gentamycin followed by a treatment with 0.3% Triton-X (#2), and centrifuged at 400g for 1 minute to remove tissue fragments. The supernatant was sample #3. The tissue pellet was resuspended in PBS (sample #4). The rest of the supernatant was centrifuged at 21000g for 5 minutes, and the pellet was resuspended in PBS (sample #5). Serial dilutions of the samples #2–#5 were spotted on an LB plate. FIG. 17 is a schematic representation of amplification of a payload (e.g., a nucleic acid encoding a prodrug converting enzyme and/or a detection marker) in target cells using self-amplifying RNA. The construct includes the functional components of RNA- dependent RNA polymerase (RdRP) or viral genome replication apparatus: nsp1, nsp2, nsp3 and nsp4, and mRNA of a payload sequence and a poly(A) tail. As shown, upon translation of Attorney Docket No. MMS.003WO the nsp1-4 genes present on the (+) strand of the construct, an RdRP is produced, which replicates the construct to produce (-) and (+) strands. Transcription of the (-) strand produces payload mRNA, which is translated to the payload protein. FIG. 18 is a schematic representation of improvement of signal from RNA transfer of payload using self-amplifying RNA. The cell on the left side shows conventional RNA transfer. The cell on the right side shows amplification of the payload in target cells using self-amplifying RNA. FIG. 19 is a graph showing the results of an in vitro invasion assay with mNeonGreen expressing bacteria displaying invasin or invasintrunc on HCT116 cells. Data points represent three independent biological replicates with the standard of deviation shown. FIG. 20 is a graph showing the results of an in vitro gentamycin protection assay. Data represent the average of three biological replicates with the standard of deviation shown. FIG.21 is a graph showing the results of an in vivo gentamycin protection assay. Data represent the average of five biological replicates with the standard of deviation shown. P values are for a paired student t-test between groups indicated. FIG.22 shows a schematic of a strain for protein transfer. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG. 22 (top panel)). This strain carries a plasmid containing a payload gene under a prokaryotic promoter and selection based on a wild-type alr gene (FIG.22 (bottom panel)). FIG.23 is a graph showing the conversion of 5-FC to 5-FU by engineered EcN measured by mass spectrometry over time. FIG.24 is a graph showing the dose response curves for the killing of wild type HCT116 cells by engineered bacteria in the presence of varying concentrations of 5-FC. Each concentration was normalized to the total number of living cells in the wells lacking 5-FC. Data points are representative of three biological replicates with error bars denoting the standard of deviation. FIG.25 is a graph showing the dose response curves for the killing of wild type HCT116 cells and β1 integrin knockout cells by engineered bacteria in the presence of varying concentrations of 5-FC. Each concentration was normalized to the total number of living cells Attorney Docket No. MMS.003WO in the wells lacking 5-FC. Data points are representative of three biological replicates with error bars denoting the standard of deviation. FIG.26 is a schematic showing a timeline of a treatment regimen for mice with induced dysplasia. FIG.27 is a series of representative images showing a dissected colon. Areas of dysplasia are outlined in white. FIG.28 is a graph showing the quantification of adenoma surface area in mouse cohorts. Data was generated over 3 separate experiments. Each point represents an individual animal with bars representing the average and error bars representing the standard of deviation. Asterisks indicate p-values for a one way ANOVA test with **** <0.0001, *** <0.001, ** < 0.01, and * <0.05. FIG. 29 is a graph showing the quantification of adenoma surface area in experiments comparing engineered EcN expressing invasin or invasintrunc. Each point represents an individual animal with bars representing the average and error bars representing the standard of deviation. * indicates a p value < 0.05 by a one way ANOVA test. FIG. 30 is a series of images showing representative histology for cleaved caspase 3 in mice undergoing different treatments. Arrows indicate sites of caspase 3 cleavage. FIG. 31 is a graph showing the quantification of intestinal crypts showing caspase 3 cleavage as a marker for apoptosis. Each point represents an analysis of 45 crypts across n = at least 7 animals per group with bars representing the average and error bars representing the standard of deviation. **** indicates a p value < 0.0001 by a one way ANOVA test. FIG.32 is a graph showing dose response curves for the killing of β1 integrin knockout HCT116 cells by engineered bacteria in the presence of varying concentrations of 5- FC. Each concentration was normalized to the total number of living cells in the wells lacking 5-FC. Data points are representative of three biological replicates with error bars denoting the standard of deviation. DETAILED DESCRIPTION The present technology relates to genetically engineered microorganisms and associated compositions and methods of use. In some embodiments, for example, a method for treating a subject includes administering a genetically engineered microorganism to the subject. Attorney Docket No. MMS.003WO The genetically engineered microorganism can include a targeting gene encoding a targeting element that binds to a surface marker on a target cell and, optionally, promotes internalization of the genetically engineered microorganism by the target cell. The genetically engineered microorganism can include a payload gene encoding a therapeutic agent, such as a prodrug converting enzyme that converts an inactive prodrug to an active drug. The method can further include administering the prodrug to the subject. Optionally, the genetically engineered microorganism can include a second payload gene encoding a detection marker. The present technology can provide numerous advantages compared to conventional techniques for treating and/or diagnosing a subject having a disease such as cancer. For example, conventional techniques for diagnosing abnormally growing cells in the gastrointestinal (GI) tract are typically based upon invasive colonoscopies that are not comfortable for the patient and not always successful in detection of diseased cells. The ability to visualize and remove abnormal cells and diseased tissue varies depending on the skills of the surgeon and prominence of the polyps or tumors. Certain abnormally growing cells are flat or small in number and therefore, not visualized and removed by even skilled surgeons. Further, for patients that have a condition that involves increased risk of colorectal cancer, frequent colonoscopies are a substantial burden. Moreover conventional chemotherapeutic agents used for treating cancer typically lack any mechanism for differentiating between diseased and healthy cells, and thus may produce severe side effects due to off-target activity. In contrast, the genetically engineered microorganisms described herein allow a therapeutic payload to be delivered to diseased cells with a high degree of specificity, thus reducing the incidence of side effects due to off-target delivery. Moreover, the genetically engineered microorganisms herein can differentiate between diseased and healthy cells at an early stage of cancer development, thus allowing for improved patient outcomes due to earlier detection and/or treatment. Embodiments of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings in which like numerals represent like elements throughout the several figures, and in which example embodiments are shown. Embodiments of the claims may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. The examples set forth herein are non-limiting examples and are merely examples among other possible examples. The headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed present technology. Embodiments under any one heading may be used in conjunction with embodiments under any other heading. Attorney Docket No. MMS.003WO I. Anatomy and Pathology of the GI Tract and Associated Tissues The GI tract includes the mouth, esophagus, stomach, small intestine, large intestine, and rectum. The GI wall surrounding the lumen of the GI tract is made up of four concentric layers, arranged from the lumen outwards: (1) mucosa, (2) submucosa, (3) muscular layer, and (4) serosa (if the tissue is intraperitoneal) or adventitia (if the tissue is retroperitoneal) The characteristics of the mucosa depends on the organ. For example, the stomach mucosal epithelium is simple columnar, and is organized into gastric pits and glands to deal with secretion. The small intestinal mucosa, which is made of glandular epithelium intermixed with secretory cells (e.g., goblet cells and Paneth cells) and immune cells (e.g., dendritic cells and M cells of the gut-associated lymphoid tissue (GALT)) arranged into villi, creating a brush border and increasing the area for absorption. The epithelial cells of the GI tract form a polarized continuous layer. The epithelial cells are connected by tight and adherens junctions, creating a barrier at the apical surface, which controls the selective diffusion of solutes, ions, and proteins between the apical and basal tissue compartments. The apical surface of the cells faces the GI tract lumen, and the basolateral surface sits adjacent to an internal-facing basement membrane. The basement membrane is an extracellular matrix (ECM) that includes laminins, collagen IV, proteoglycans, and nidogen. The epithelial cells interact with the ECM through integrins and the transmembrane proteoglycan dystroglycan, which are integral membrane proteins that bind to ECM components as well as intracellular proteins. β1 integrins, which are widely expressed in epithelial cells, have a central role in establishing their polarity. For example, the binding of integrin to ECM components activates signaling by the integrins, which influences the organization of the cytoskeleton, which contributes to cellular polarity. Disruption of the polarity and barrier function can cause disease. For example, following inactivation of the tumor suppressor gene APC, tissue polarity is lost very early during cancer progression. Thus, the mislocalization of integrins at the opposing basal surface domain has been correlated with loss of epithelial architecture and cancer development. Similarly, pathogens such as enteropathogenic E. coli and Y. pseudotuberculosis may disrupt cell polarity and enable the apical migration of basolateral membrane proteins. Moreover, diseases such as Crohn’s disease, untreated celiac disease, irritable bowel syndrome, and inflammatory bowel disease may feature disruption of the barrier function. Therefore, the detection of mislocalized and/or aberrantly expressed cell surface molecules can have great diagnostic value, as described further herein. Attorney Docket No. MMS.003WO Tissues that are associated with the GI tract include, for example, the bile ducts, the gallbladder, the pancreatic duct, and the pancreas. A bile duct is a long tube-like structure that carries bile. Small bile ducts are visible in portal triads of liver lobules, which also contain a small hepatic artery branch, and a portal vein branch. The small bile ducts fuse to form larger bile ducts. The larger bile ducts in the hepatic triads coalesce to intrahepatic bile ducts that become the right and left hepatic ducts that fuse at the undersurface of the liver to become the common bile duct. About halfway down the common bile duct, the cystic duct (carrying bile to and from the gallbladder) branches off to the gallbladder. The common bile duct opens into the intestine. The intrahepatic ducts, cystic duct, and the common bile duct are lined by a tall columnar epithelium. The gallbladder stores bile excreted from the liver. The columnar mucosa is arranged in folds over the lamina propria, allowing expansion. Beneath the lamina propria is a muscularis, and surrounding the gallbladder is a connective tissue layer and serosa. The gallbladder mucosa transports out sodium in the bile, passively followed by chloride and water. Thus, bile excreted by the liver and stored in the gallbladder becomes more concentrated. The muscularis of the gallbladder contracts under the influence of the hormone cholecystokinin excreted by enteroendocrine cells of the small intestine. The pancreatic duct, or duct of Wirsung (also known as the major pancreatic duct), is a duct joining the pancreas to the common bile duct. The pancreatic duct joins the common bile duct just prior to the ampulla of Vater, after which both ducts perforate the medial side of the second portion of the duodenum at the major duodenal papilla. There are many rare anatomical variants as well. Pancreatic ducts are lined by columnar cells with luminal microvilli and glycocalyx and small apical cytoplasmic mucin droplets. In large pancreatic ducts, many epithelial cells also have cilia, which function to aid the downstream movement of exocrine secretions. The genetically engineered microorganisms described herein can be used to detect and/or treat many different diseases of the GI tract and/or associated tissues. In some embodiments, the disease is a cancer or a precancerous syndrome, such as squamous cell carcinoma of anus, colorectal cancer (including colorectal adenocarcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer), colorectal polyposis (including Peutz-Jeghers syndrome, juvenile polyposis syndrome, MUTYH-associated polyposis, familial adenomatous polyposis/Gardner’s syndrome, and Cronkhite-Canada syndrome), carcinoid tumor, pseudomyxoma peritonei, duodenal adenocarcinoma, distal bile Attorney Docket No. MMS.003WO duct carcinomas, pancreatic ductal adenocarcinomas, gastric carcinoma, signet ring cell carcinoma (SRCC), gastric lymphoma (MALT lymphoma), linitis plastic (Brinton’s disease), or squamous cell carcinoma of esophagus and adenocarcinoma. Colorectal cancer (CRC) is a common and often lethal cancer that originates in the large intestine. Disease recognition at an early stage is advantageous to devise suitable preventive cancer strategies. Colorectal adenoearyma is the most frequent precancerous lesion. Colorectal adenoma is an asymptomatic lesion often found incidentally during colonoscopy performed for unrelated symptoms or for CRC screening. About 25% of men and 15% of women who undergo colonoscopic screening have one or more adenomas. Up to 40% of people over the age of 60 harbor colorectal adenomatous polyps as shown in the colonoscopy examinations, although not all colonic polyps are adenomas and more than 90% of adenomas do not progress to cancer. Other potentially premalignant conditions include chronic inflammatory bowel diseases and hereditary syndromes, such as familial adenomatous polyposis (FAP), Peutz- Jeghers syndrome, and juvenile polyposis. These conditions can involve different sites of the GI tract. In some embodiments, the disease is polyposis (including FAP and attenuated FAP (AFAP), which are caused by pathogenic variants in the APC gene; and MUTYH-associated polyposis, which is caused by pathogenic variants in the MUTYH gene); and/or Lynch syndrome (often referred to as hereditary nonpolyposis colorectal cancer), which is caused by germline pathogenic variants in DNA MMR genes (MLH1, MSH2, MSH6, and PMS2) and EPCAM. Other CRC syndromes and their associated genes include oligopolyposis (POLE, POLD1), NTHL1, juvenile polyposis syndrome (BMPR1A, SMAD4), Cowden syndrome (PTEN), and Peutz-Jeghers syndrome (STK11). Lynch syndrome, also known as hereditary non-polyposis colon cancer (HNPCC), accounts for 2% to 4% of all CRC cases. Individuals with HNPCC have about a 75% lifetime risk of developing CRC, and are predisposed to several types of cancer. Colon cancers and polyps arise in Lynch syndrome patients at a younger age than in the general population with sporadic neoplasias, and the tumors develop at a more proximal location. These cancers are often poorly differentiated and mucinous. Muir-Torre syndrome is a variant of Lynch syndrome that presents additional predisposition to certain skin tumors. FAP, having a prevalence of 1 in 10,000 individuals, is the second most common genetic syndrome predisposing to CRC. The lifetime risk of developing CRC for Attorney Docket No. MMS.003WO individuals suffering from FAP without prophylactic colectomy approaches 100%. The characteristic features of FAP include the development of hundreds to thousands of colonic adenomas beginning in early adolescence. The average age of CRC diagnosis (if untreated) in FAP patients is 40 years; 7% develop a tumor by the age of 20 and 95% by the age of 50. AFAP is a less severe form of the disease, with an average lifetime risk of CRC of 70%. In this group, approximately 30 adenomatous polyps develop in the colon, colonic neoplasms tend to be located in the proximal colon, and cancer occurs at an older age. Gardner’s syndrome and Turcot’s syndrome are rare variants of FAP. In addition to polyps, Gardner’s syndrome causes extra-colonic symptoms like epidermoid cysts, osteomas, dental abnormalities, and/or desmoid tumors. Turcot’s syndrome causes colorectal adenomatous polyps and predisposition to developing malignant tumors of the central nervous system, such as medulloblastoma. The genetic conditions MUTYH-associated polyposis, Peutz-Jeghers syndrome, and juvenile polyposis syndrome are other rarer syndromes that cause colon polyps, and predisposition to cancer. Patients with MUTYH-associated polyposis (MAP) develop adenomatous polyposis of the colorectum and have an 80% risk of CRC. Peutz-Jeghers and juvenile polyposis syndromes exhibit an increased risk for colorectal and other malignancies with the lifetime risk of CRC is approximately 40%. Biliary tract cancers, also called cholangiocarcinomas, refer to those malignancies occurring in the organs of the biliary system, including pancreatic cancer, gallbladder cancer, and cancer of bile ducts. Approximately 7,500 new cases of biliary tract cancer are diagnosed each year. These cancers include about 5,000 gallbladder cancers, and between 2,000 and 3,000 bile duct cancers. The preneoplastic and neoplastic lesions of the bile duct and pancreas share analogies in terms of molecular, histological, and pathophysiological features. Intraepithelial neoplasms are reported in the biliary tract, as biliary intraepithelial neoplasm (BilIN), and in the pancreas, as pancreatic intraepithelial neoplasm (PanIN). Both can evolve to invasive carcinomas, respectively cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC). BilINs are usually encountered in the epithelium lining the extrahepatic bile ducts (EHBDs), and large intrahepatic bile ducts (IHBDs), and may also be found in the gallbladder. BilINs are microscopic lesions, with a micropapillary, pseudopapillary, or flat growth pattern, involved in the process of multistep cholangiocarcinogenesis. Based on the degree of cellular and architectural atypia, BilINs have been classified into three categories: BilIN-1 (low grade dysplasia) showing the mildest changes compared to non-neoplastic Attorney Docket No. MMS.003WO epithelium of the bile ducts; BilIN-2 (intermediate grade dysplasia) with increased nuclear atypia and focal anomalies of cellular polarity as compared to BilIN-1; and BilIN-3 (high grade dysplasia or carcinoma in situ), which are usually identified in proximity of cholangiocarcinoma areas. About 30,000 new cases of pancreatic cancer are diagnosed in the United States each year. Because the early symptoms are vague, and there are no screening tests to detect it, early diagnosis is difficult. PanINs are defined as microscopic flat or micropapillary noninvasive lesions. These lesions are frequently less than 5 mm in size, and considered the most common malignant precursors of PDAC. A lower proportion of cases of PDAC also originate from the intraductal papillary mucinous neoplasms of the pancreas (IPMNs) and mucinous cystic neoplasms (MCNs). PanINs have also been classified, according to the degree of cellular and architectural atypia, into low grade (previously classified as PanIN-1 and PanIN- 2) with mild-moderate cytological atypia and basally located nuclei, and high grade (previously classified as PanIN-3) with severe cytological atypia, loss of polarity, and mitoses. In some embodiments, the genetically engineered microorganisms disclosed herein are used to detect and/or treat an inflammatory bowel disease. Inflammatory bowel disease (IBD) is a group of nonspecific chronic inflammatory diseases of the gut, which includes Crohn’s disease, ulcerative colitis (UC), and indeterminate colitis. The pathogenesis of IBD remains unclear, and it is characterized by long-lasting and relapsing intestinal inflammation. The incidence of UC in the United States is estimated to be between 9 and 12 per 100,000 persons with a prevalence of 205 to 240 per 100,000 persons. The etiology of UC is unknown. However, abnormal immune responses to contents in the gut, including intestinal microbes, are thought to drive disease in genetically predisposed individuals. Colitis-associated colorectal cancer (CACC) is one of the most serious complications of IBD, particularly in UC; it accounts for approximately 15% of all causes mortality among IBD patients. Because of worse prognosis and higher mortality in CACC than in sporadic CRC, early CACC detection is crucial. Crohn’s disease is marked by inflammation of the GI tract. The inflammation can appear anywhere in the GI tract from the mouth to the anus. People with the disease often experience ups and downs in symptoms, and may even experience periods of remission. The length of diagnostic delay can represent an issue for at least a proportion of patients with Crohn’s disease. However, Crohn’s disease is a progressive disease that starts with mild Attorney Docket No. MMS.003WO symptoms and gradually gets worse. Early diagnosis is important to help prevent bowel damage such as fistulae, abscesses, or strictures. In some embodiments, the genetically engineered microorganisms disclosed herein are used to detect and/or treat irritable bowel syndrome. Irritable bowel syndrome (IBS) is a disorder which manifests as a set of chronic GI symptoms and changes in bowel habits in the absence of evident structural and biochemical abnormalities. IBS has a global prevalence of 10% to 15% and is more frequent among individuals aged < 50 years old. Altered bowel habits are the most commonly reported clinical feature, with the syndrome predominantly associated with constipation (IBS-C), diarrhoea (IBS-D) or a mixture of both conditions (IBS- M). In addition, patients with IBS often experience abdominal pain, which can be provoked by emotional stress or eating and is usually alleviated by the passing of stool. A diagnosis of IBS is confirmed according to the latest version of the Rome criteria based on the clinical experience and consensus of a committee of multinational experts. In some embodiments, the genetically engineered microorganisms disclosed herein are used to detect and/or treat Barrett’s esophagus. Barrett’s esophagus is a condition in which tissue that is similar to the lining of intestine replaces tissue lining the esophagus. People with Barrett’s esophagus may develop esophageal adenocarcinoma. The exact cause of Barrett’s esophagus is unknown, but gastroesophageal reflux disease (GERD) increases the risk of developing Barrett’s esophagus. II. Genetically Engineered Microorganisms The present technology provides genetically engineered microorganisms that can be used to detect and/or treat a disease in a subject, such as a disease characterized by the presence of diseased GI tissue. As described herein, diagnosis, and specifically early diagnosis, can be advantageous for preventing mortality and morbidity in individuals suffering from precancerous lesions, cancers of the GI tract and/or associated tissues (e.g., CRC, biliary tract cancer, pancreatic cancer), IBD (e.g., Crohn’s disease, ulcerative colitis), IBS, Barrett’s esophagus, and/or other diseases described herein. The genetically engineered microorganisms of the present disclosure can be used to detect the presence of target cells (e.g., diseased and/or abnormal cells) that are indicative of the disease at an early stage with a high level of sensitivity and specificity, thus allowing for prophylactic measures and/or providing improved treatment outcomes. Attorney Docket No. MMS.003WO In some embodiments, the genetically engineered microorganisms herein include one or more of the following: a targeting gene that encodes a targeting element that allows the microorganism to bind to and enter a target cell (e.g., as described in Section II.A below), a lysis gene that encodes a lysis element that allows the microorganism and/or payload to enter the cytoplasmic space of the target cell (e.g., as described in Section II.B below), an auxotrophic mutation (e.g., as described in Section II.C below), a selection mechanism (e.g., as described in Section II.D below), and/or a payload gene encoding a therapeutic agent to be delivered to the target cell, such as a prodrug converting enzyme (e.g., as described in Section III below). The genetically engineered microorganism of the present technology may be derived from any non-pathogenic microorganism, such as the non-pathogenic microorganisms that are normal flora of human GI tract or the microorganisms that are generally recognized as safe for human consumption via foods like yogurts, cheeses, breads and the like. In some embodiments, the genetically engineered microorganism of any one of the embodiments disclosed herein may be derived from a microorganism selected from Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Lactococcus, Pediococcus, Leuconostoc, Bacillus, or Escherichia coli. Illustrative species that are suitable for the genetically engineered microorganism of any one of the embodiments disclosed herein include Bacillus coagulans, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium essencis, Bifidobacterium faecium, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium longum subsp. infantis, Bifidobacterium pseudolungum, Lactobacillus acidophilus, Lactobacillus boulardii, Lactobacillus breve, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus delbrueckii ssp. Bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus rhamnosus GG, Lactobacillus salivarius, Lactococcus lactis, Streptococcus thermophilus, Pediococcus acidilactici, Enterococcus faecium, Leuconostoc, Carnobacterium, Proprionibacterium, Saccharomyces boulardii, and Escherichia coli. In some embodiments, the genetically engineered microorganism of any one of the embodiments disclosed herein may be derived from a probiotic Escherichia coli strain, such as Escherichia coli Nissle 1917, Escherichia coli Symbioflor2 (DSM 17252), Escherichia coli strain A034/86, or Escherichia coli O83 (Colinfant). In some embodiments, the genetically Attorney Docket No. MMS.003WO engineered microorganism of any one of the embodiments disclosed herein is Escherichia coli Nissle 1917 or a derivative thereof. Escherichia coli Nissle 1917 contains two naturally occurring, stable, cryptic plasmids pMUT1 (GenBank Accession No. MW240712) and pMUT2 (GenBank Accession No. CP023342). In some embodiments, the Escherichia coli Nissle 1917 or the derivative thereof harbors a plasmid pMUT1 and/or a plasmid pMUT2, and/or one or more derivatives thereof. In some embodiments, the Escherichia coli Nissle 1917 or the derivative thereof is cured of the plasmid pMUT1 and/or the plasmid pMUT2. In some embodiments, the Escherichia coli Nissle 1917 or the derivative thereof includes an exogenous plasmid (e.g., a plasmid based on pUC18/19, a pBR322, R6K, 15A or a pSC10). In some embodiments, an Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT1 having a wild type alr gene as a selection mechanism. In some embodiments, the Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT1 having a wild type alr gene as a selection mechanism, and genes encoding invasin and/or listeriolysin, or a mutant derivative thereof. In some embodiments, the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of plasmid pMUT1 having a wild type alr gene under its own promoter as a selection mechanism, and optionally, genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof. In some embodiments, an Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT2 having a wild type alr gene under the control of its own promoter as a selection mechanism. In some embodiments, the Escherichia coli Nissle 1917 derivative harbors a derivative of plasmid pMUT2 having wild type alr gene under the control of its own promoter as a selection mechanism, and genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof. In some embodiments, the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of plasmid pMUT2 having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof. In some embodiments, an Escherichia coli Nissle 1917 derivative harbors an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism. The exogenous plasmid may be based on any plasmid that replicates in the microorganism (e.g., a plasmid based on pUC18/19, a pBR322, R6K, 15A or a pSC101). In some embodiments, the Escherichia coli Nissle 1917 derivative harbors a derivative of the exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and genes encoding invasin and/or listeriolysin O, or a mutant derivative Attorney Docket No. MMS.003WO thereof. In some embodiments, the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of the exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, genes encoding invasin and/or listeriolysin O, or a mutant derivative thereof. In some embodiments, the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of the exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and, optionally, comprising genes encoding a payload (e.g., a detection marker and/or a therapeutic agent). In some embodiments, an Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of pMUT1, pMUT2, or an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, a gene encoding a payload (e.g., a detection marker and/or a therapeutic agent). In some embodiments, the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of pMUT1, pMUT2, or an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, a gene encoding invasin and/or listeriolysin O, or a mutant derivative thereof. In some embodiments, the Escherichia coli Nissle 1917 derivative having null mutations in the alr and dadX genes harbors a derivative of pMUT1, pMUT2, or an exogenous plasmid having a wild type alr gene under the control of its own promoter as a selection mechanism, and optionally, genes encoding one or more of a payload (e.g., a detection marker and/or a therapeutic agent), invasin, listeriolysin O, or a mutant derivative thereof. The complete genome sequence of Escherichia coli Nissle 1917 is known. In some embodiments, the targeting gene (e.g., a gene encoding a surface protein such as invasin) is integrated at a first genomic site of the Escherichia coli Nissle 1917 derivative. Additionally or alternatively, in some embodiments, a lysis gene (e.g., a gene encoding a lysin such as listeriolysin O) is integrated at the same site or a second genomic site of the Escherichia coli Nissle 1917 derivative. In some embodiments, the target gene and the lysis gene are integrated at a single genomic site, optionally, the single genomic site is an integration site of a bacteriophage. In some embodiments, the bacteriophage is bacteriophage Lambda. Alternatively, the target gene and/or lysis gene are inserted into a plasmid, which is optionally a naturally occurring plasmid. Thus, in some embodiments, the one or more payload genes may be inserted on a natural endogenous plasmid from Escherichia coli Nissle 1917 (e.g., pMUT1, pMUT2, and/or a derivative thereof). In some embodiments, the plasmid includes a selection Attorney Docket No. MMS.003WO mechanism (e.g., an auxotrophic marker such as alr as described). In some embodiments, the targeting gene is inserted on a plasmid. In some embodiments, the lysis gene is inserted on the plasmid. A. Targeting Elements In some embodiments, the genetically engineered microorganism includes at least one targeting gene encoding a targeting element that, when expressed, causes the microorganism to specifically interact with a target cell (e.g., a diseased and/or abnormal cell). For example, the targeting element can promote binding of the microorganism to the target cell and/or entry of the microorganism into the target cell. The targeting element can be selected so that the microorganism has little or no interaction with non-target cells (e.g., non-diseased and/or normal cells). In some embodiments, the targeting element is a protein or peptide expressed on the surface of the microorganism that specifically interacts with one or more surface markers on the target cell. The surface marker can be a protein (e.g., a cell membrane receptor), peptide, glycoprotein, carbohydrate, or any other cell surface molecule that is expressed by or is otherwise characteristic of the target cell. For example, the surface marker can be found on the target cell but not on non-target cells. Alternatively, the surface marker can be found on both target and non-target cells, but can be localized differently in target and non-target cells such that the surface marker is accessible to the microorganism on the target cell but not accessible to the microorganism on the non-target cell. For instance, the surface marker can be a mislocalized and/or aberrantly expressed protein found in cancerous tissues and/or precancerous lesions (e.g., polyps or adenomas), tears and erosions (Barrett’s Esophagus), and/or cells indicative of inflammatory diseases. In some embodiments, the targeting gene is an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptors. In some embodiments, the cell membrane receptor is not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, or common bile duct, etc., but is exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, or common bile duct, etc., in the subject suffering from a disease. Thus, the surface protein can promote binding and invasion of the microorganism in the diseased epithelial cells, but not binding and invasion into normal cells. By virtue of the identity of the surface protein, the genetically engineered microorganism may Attorney Docket No. MMS.003WO mimic the affinity of the native surface protein. In some embodiments, the genetically engineered microorganism specifically binds to one or more of oral epithelial cells, buccal epithelial cells of the tongue, pharyngeal epithelial cells, mucosal epithelial cells, endothelial cells of the stomach, intestinal epithelial cells, colon epithelial cells, etc. In some embodiments, the targeting element is or includes an invasin, or a fragment thereof. The invasin can be selected from Yersinia enterocolitica invasin, Yersinia pseudotuberculosis invasin, Salmonella enterica PagN, Candida albicans Als3. In some embodiments, the targeting element is or includes an intimin, or a fragment thereof. The intimin can be selected from Escherichia albertii intimin (e.g., NCBI accession no. WP_113650696.1), Escherichia coli intimin (e.g., NCBI accession no. WP_000627885), or Citrobacter rodentium intimin (e.g., NCBI accession no. WP_012907110.1). In some embodiments, the targeting element is or includes an adhesin, or a fragment thereof. In some embodiments, the targeting element is or includes a flagellin, or a fragment thereof. In some embodiments, the targeting element includes one or more of the following: invasin, YadA (Yersinia enterocolitica plasmid adhesion factor), Rickettsia invasion factor RickA (actin polymerization protein), Legionella RaIF (guanine exchange factor), one or more Neisseria invasion factors (e.g., NadA (Neisseria adhesion/invasion factor), OpA, and/or OpC (opacity-associated adhesions)), Listeria InlA and/or InlB, one or more Shigella invasion plasmid antigens (e.g., IpaA, IpaB, IpaC, IpgD, IpaB-IpaC complex, VirA, and/or IcsA), one or more Salmonella invasion factors (e.g., SipA, SipC, SpiC, SigD, SopB, SopE, SopE2, and/or SptP), Staphylococcus FnBPA and/or FnBPB, one or more Streptococcus invasion factors (e.g., ACP, Fba, F2, Sfb1, Sfb2, SOF, and/or PFBP), an intimin, and/or Porphyromonas gingivalis FimB (integrin binding protein fibriae). In some embodiments, the targeting element is or includes a type III secretion system or a component thereof. In some embodiments, the targeting element is or includes a peptide or protein that specifically binds to the surface of target cells, such as a leptin, an antibody, or a fragment thereof (e.g., sdAb (also known as a Nanobody®) or an scFv fragment). In some embodiments, the targeting element is or includes a fusion protein of any two or more of the aforementioned proteins or fragments thereof. For example, the targeting element can be or include a fusion protein of invasin and intimin. In some embodiments, the targeting element includes an active fragment of one or more of invasin, YadA, RickA, RaIF, NadA, OpA, OpC, InlA, InlB, IpaA, IpaB, IpaC, IpgD, IpaB-IpaC, VirA, Attorney Docket No. MMS.003WO IcsA, SipA, SipC, SpiC, SigD, SopB, SopE, SopE2, SptP, FnBPA, FnBPB, ACP, Fba, F2, Sfb1, Sfb2, SOF, PFBP, or FimB. In some embodiments, the fragment(s) are expressed on the surface of the engineered microorganism disclosed herein, e.g., on an adhesion scaffold. Representative examples of targeting elements and their associated polynucleotide sequences are provided in Table 1 below. Table 1: Targeting Elements
Figure imgf000022_0001
Attorney Docket No. MMS.003WO
Figure imgf000023_0001
Attorney Docket No. MMS.003WO
Figure imgf000024_0001
In some embodiments, the genetically engineered microorganism produces a targeting element having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the genetically engineered microorganism includes a targeting element of SEQ ID NO: 1. In some embodiments, the genetically engineered microorganism produces a targeting element that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or the coding region thereof (e.g., residues 256–3213). In some embodiments, the genetically engineered microorganism includes a targeting element that is encoded by the sequence of SEQ ID NO: 2 or the coding region thereof. In some embodiments, the genetically engineered microorganism includes a single targeting gene encoding a targeting element that facilitates both binding to the target cell and entry into the target cell. Alternatively, the genetically engineered microorganism can include multiple targeting genes expressing multiple targeting elements, such as a first targeting gene encoding a first targeting element that facilitates binding to the target cell, and a second targeting gene encoding a second targeting element that facilitates entry into the target cell. In such embodiments, the multiple targeting elements can be expressed as multiple separate proteins, or can be expressed as a single fusion protein. Optionally, if the target cell is capable of internalizing the genetically engineered microorganism without aid of a targeting element, the genetically engineered microorganism can include a targeting gene encoding a targeting element that facilitates binding to the target cell, without any targeting elements to facilitate entry into the target cell. The targeting gene(s) can be incorporated in a genomic site of the genetically engineered microorganism (e.g., a chromosome), a non-genomic site of the genetically engineered microorganism (e.g., a plasmid such as an episome, which may be a natural endogenous plasmid or an exogenous plasmid), or suitable combinations thereof. For example, in embodiments where the genetically engineered microorganism is Escherichia coli Nissle 1917 or a derivative thereof, one or more targeting genes can be integrated into a genomic site Attorney Docket No. MMS.003WO of the Escherichia coli Nissle 1917 or derivative thereof, which can be the integration site of a bacteriophage (e.g., bacteriophage Lambda). Alternatively or in combination, one or more targeting genes can be inserted into a plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The targeting gene(s) can be incorporated at the same site or different site as the other genetic elements described herein (e.g., lysis gene(s), payload gene(s), and/or selection mechanism(s)). In some embodiments, the targeting gene(s) are integrated at a first genomic site, and the lysis gene(s) are integrated at the first genomic site or a second, different genomic site. In some embodiments, the target gene(s) and the lysis gene(s) are integrated at a single genomic site, which can be an integration site of a bacteriophage (e.g., bacteriophage Lambda). In some embodiments, the targeting gene(s) are integrated at a first genomic site; the lysis gene(s) are integrated at the first genomic site or a second, different genomic site; and the payload gene(s) are integrated at the first genomic site, the second genomic site, or a third, different genomic site. In some embodiments, the target gene(s), the lysis gene(s), and the payload gene(s) are integrated at a single genomic site, which can be an integration site of a bacteriophage (e.g., bacteriophage Lambda). In some embodiments, the targeting gene(s) are integrated at a first genomic site, the lysis gene(s) are integrated at the first genomic site or a second, different genomic site, and the payload gene(s) is inserted in a plasmid. In some embodiments, the targeting gene(s) are integrated at a first genomic site, the lysis gene(s) are integrated at the first genomic site or a second, different genomic site, and the payload gene(s) and selection mechanism(s) are inserted in a plasmid. The plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The plasmid may be a single copy of a multi-copy plasmid. In some embodiments, the targeting gene(s) are integrated at a first genomic site, and the lysis gene(s) are inserted into a first plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The first plasmid may be a single copy of a multi-copy plasmid. The payload gene(s) can be integrated into the first genomic site, or a second, different genomic site; or can be inserted into the first plasmid or a second, different plasmid. The second plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The second plasmid may be a single copy of a multi-copy plasmid. Attorney Docket No. MMS.003WO In some embodiments, the lysis gene(s) are integrated at a first genomic site, and the targeting gene(s) are inserted into a first plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The first plasmid may be a single copy of a multi-copy plasmid. The payload gene(s) can be integrated into the first genomic site or a second, different genomic site; or can be inserted into the first plasmid or a second, different plasmid. The second plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The second plasmid may be a single copy of a multi-copy plasmid. In some embodiments, the targeting gene(s) are inserted into a first plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The first plasmid may be a single copy of a multi-copy plasmid. The lysis gene(s) can be inserted into the first plasmid or a second, different plasmid. The second plasmid can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The second plasmid may be a single copy of a multi-copy plasmid. The payload gene(s) can be inserted into the first plasmid or the second plasmid. The first plasmid and/or the second plasmid can include the selection mechanism(s). In some embodiments, the targeting gene(s), lysis gene(s), payload gene(s), and selection mechanism(s) are all inserted in the same plasmid. B. Lysis Elements In some embodiments, upon binding to the target cell, the genetically engineered microorganism is endocytosed (e.g., phagocytosed) by the target cell and thus becomes internalized within an endocytic vacuole (e.g., phagosome) the target cell. Accordingly, the genetically engineered microorganism can include at least one lysis gene encoding a lysis element that, when expressed, lyses the endocytic vacuole to allow the genetically engineered microorganism and/or payload to escape into the intracellular space. For example, the lysis element can be a lysin that lyses the endocytotic vacuole, and thereby contributes to pore formation, breakage, and/or degradation of the phagosome. In some embodiments, the lysin is a cholesterol-dependent cytolysin. In some embodiments, the lysin is selected from one or more of listeriolysin O, ivanolysin O, streptolysin, perfringolysin, botulinolysin, or leukocidin, or fragment or a mutant derivative thereof. In some embodiments, the lysin is listeriolysin O or a mutant derivative thereof (e.g., a derivative lacking a periplasmic secretion signal). Representative examples of lysis elements are provided in Table 2 below. Attorney Docket No. MMS.003WO Table 2: Lysis Elements
Figure imgf000027_0001
In some embodiments, the genetically engineered microorganism produces a lysis element having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the genetically engineered microorganism includes a lysis element of any one of SEQ ID NO: 3 or SEQ ID NO: 4. In some embodiments, the genetically engineered microorganism includes a single lysis gene encoding a single lysis element. Alternatively, the genetically engineered microorganism can include multiple lysis genes expressing multiple lysis elements, such as a first targeting gene encoding a first lysin and a second targeting gene encoding a second, different lysin. In such embodiments, the multiple lysis elements can be expressed as multiple separate proteins, or can be expressed as a single fusion protein. The lysis gene(s) can be incorporated in a genomic site of the genetically engineered microorganism (e.g., a chromosome), a non-genomic site of the genetically engineered microorganism (e.g., a plasmid such as an episome, which may be a natural endogenous plasmid or an exogenous plasmid), or suitable combinations thereof. For example, Attorney Docket No. MMS.003WO in embodiments where the genetically engineered microorganism is Escherichia coli Nissle 1917 or a derivative thereof, one or more lysis genes can be integrated into a genomic site of the Escherichia coli Nissle 1917 or derivative thereof, which can be the integration site of a bacteriophage (e.g., bacteriophage Lambda). Alternatively or in combination, one or more lysis genes can be inserted into a plasmid, which can be a naturally occurring plasmid (e.g., pMUT1 and/or pMUT2) or an exogenous plasmid. The lysis gene(s) can be incorporated at the same site or different site as the other genetic elements described herein (e.g., targeting gene(s), payload gene(s), and/or selection mechanism(s)). For example, in some embodiments, the lysis gene(s) are integrated at a first genomic site, the targeting gene(s) are integrated at the first genomic site or a second, different genomic site, and the payload gene(s) and selection mechanism(s) are inserted in a plasmid. Alternatively, the lysis gene(s), targeting gene(s), payload gene(s), and selection mechanism(s) can all be inserted in the same plasmid. In some embodiments, the lysis gene(s) are constitutively expressed. Alternatively, the lysis gene(s) can be conditionally expressed. For instance, expression of the lysis gene(s) can be induced after endocytosis, e.g., using a promoter that responds to changes in the environment indicative of endosomal localization, such as pH, temperature, and/or addition of a small molecule. Expression of the lysis gene(s) can occur from a plasmid (e.g., an episome) or from a genomic site (e.g., a chromosome, at the same location as the targeting gene(s) or at a different location). In other embodiments, however, the lysis gene(s) are optional and may be omitted. C. Auxotrophic Mutations In some embodiments, the genetically engineered microorganism harbors at least one auxotrophic mutation, such as a nutritional auxotrophic mutation. The auxotrophic mutation can be used to inhibit proliferation of the genetically engineered microorganism after administration to the subject (e.g., for containment purposes). For example, the auxotrophic mutation can be selected from one or more of dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF. The mutation can be a partial or complete deletion of the gene, or any other modification that causes inactivation of the gene. In some embodiments, the genetically engineered microorganism includes a single auxotrophic mutation (e.g., a partial or complete deletion of a single gene), while in other embodiments, the genetically engineered Attorney Docket No. MMS.003WO microorganism can include multiple auxotrophic mutations (e.g., a partial or complete deletion of multiple genes). In some embodiments, the auxotrophic mutation includes a deletion, inactivation, or decreased expression or activity of at least one gene involved in the synthesis of a metabolite (e.g., a non-genetically encoded amino acid) required for cell wall synthesis. For example, the gene can be required for synthesis of D-alanine or diaminopimelic acid. In some embodiments, the engineered microorganism has an inactivation of the dapA gene, which can be caused by a partial or complete deletion. Alternatively or in combination, the engineered microorganism can have an inactivation of the alr and dadX genes, which can be caused by a partial or complete deletion. In some embodiments, the engineered microorganism harbors a combination of dapA, alr, and dadX auxotrophic mutations. In some embodiments, the auxotrophic mutation facilitates lysis of the microorganism inside the target cell upon invasion, which can enhance efficacy of payload delivery to the intracellular space of the target cell. For example, the dapA auxotrophic mutation causes a defect in cell wall synthesis that can facilitate lysis of the microorganism inside the target cell upon invasion. In other embodiments, however, an auxotrophic mutation that facilitates lysis of the microorganism is optional and may be omitted. Optionally, the auxotrophic mutation can be used as a selection mechanism (e.g., an antibiotic-free selection mechanism). For example, the genetically engineered microorganism can include a plasmid that is selected by complementation of the at least one auxotrophic mutation (e.g., alr and dadX mutations) by at least one corresponding functional gene present on the plasmid (e.g., a functional alr gene). In such embodiments, the plasmid can also incorporate any of the other genetic elements described herein such as the targeting gene(s), lysis gene(s) and/or payload gene(s). Representative examples of polynucleotide sequences for selection markers for complementation of an auxotrophic mutation are provided in Table 3 below. Table 3: Selection Markers
Figure imgf000029_0001
Attorney Docket No. MMS.003WO
Figure imgf000030_0001
In some embodiments, the genetically engineered microorganism includes a selection marker that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or the coding region thereof (e.g., residues 191–1270). In some embodiments, the genetically engineered microorganism includes a selection marker that is encoded by the sequence of SEQ ID NO: 5 or the coding region thereof. D. Selection Mechanisms In some embodiments, the genetically engineered microorganism includes one or more plasmids having a selection mechanism. The selection mechanism can be used to ensure that the plasmid(s) having one or more of the genetic elements described herein (e.g., payload gene(s), targeting gene(s), and/or lysis gene(s)) are maintained within the genetically engineered microorganism. As described elsewhere herein, the plasmid(s) can include an endogenous plasmid (e.g., pMUT1 and/or pMUT2 for Escherichia coli Nissle 1917), an exogenous plasmid, or suitable combinations thereof. In some embodiments, the genetically engineered microorganism includes a single selection mechanism, while in other embodiments, Attorney Docket No. MMS.003WO the genetically engineered microorganism can include multiple selection mechanisms. Examples of selection mechanisms that can be used include, but are not limited to, an antibiotic resistance marker, a toxin-antitoxin system, a marker causing complementation of a mutation in an essential gene (e.g., complementation of an auxotrophic mutation as described in Section II.C above), or a cis acting genetic element, or a combination of any two or more thereof. In some embodiments, the selection mechanism is a resistance marker to an antibiotic, such as an antibiotic that is not used or is rarely used in humans or animals for therapy. In some embodiments, the selection mechanism is an antibiotic resistance marker selected from a kanamycin resistance gene or a tetracycline resistance gene, or a combination thereof. In some embodiments, the selection mechanism may not require an antibiotic for plasmid maintenance. For example, the selection mechanism can be a toxin-antitoxin system selected from a hok/sok system of plasmid
Figure imgf000031_0001
system of plasmid RK2, ccdAB of F plasmid, flmAB of F plasmid, kis/kid system of plasmid R1, XCV2162- ptaRNA1 of Xanthomonas campestris, ataT-ataR of enterohemorragic E. coli or Klebsiella, toxIN system of Erwinia carotovora, parE-parD system of Caulobacter crescentus, or fst-RNAII from Enterococcus faecalis plasmid AD1, ε-ζ system of Bacillus subtilis plasmid pSM19035, or a combination of any two or more thereof. As another example, the selection mechanism can be an essential gene encoding an enzyme involved in biosynthesis of an essential nutrient or a substrate (e.g., an amino acid). The essential nutrient or substrate can be required for cell wall synthesis and/or a house- keeping function. Representative examples of amino acids required for cell wall synthesis include D-alanine and diaminopimelic acid. In some embodiments, the essential gene is selected from dapA, dapD, murA, alr, dadX, murI, dapE, or thyA, or a combination of any two or more thereof. In some embodiments, the essential genes are a combination of alr and dadX (both of which encode for alanine racemases). In some embodiments, the essential genes are a combination of alr and dadX, and the plasmid(s) are selected using a functional alr gene (alr+, e.g., a wild type alr gene) as a selection marker. In some embodiments, the plasmid(s) are selected by complementation of the alr and dadX mutations by a functional alr gene present on the plasmid and/or the second plasmid. In some embodiments, the house-keeping function is selected from infA, a gene encoding a subunit of an RNA polymerase, a DNA polymerase, an rRNA, a tRNA, a cell division protein, or a chaperon protein, or a combination of any two or more thereof. Attorney Docket No. MMS.003WO In some embodiments, the genetically engineered microorganism includes a selection marker that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or the coding region thereof. In some embodiments, the genetically engineered microorganism includes a selection marker that is encoded by the sequence of SEQ ID NO: 5 or the coding region thereof. In a further example, the selection mechanism can be a cis acting genetic element, such as ColE1 cer locus or par from pSC101. The selection mechanism can be incorporated in the same plasmid(s) as the other genetic elements described herein (e.g., targeting gene(s), payload gene(s), and/or selection mechanism(s)). For example, in some embodiments, the targeting gene(s) and/or lysis gene(s) are inserted in a first plasmid, the payload gene(s) are inserted in a second plasmid, and the selection mechanism is inserted in both the first and second plasmids. Alternatively, the lysis gene(s), targeting gene(s), payload gene(s), and selection mechanism(s) can all be inserted in the same plasmid. III. Payload Delivery Using Genetically Engineered Microorganisms In some embodiments, the genetically engineered microorganisms of the present disclosure deliver at least one payload to a target cell (e.g., a diseased and/or abnormal cell). The payload can be a nucleic acid (e.g., DNA, RNA), a peptide, a protein, or a combination thereof. The payload can be delivered to the target cell in order to treat and/or detect a disease, such as any of the diseases described herein. For example, the payload can facilitate treatment and/or detection of a disease characterized by the presence of diseased GI tissue in subject in need thereof, such as precancerous lesions (e.g., adenomas), GI tract cancers (e.g., CRC), IBD (e.g., UC, Crohn’s disease), IBS, or Barrett’s esophagus. FIGS. 1A–1E are schematic illustrations of a method for delivering a payload to diseased GI tissue using genetically engineered microorganisms, in accordance with embodiments of the present technology. Referring first to FIG.1A, certain types of GI diseases (e.g., GI cancer and/or precancerous syndromes) can be characterized by the presence of diseased GI epithelium having mislocalized and/or aberrantly expressed cell surface molecules. In some instances, certain types of cell membrane receptors (e.g., integrins such as β1 integrins) are present only on the basolateral surfaces of healthy GI epithelial cells, and are not present on the apical surfaces facing the GI tract lumen. In contrast, in diseased GI epithelial cells, at Attorney Docket No. MMS.003WO least some of the cell membrane receptors can be mislocalized and displayed on the apical surface facing the GI tract lumen. Alternatively or in addition, diseased GI epithelial cells can also exhibit novel mammalian membrane receptors, which may be receptors that are normally not found in healthy cells or receptors that are formed by translocations and other genomic rearrangement. Referring next to FIG. 1B, the genetically engineered microorganisms of the present disclosure (e.g., genetically engineered Escherichia coli Nissle 1917) can be administered to a subject having a disease characterized by the presence of diseased GI epithelium. The genetically engineered microorganisms can be introduced into the lumen of the GI tract of the subject and thus be exposed to diseased and healthy cells of the GI epithelium. The genetically engineered microorganisms can include one or more targeting genes encoding one or more targeting elements that facilitate binding and/or entry into the diseased GI cells, while avoiding binding and/or entry into the healthy GI cells, as described herein. In some embodiments, for example, the targeting element(s) expressed by the genetically engineered microorganism bind to the diseased cells via the mislocalized and/or aberrantly expressed molecules on the apical surfaces of the diseased cells, and undergo internalization. For example, the genetically engineered microorganisms can express invasin, which can bind to mislocalized integrins on the apical surfaces of the diseased cells and can facilitate endocytosis of the microorganisms by the diseased cells. The genetically engineered microorganisms can exhibit little or no binding affinity to the healthy cells due to lack of mislocalized and/or aberrantly expressed cell surface molecules on the apical surfaces of the healthy cells (cell surface molecules present on the basolateral surfaces of the healthy cells may be inaccessible to microorganisms within the GI tract lumen). As shown in FIG. 1C, upon internalization, the genetically engineered microorganism can initially be sequestered in a phagosome of the diseased cell. The genetically engineered microorganism can undergo lysis due to an auxotrophic mutation (e.g., a dapA attenuation mutation) that causes a defect in cell wall synthesis. The genetically engineered microorganism can also include one or more lysis genes encoding one or more lysis elements (e.g., Listeriolysin O), as described herein. The lysis element(s) can be released upon lysis of the microorganism and/or can be naturally exported by the microorganism. The lysis element(s) can lyse the phagosome of the diseased cell to allow the contents of the lysed microorganism to escape into the cytoplasmic space of the diseased cell. Attorney Docket No. MMS.003WO In some embodiments, the genetically engineered microorganism carries a DNA payload (e.g., plasmid DNA or other DNA molecule) that is transferred into the cytoplasmic space of the diseased cell upon lysis of the microorganism and the phagosome. The DNA can undergo nuclear localization, which can occur naturally or be guided by binding of a protein that includes a nuclear localization signal. Subsequently, the DNA can be transcribed to RNA, and the RNA can be translated to produce protein expression. The protein can remain within the intracellular space of the diseased cell, can be transported to and incorporated into the cellular membrane, and/or can be secreted. The protein can be a therapeutic agent, a detection marker, or a combination thereof, as described in further detail below. Referring to FIG. 1D, the genetically engineered microorganism can alternatively or additionally carry an RNA payload (e.g., messenger RNA or other RNA molecule) that is transferred into the cytoplasmic space of the diseased cell upon lysis of the microorganism and the phagosome. The RNA can be translated to produce protein expression. The protein can remain within the intracellular space of the diseased cell, can be transported to and incorporated into the cellular membrane, and/or can be secreted. The protein can be a therapeutic agent, a detection marker, or a combination thereof, as described in further detail below. Referring to FIG. 1E, the genetically engineered microorganism can alternatively or additionally carry a protein payload. The protein can be a therapeutic agent, a detection marker, or a combination thereof, as described in further detail below. The protein can be expressed within the genetically engineered microorganism and subsequently delivered to the cell. In the illustrated embodiment, the protein is transferred into the cytoplasmic space of the diseased cell upon lysis of the microorganism and the phagosome. The protein can remain within the intracellular space of the diseased cell, can be transported to and incorporated into the cellular membrane and/or other cellular compartments, and/or can be secreted. In other embodiments, however, the protein may not be transferred into the cytoplasmic space, e.g., the protein may remain within the phagosome, such that the genetically engineered microorganism may not include a lysis gene encoding a lysis element (e.g., listeriolysin O) that facilitates lyses of the endocytic vacuole. Moreover, the protein may remain within the microorganism and/or can be present on the surface of the microorganism, such that the microorganism may not include an auxotrophic mutation that facilitates lysis of the microorganism (e.g., a dapA auxotrophic mutation). This approach can be used in embodiments where the protein is capable of exerting a desired effect without being transported Attorney Docket No. MMS.003WO into the cytoplasmic space (e.g., if the protein is an enzyme that acts upon a small molecule that is capable of crossing the cell membrane and bacterial cell wall, as discussed further below). Optionally, in some embodiments, the genetically engineered microorganism may not need to be internalized by the target cell, and may instead remain external to the target cell (e.g., attached to an external surface of the target cell). This approach can be used in embodiments where the protein is capable of exerting a desired effect without being delivered into the target cell (e.g., if the protein is an enzyme that acts upon a small molecule that is capable of crossing the cell membrane and bacterial cell wall, as discussed further below). A. Therapeutic Agents In some embodiments, the genetically engineered microorganisms of the present disclosure include one or more payload genes encoding at least one gene product that can be used to treat a target cell in a subject (e.g., a diseased and/or abnormal cell that is indicative of a disease). For example, the gene product(s) can be or include at least one therapeutic agent, such as a protein peptide, or nucleic acid molecule (e.g., an RNA molecule such as a catalytic RNA, a non-coding RNA, etc.). The therapeutic agent can be any agent that exerts a therapeutic effect in the subject, such as obtaining a beneficial or intended clinical result including alleviation of symptoms, a reduction in the severity of the disease, inhibiting an underlying cause of the disease, steadying the disease in a non-advanced state, delaying the progress of the disease, and/or improvement or alleviation of disease symptoms. In some embodiments, the therapeutic agent exhibits therapeutic activity in the target cell, such as by directly killing the target cell, mediating killing of the target cell by another component, or inhibiting or otherwise interfering with cellular processes in the target cell. The therapeutic agent may be a prodrug converting enzyme, a direct cytotoxic agent, or a combination thereof. 1. Prodrug Converting Enzymes In some embodiments, the therapeutic agent is or includes an enzyme that acts upon a substrate to produce a therapeutic effect. For example, the enzyme can be a prodrug converting enzyme. A prodrug converting enzyme can be a protein having a function of converting an inactive prodrug into an active drug (e.g., an active metabolite) through an enzymatic reaction. In some embodiments, the prodrug converting enzyme enzymatically converts an inactive prodrug into an active drug capable of directly or indirectly having therapeutic effect. For example, the active drug can be an anti-cancer drug, a cytotoxic drug, Attorney Docket No. MMS.003WO anti-bacterial drug, an anti-parasite drug, a hormone, an immunosuppressive drug, or a combination thereof. In some embodiments, the prodrug converting enzyme activates a compound with little or no cytotoxicity into a therapeutically active product. In some embodiments, the prodrug converting enzyme activates a compound with little or no cytotoxicity into a toxic product. In some embodiments, the prodrug converting enzyme sensitizes the target cell therapy. For example, the prodrug converting enzyme can increase cell permeability to another therapeutic agent, restore hormonal responsiveness, and/or render the cell more sensitive to radiotherapy and/or chemotherapeutics. In some embodiments, the prodrug converting enzyme converts a precursor of 5-fluoruracil (5-FU) into 5-FU. 5-FU is a chemotherapeutic agent that acts as a thymidylate synthase inhibitor, thus blocking DNA replication. In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5-FU, such as a cytosine deaminase or a derivative (e.g., fusion protein) thereof. For example, FIG.2 illustrates the conversion of 5-FC into 5-FU by cytosine deaminase (CD). In some embodiments, the prodrug converting enzyme is selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450 (e.g., that acts on the prodrug as a substrate), an uracil phosphoribosyltransferase (UPRT), or a combination thereof, or a fusion protein comprising two or more thereof. In some embodiments, the enzyme is a fusion protein of a cytosine deaminase and a UPRT (CD:UPRT). In some embodiments, the cytosine deaminase is a yeast cytosine deaminase (yCD) and/or the UPRT is a yeast or bacterial URPT (yURPT or UPRT). In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5- fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU. In some embodiments, the enzyme is a carboxylesterase. In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU. In some embodiments, the prodrug converting enzyme is a β-glucuronidase. In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 6-methylpurine-deoxyriboside (MePdR) into 6-methylpurine. In some embodiments, the enzyme is a purine nucleoside phosphorylase (PNP). Attorney Docket No. MMS.003WO In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate (fludarabine monophosphate) into fluoroadenine. In some embodiments, the enzyme is a purine nucleoside phosphorylase (PNP). In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil into 1-(2-deoxy-2- fluoro-beta-D-arabinofuranosyl) 5-methyluracil monophosphate (FMAUMP). In some embodiments, the enzyme is a thymidine kinase (TK) and/or a thymidylate synthase (TS). In some embodiments, the prodrug is 5-fluorocytosine (5-FC), pentyl N-[1- [(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4- yl]carbamate (capecitabine), 5-FU glucuronide, 6-methylpurine-deoxyriboside (MePdR), fludarabine monophosphate, 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, or a combination of any two or more thereof. In some embodiments, the prodrug converting enzyme is a carboxypeptidase (e.g., a Pseudomonas sp. Carboxypeptidase G2 (CPG2)). In some embodiments, the prodrug converting enzyme is a β-lactamase (e.g., an Enterobacter cloacae β-lactamase). In some embodiments, the prodrug is ganciclovir, and the prodrug converting enzyme is a thymidine kinase (e.g., a viral thymidine kinase such as herpes simplex thymidine kinase). In some embodiments, the prodrug and active drug are small molecules that are capable of crossing the cell membrane of the target cell and the cell wall of the genetically engineered microorganism. One example of a prodrug/active drug combination that is capable of crossing cell membranes and cell walls is 5-FC/5-FU. In such embodiments, the prodrug converting enzyme does not need to be present in the cytoplasmic space of the target cell to convert the prodrug into the active drug. Accordingly, the genetically engineered microorganism may not include a lysis gene encoding a lysis element that facilitates lysis of the endocytic vacuole of the target cell and/or may not include an auxotrophic mutation that facilitates lysis of the microorganism. Additional examples of prodrug converting enzymes are provided in International Publication No. WO 1997/038087, U.S. Patent Application Publication No. 2020/0268794, U.S. Patent Application Publication No. 2020/0215111, U.S. Patent Application Publication No. 2020/0157518, U.S. Patent Application Publication No. Attorney Docket No. MMS.003WO 2019/0038727, U.S. Patent Application Publication No. 2017/0173092, the disclosures of which are incorporated by reference herein in their entirety. Additional examples of prodrug converting enzymes are also provided in Aghi et al., Prodrug activation enzymes in cancer gene therapy, J Gene Med 2: 148-64 (2000); and Malekshah et al., Enzyme/Prodrug Systems for Cancer Gene Therapy, Curr Pharmacol Rep.2016; 2(6): 299–308, the disclosures of which are incorporated by reference in their entirety. The use of a prodrug converting enzyme as a therapeutic agent may provide various advantages, such as reducing off-target cytotoxicity (e.g., killing of nontarget cells). For example, a payload gene encoding a prodrug converting enzyme (e.g., a cytosine deaminase can be selectively delivered to target cells (e.g., diseased and/or abnormal cells of the GI tract) using a genetically engineered microorganism, as described herein. Accordingly, the prodrug converting enzyme may be selectively expressed in the target cells, while nontarget cells may exhibit little or no expression of the prodrug converting enzyme. As another example, the prodrug converting enzyme can be expressed by a genetically engineered microorganism that is selectively internalized by and/or selectively binds to the surface of target cells, as described herein. Subsequently, a prodrug (e.g., 5-FC) can be administered to the subject. The prodrug converting enzyme present in and/or proximate to the target cells can convert the inactive prodrug into an active form (e.g., 5-FU) that results in killing of the target cells. In contrast, the prodrug remains in its inactive form in nontarget cells lacking the prodrug converting enzyme, such that the nontarget cells are substantially unaffected. Representative examples of prodrug converting enzymes and their associated polynucleotide sequences are provided in Table 4 below. Table 4: Prodrug Converting Enzymes
Figure imgf000038_0001
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Figure imgf000039_0001
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Figure imgf000040_0001
In some embodiments, the genetically engineered microorganism produces a therapeutic agent having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the genetically engineered microorganism includes a therapeutic agent of SEQ ID NO: 6. In some embodiments, the genetically engineered microorganism includes a therapeutic agent that is encoded by a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO: 8 or the coding region thereof. In some embodiments, the genetically engineered microorganism includes a therapeutic agent that is encoded by the sequence of any one of SEQ ID NO: 7 or SEQ ID NO: 8 or the coding region thereof. 2. Direct Cytotoxic Agent In some embodiments, the therapeutic agent is a cytotoxic agent (e.g., a cytotoxic protein or peptide) that is capable of directly killing the target cell, also referred to herein as a “direct cytotoxic agent.” A direct cytotoxic agent can kill the target cell by interfering with cellular processes, causing damage to cellular components, or suitable combinations thereof. Examples of direct cytotoxic agents that may be used include microbial toxins, such as a clostridial toxin (e.g., botulinum neurotoxin, tetanus toxin, perfringolysin, α toxin, β toxin), a staphylococcal toxin, a Shiga toxin, an anthrax toxin, a diphtheria toxin, a pertussis toxin, a cholera toxin; caspases (e.g., constitutively active caspase-3); and suitable derivatives and combinations thereof. In some embodiments, the direct cytotoxic agent exhibits enhanced activity in target cells compared to the nontarget cells, e.g., direct cytotoxic agents that interfere with DNA replication may have an enhanced cytotoxic effect in rapidly dividing tumor cells compared to normal cells. Alternatively, the direct cytotoxic agent may exhibit the same or similar activity in target cells versus nontarget cells. In either case, off-target effects (e.g., killing of nontarget cells) may be reduced by using a genetically engineered microorganism to selectively deliver a payload gene encoding a direct cytotoxic agent to target cells, or to selectively deliver the direct cytotoxic agent to target cells, as described herein. Accordingly, the direct cytotoxic agent may be selectively expressed in and/or delivered to the target cells, Attorney Docket No. MMS.003WO thus resulting in selective killing of the target cells. In contrast, nontarget cells may exhibit little or no expression and/or receive little or no delivery of the direct cytotoxic agent and thus may remain substantially unaffected. 3. Associated Sequence Elements In some embodiments, the therapeutic agent (e.g., prodrug converting enzyme or direct cytotoxic agent) remains in the cytoplasm of the target cell. In some embodiments, the therapeutic agent is displayed on the surface of the target cell, e.g., the therapeutic agent can be attached to the surface of the target cell (e.g., by a GPI anchor). In some embodiments, the therapeutic agent is secreted by the target cell. In such embodiments, the nucleic acid molecule encoding the therapeutic agent can include a leader sequence for secretion of the therapeutic agent. In some embodiments, the therapeutic agent (e.g., prodrug converting enzyme or direct cytotoxic agent) is expressed from a mammalian promoter. In some embodiments, the mammalian promoter directs GI tract epithelial cell-specific expression. In some embodiments, a nucleic acid from the genetically engineered microorganism enters the nucleus of at least one target cell (e.g., a diseased and/or abnormal cell). In some embodiments, the nucleic acid is a DNA molecule (e.g., a plasmid). In some embodiments, the therapeutic agent (e.g., prodrug converting enzyme or direct cytotoxic agent) is expressed from a microbial promoter, and an RNA molecule (e.g., an mRNA) encoding the therapeutic agent is translated in the target cell (e.g., a diseased and/or abnormal cell). In some embodiments, the genetically engineered microorganism has a deletion of an RNase gene to increase stability of the RNA molecule. In some embodiments, the RNase gene is RNase III (rnc). In some embodiments, the payload gene(s) encoding the therapeutic agent is present in a cassette that directs RNA self-amplification of the cassette in the target cells. In some embodiments, the cassette includes an RNA viral genome replication apparatus. In some embodiments, the RNA viral genome replication apparatus encodes an RNA- dependent RNA polymerase (RdRP). In some embodiments, the RdRP is an alphavirus RdRP. In some embodiments, the alphavirus RdRP is nsp1, nsp2, nsp3, and/or nsp4. In some embodiments, the RNA molecule with the payload gene(s) encoding the therapeutic agent further includes an internal ribosome entry site (IRES). In some embodiments, the payload gene(s) are operably linked to a sequence element that causes the therapeutic agent (e.g., prodrug converting enzyme or direct cytotoxic Attorney Docket No. MMS.003WO agent) to be selectively expressed in the target cell, while producing little or no expression of the therapeutic agent in nontarget cells, thus reducing off-target effects of the therapeutic agent. For instance, the sequence element can be a promoter and/or a cis-regulatory element (e.g., an enhancer) that is activated in the target cells due to the presence of transcription factors that are specifically present in the target cells, but is not activated in nontarget cells due to the absence of such transcription factors. For instance, the transcription factors can be selectively expressed and/or overexpressed in cancerous and/or precancerous cells. In some embodiments, the payload gene(s) encoding the therapeutic agent includes one or more introns (e.g., one or more spliceosomal introns) such that the therapeutic agent is expressed in a first, active form (e.g., a catalytically active form) only in the target cells (e.g., diseased and/or abnormal cells), and is expressed in a second, nonactive form in nontarget cells (e.g., the genetically engineered microorganism and/or healthy cells). For example, the first form can be a protein or peptide produced from a spliced RNA molecule, and the second form can be a protein or peptide produced from the unspliced RNA molecule. As another example, the first form can be a protein or peptide produced from a first splice variant of an RNA molecule, and the second form can be a protein or peptide produced from a second splice variant of the RNA molecule (e.g., a variant produced by alternative splicing of the same RNA molecule). The inclusion of introns in the payload gene can reduce off-target effects by ensuring that the therapeutic agent only exhibits significant activity when expressed in diseased and/or abnormal cells, and exhibits little or no activity when expressed by the genetically engineered microorganism (e.g., due to leaky expression, background activity of mammalian promoters in the microorganism) and/or when expressed by other nontarget cells (e.g., due to nonspecific binding of the microorganism to nontarget cells). For example, in embodiments where the therapeutic agent is a prodrug converting enzyme, the prodrug converting enzyme can be capable of converting the inactive prodrug into the active drug only when the prodrug converting enzyme is expressed in a spliced form or as a splice variant produced specifically by the target cell. As another example, in embodiments where the therapeutic agent is a direct cytotoxic agent, the direct cytotoxic agent may exhibit therapeutic activity only when expressed in a spliced form or as a splice variant produced specifically by the target cell. In other embodiments, however, the payload gene may not include any introns, e.g., if the payload gene is to be expressed in the microorganism for delivery of a protein payload to the target cell. Attorney Docket No. MMS.003WO B. Detection Markers In some embodiments, the genetically engineered microorganisms of the present disclosure include one or more payload genes encoding at least one gene product that can be used to detect a target cell in a subject (e.g., a diseased and/or abnormal cell that is indicative of a disease). For example, the gene product(s) can be or include at least one detection marker, such as a protein, peptide, and/or nucleic acid. The detection marker can be an intracellular biomarker that is present within the target cell, a surface biomarker that is located on the surface of the target cell, a secretable biomarker that is exported by the target cell, or suitable combinations thereof. The presence or absence of the detection marker can be correlated to presence or absence of the target cell in the patient, which in turn can be correlated to a positive or negative diagnosis, respectively. The detection marker can be measured using any suitable technique. The appropriate technique can be selected based on the type of detection marker (e.g., protein, peptide, RNA, or DNA), whether the detection marker exhibits functional activity and/or functional properties (e.g., enzymatic activity, fluorescence, luminescence), and/or other relevant considerations. For example, the detection marker can be measured using one or more of the following: agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, circular dichroism (CD), chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, flow cytometry, fluorimetry, electrophoretic assay, enzymatic assay, enzyme linked immunosorbent assay (ELISA), gas chromatography, gravimetry, high pressure liquid chromatography (HPLC), immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, reverse transcription-polymerase chain reaction (RT-PCR), multiplex RT-PCR, quantitative PCR (qPCR), reverse transcription-qPCR (RT- qPCR), digital PCR (dPCR), droplet digital PCR (ddPCR), semi-quantitative PCR, semi- quantitative RT-PCR, gel electrophoresis, RNA sequencing, fluorescent in situ hybridization (FISH, including mRNA fluorescent in situ hybridization (RNA-FISH)), or chromogenic in situ hybridization (CISH), or suitable combinations thereof. Optionally, in embodiments where multiple types of detection markers are used, some or all of the detection markers can be measured using different detection techniques. In some embodiments, the detection marker is present in a biological sample collected from the subject, such as feces, blood, serum, plasma, mucus, urine, saliva, and/or a biopsy sample. Accordingly, the subject can be diagnosed by collecting the biological sample Attorney Docket No. MMS.003WO and analyzing the biological sample for the presence of the detection marker. In some embodiments, the detection marker is collected and measured in a noninvasive manner (e.g., without performing colonoscopy or endoscopy on the subject). Optionally, the detection marker can be measured without the use of a clinical laboratory instrument selected from a colonoscope, an endoscope, a radiography instrument (e.g., an X-ray machine), a computed tomography (CT) scanner, a magnetic resonance imaging (MRI) machine, a blood gas analyzer, and/or a urine chemistry analyzer. In other embodiments, however, the detection marker can be measured using invasive techniques (e.g., via colonoscopy or endoscopy) and/or using one or more of the clinical laboratory instruments provided above. 1. Protein/Peptide-Based Detection In some embodiments, the detection marker is or includes at least one protein or peptide. The protein or peptide can be an intracellular protein or peptide that is present within the target cell, a surface protein or peptide that is located on the surface of the target cell, a secretable protein or peptide that is exported by the target cell, or suitable combinations thereof. The protein or peptide can be any protein or peptide that is not endogenously produced by the cells of the subject (e.g., by target cells and/or by non-target cells within the subject). For instance, the protein or peptide can be a naturally occurring protein or peptide that is endogenously produced by a different type of organism than the subject (e.g., a non- mammalian organism), or can be a derivative of a naturally occurring protein or peptide. Optionally, the protein or peptide can be a synthetic protein or peptide. Synthetic proteins and peptides can be generated in various ways, such as by modifying the sequence of a naturally occurring protein or peptide (e.g., to increase stability, modify localization) or via de novo design. In some embodiments, a library of synthetic proteins or peptides are generated and screened for the desired activity. In some embodiments, the protein or peptide serves as the sole detection marker for determining the presence or absence of target cells in the subject. In other embodiments, however, the protein or peptide can be used in combination with another detection marker for determining the presence or absence of target cells in the subject, such as the RNA molecule encoding the protein or peptide, or a different RNA molecule. In some embodiments, the protein or peptide that serves as the detection marker is selected from a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT Attorney Docket No. MMS.003WO contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination of any two or more these. In some embodiments, the detection marker is a protein or peptide that is easily detectable, e.g., based on binding to an antibody or other binding protein, via a noninvasive or invasive imaging technique, via an assay performed on a biological sample collected from the subject, etc. In some embodiments, the detection marker is a fluorescent protein. The fluorescent protein can be selected from GFP, RFP, YFP, Sirius, Sandercyanin, shBFP- N158S/L173I, Azurite, EBFP2, mKalama1, mTagBFP2, TagBFP, shBFP, ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquoise, mTurquoise2, TagCFP, mTFP1, monomeric Midoriishi-Cyan, Aquamarine, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, Monomeric Azami Green, mWasabi, Clover, mNeonGreen, NowGFP, mClover3, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, lanRFP-ΔS83, mPapaya1, mCyRFP1, Monomeric Kusabira-Orange, mOrange, mOrange2, mKOκ, mKO2, TagRFP, TagRFP-T, RRvT, mRuby, mRuby2, mTangerine, mApple, mStrawberry, FusionRed, mCherry, mNectarine, mRuby3, mScarlet, mScarlet-I, mKate2, HcRed-Tandem, mPlum, mRaspberry, mNeptune, NirFP, TagRFP657, TagRFP675, mCardinal, mStable, mMaroon1, mGarnet2, cyOFP, iFP1.4, iRFP713 (iRFP), iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, miRFP703, miRFP709, or miRFP670. In some embodiments, the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713, iRFP720, or iSplit. In some embodiments, the detection marker is a fluorescent protein that becomes fluorescent upon contact with a metabolite found only in the mammalian cytoplasm. For example, the detection marker can be a near-infrared fluorescent protein that utilizes biliverdin as a cofactor to fluoresce, such as iRFP or iRFP670. As another example, the detection marker can be Japanese freshwater eel (Anguilla japonica) UnaG protein, which fluoresces only upon binding to bilirubin. Accordingly, in some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one fluorescent protein to the target cells (e.g., diseased epithelial cells), the target cells express the fluorescent protein, and the target cells are detected based on fluorescence of the fluorescent protein. Optionally, the payload gene(s) can include one or more introns such that the fluorescent protein is expressed in a detectable form (e.g., a fluorescent form) only in the target cells, as described herein. Attorney Docket No. MMS.003WO In some embodiments, the fluorescent protein is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay. Alternatively or in combination, the fluorescent protein can be detected in situ in the subject using an endoscopic procedure or colonoscopic procedure (e.g., a white light endoscopic procedure or Laser-Induced Fluorescence Endoscopy (LIFE)). In some embodiments, the detection marker is a bioluminescent protein. The bioluminescent protein can be selected from a Ca+2 regulated photoprotein (e.g., aequorin, symplectin, Mitrocoma photoprotein, Clytia photoprotein, and Obelia photoprotein), North American firefly luciferase, Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina luciferase, Metridina luciferase, Metrida luciferase, OLuc protein, red firefly luciferase, bacterial luciferase, or active variants thereof. Accordingly, in some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one bioluminescent protein to the target cells (e.g., diseased epithelial cells), the target cells express the bioluminescent protein, and the target cells are detected based on luminescence of the bioluminescent protein. Optionally, the payload gene(s) can include one or more introns such that the bioluminescent protein is expressed in a detectable form (e.g., a luminescent form or a form that acts on a luminescent substrate) only in the target cells, as described herein. In some embodiments, the bioluminescent protein is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay. Alternatively or in combination, the bioluminescent protein can be detected in situ within the subject using an endoscopic procedure or colonoscopic procedure. In some embodiments, a substrate of the bioluminescent protein is administered during and/or before the endoscopic procedure or colonoscopic procedure. Illustrative substrates include luciferin, or a pharmaceutically acceptable analog, derivative, or salt thereof. In some embodiments, the administration of the substrate of the bioluminescent protein is started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection. In some embodiments, the detection marker is an enzyme reporter. In some embodiments, the enzyme reporter catalyzes a reaction, which may be detected on the basis of Attorney Docket No. MMS.003WO change in, e.g., color, fluorescence, or luminescence. Such reactions may use chromogenic, fluorogenic, or luminogenic substrates, which may be used in an in vitro assay to detect the target cells in a biological sample collected from the subject, and/or may be provided locally or systemically to the subject at the time of detection of the target cells. In some embodiments, the enzyme substrate is colorigenic, luminogenic, and/or fluorogenic. The enzyme reporter can be selected from beta-galactosidase, chloramphenicol acetyltransferase, horseradish peroxidase, alkaline phosphatase, acetylcholinesterase, or catalase. In some embodiments, the enzyme reporter is beta-galactosidase, and the substrate is selected from resorufin β-D-galactopyranoside, 5-dodecanoylaminofluorescein di-β-D- galactopyranoside, 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-Gal), or GALACTO-LIGHT PLUS. In some embodiments, the enzyme reporter is horseradish peroxidase, and the substrate is selected from 3,3′,5,5′-Tetramethylbenzidine (TMB), 3,3’- Diaminobenzidine (DAB), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), or 5-Amino-2,3-dihydrophthalazine-1,4-dione (luminol). In some embodiments, the enzyme reporter is chloramphenicol acetyltransferase, and the substrate is BODIPY FL-1- deoxychloramphenicol. Accordingly, in some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one enzyme reporter to the target cells (e.g., diseased epithelial cells), the target cells express the enzyme reporter, and the target cells are detected based on enzymatic activity of the enzyme reporter. Optionally, the payload gene(s) can include one or more introns such that the enzyme reporter is expressed in a detectable form (e.g., an enzymatically active form) only in the target cells, as described herein. In some embodiments, the enzymatic reporter is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay. Alternatively or in combination, the enzymatic reporter can be detected in situ within the subject using an endoscopic procedure or colonoscopic procedure. In some embodiments, the substrate of the enzymatic reporter is administered during and/or before the endoscopic procedure or colonoscopic procedure. In some embodiments, the administration of the substrate is started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days prior to marker detection. Attorney Docket No. MMS.003WO In some embodiments, the detection marker is a secretable biomarker that is excreted in a biological sample collected from the subject. For example, the biological sample can be a biological fluid selected from blood, serum, plasma, mucus, urine, and/or saliva. Alternatively or in combination, the biological sample can be a biopsy sample collected from the subject. In some embodiments, the secretable biomarker is an enzyme, a peptide hormone, or a protein or peptide antigen. For instance, the secretable biomarker can be a secreted protein (or a subunit of peptide therefrom) selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2 (CCSP-2), Cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), or an artificial protein capable of tight binding with high specificity to a detection antibody or protein, or a subunit or fragment thereof, or a combination of any two or more thereof. In some embodiments, the secretable biomarker is a human chorionic gonadotropin (hCG). hCG is a hormone composed of two subunits: the α and β subunits. In some embodiments, the hCG is an α subunit of human chorionic gonadotropin (αhCG). In some embodiments, the hCG is a β subunit of human chorionic gonadotropin (βhCG). hCG is primarily catabolized by the liver, although about 20% is excreted in the urine. The β subunit is degraded in the kidney to make a core fragment which is measured by urine hCG tests. The pattern of excretion of hCG in urine throughout pregnancy is well established. Excretion of hCG in saliva has also been reported. Accordingly, in some embodiments, the hCG is measured using blood, serum, plasma, urine, and/or saliva. In some embodiments, the hCG (e.g., βhCG) is optimized for distribution in body fluids (e.g., decreased or increased excretion, and increased stability). In some embodiments, the hCG is optimized for distribution in the blood. In some embodiments, the hCG is optimized for distribution in the urine. Accordingly, in some embodiments, the hCG is optimized for increased excretion (e.g., in urine or feces). In alternative embodiments, the hCG (e.g., βhCG) is optimized for decreased excretion, leading to accumulation of the hCG in the blood. In some embodiments, the hCG is optimized for increased stability (e.g., improved protein folding and/or stability against proteolysis) leading to increased accumulation. In some embodiments, the hCG (e.g., βhCG) is optimized for increased stability and decreased excretion, leading to accumulation in the blood. In some embodiments, the hCG (e.g., βhCG) has increased stability and increased excretion, leading to accumulation in the urine. Attorney Docket No. MMS.003WO In some embodiments, the hCG is measured using one anti-hCG antibody, or a fragment thereof. In some embodiments, the hCG is measured using two anti-hCG antibodies, or fragment(s) thereof. In some embodiments, the two anti-hCG antibodies, or fragment(s) thereof bind different epitopes. In some embodiments, the hCG is measured using an immunometric assay such as an ELISA. In some embodiments, the hCG is measured using an ELISA selected from direct ELISA, indirect ELISA, sandwich ELISA, or competitive ELISA. In some embodiments, a commercially available hCG detection kit is used. In some embodiments, a method for detecting diseased epithelial tissue includes: (i) administering to the target epithelial tissue (such as target tissue of the GI tract) of a subject in need thereof, a genetically engineered microorganism of any of the embodiments disclosed herein including an exogenous gene encoding an hCG or a fragment thereof; (ii) obtaining a biological sample from the subject selected from blood, serum, plasma, urine, or saliva; and (iii) measuring the hCG or a fragment thereof in the biological sample obtained from the subject. In some embodiments, the detection is performed using an agglutination assay, chemiluminescence, colorimetry, fluorimetry, a quantitative immunoassay, a dipstick assay, lateral flow immunoassay, ELISA, or a combination of any two or more thereof. In some embodiments, when the hCG or a fragment thereof is detected in the biological sample, additional testing, (e.g., colonoscopy) is indicated. In some embodiments, the expression of hCG or a fragment thereof is detected in a biopsy sample using a technique selected from RT- PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH. In some embodiments, the expression of hCG is used for localizing diseased epithelial or GI tissue and/or epithelium of the ducts that are connected with GI (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct). In some embodiments, the secretable biomarker is an alkaline phosphatase. In some embodiments, the alkaline phosphatase is secreted alkaline phosphatase (SEAP). Alkaline phosphatases are normally membrane-bound, thus not secreted. Without being bound by theory, introduction of a termination codon in the sequence encoding the membrane anchoring domain may yield a fully active secreted alkaline phosphatase. In some embodiments, the alkaline phosphatase (e.g., SEAP) is assayed from a blood sample. In some embodiments, the secreted alkaline phosphatase is measured using an enzymatic assay. In some embodiments, a chromogenic, fluorogenic, luminogenic and/or substrate is used in the assay. Illustrative chromogenic alkaline phosphatase substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenyl phosphate Attorney Docket No. MMS.003WO (pNPP)), and 4-Chloro-2-methylbenzenediazonium/3-Hydroxy-2-naphthoic acid 2,4- dimethylanilide phosphate. Illustrative luminogenic alkaline phosphatase substrates include, but are not limited to, dioxetane phosphates (e.g., adamantyl dioxetane phenyl phosphate and chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD)). Illustrative fluorogenic alkaline phosphatase substrates include, but are not limited to, 4-Methylumbelliferyl phosphate disodium salt (MUP), and 6,8-difluoro-7-hydroxy-4-methylcoumarin phosphate. In some embodiments, a substrate selected from p-nitrophenyl phosphate (pNPP), 4-Methylumbelliferyl phosphate disodium salt (MUP), or chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) is used to detect the alkaline phosphatase (e.g., SEAP). In some embodiments, the enzymatic assay uses a colorimetric, fluorometric and/or chemiluminescent readout. In some embodiments, pNPP is used as the substrate and the alkaline phosphatase (e.g., SEAP) is assayed from a biological sample, using a colorimetric readout. In some embodiments, 4-methylumbelliferyl phosphate disodium salt (MUP) is used as the substrate and the alkaline phosphatase (e.g., SEAP) is assayed from a biological sample using a fluorometric readout. In some embodiments, chloro-5-substituted adamantyl-1,2- dioxetane phosphate (CSPD) is used as the substrate and the alkaline phosphatase (e.g., SEAP) is assayed from a biological sample using a chemiluminescent readout. In some embodiments, a method for detecting diseased epithelial or GI tissue and and/or diseased epithelium of the ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct) includes: (i) administering to target epithelial cells (such as target cells of the GI tract) of a subject in need thereof, a genetically engineered microorganism of any of the embodiments disclosed herein including an exogenous gene encoding an alkaline phosphatase; (ii) obtaining a biological sample from the subject selected from blood, serum, or plasma; and (iii) measuring the alkaline phosphatase in the biological sample obtained from the subject. In some embodiments, the detection is performed using chemiluminescence, colorimetry, fluorimetry, a quantitative immunoassay, lateral flow immunoassay, ELISA, or a combination of any two or more thereof. In some embodiments, when the alkaline phosphatase is detected in the biological sample, additional testing, (e.g., colonoscopy) is indicated. In some embodiments, the expression of the alkaline phosphatase is detected in a biopsy sample using a technique selected from RT-PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH. In some embodiments, the expression of the alkaline phosphatase is used for localizing diseased epithelial or GI tissue Attorney Docket No. MMS.003WO and/or epithelium of the ducts that are connected with GI tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct). In some embodiments, the secretable biomarker is marker is a luciferase. In some embodiments, the luciferase is Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof. Gluc is naturally secreted from mammalian cells in an active form. Moreover, Gluc is excreted at least in urine. Accordingly, in some embodiments, the Gluc may be measured using blood, serum, plasma, urine, and/or saliva. In some embodiments, the luciferase is Gaussia luciferase (Gluc) or a derivative thereof. In some embodiments, the luciferase is measured using an enzymatic assay. In some embodiments, the luciferase is measured using a luciferin as a substrate. The luciferin, without limitation, may be firefly luciferin, Latia luciferin, bacterial luciferin, dinoflagellate luciferin, vargulin, or foxfire. In some embodiments, ATP may be a cofactor. In some embodiments, the luciferin is coelenterazine. In some embodiments, the luciferase is measured using a luminescence readout. In some embodiments, the biological sample is selected from blood, urine, or saliva. Gaussia luciferase (Gluc) is a small luciferase with numerous disulfide bonds. Gluc is stable in serum, however, it is not active in E. coli cytoplasm because the bacterial environment does not allow the disulfide bonds to form. Accordingly, in some embodiments, expression of Gluc is indicative of production and secretion of Gluc by diseased human cells. In some embodiments, the gene encoding Gluc comprises an intron to ensure production and secretion of Gluc by diseased human cells. Gluc does not require any cofactors for activity (e.g., ATP) and catalyzes the oxidation of the substrate coelenterazine in a reaction that leads to emission of blue light (480 nm). Accordingly, in some embodiments, the Gluc may be measured using luminescence. In other embodiments, the Gluc may be measured using an immunoassay such as a quantitative immunoassay, a lateral flow immunoassay, or an ELISA. In some embodiments, the Gluc is optimized for distribution in body fluids (e.g., decreased or increased excretion, and increased stability). In some embodiments, the Gluc is optimized for distribution in the blood. In some embodiments, the Gluc is optimized for distribution in the urine. Accordingly, in some embodiments, the Gluc is optimized for increased excretion (e.g., in urine or feces). In some embodiments, the Gluc is optimized for decreased excretion, leading to accumulation of the Gluc in the blood. In some embodiments, Attorney Docket No. MMS.003WO the Gluc is optimized for increased stability (e.g., improved protein folding and/or stability against proteolysis) leading to increased accumulation. In some embodiments, the Gluc is optimized for increased stability and decreased excretion, leading to accumulation in the blood. In some embodiments, the Gluc has increased stability and increased excretion, leading to accumulation in the urine. In some embodiments, a method for detecting diseased epithelial or GI tissue and/or diseased epithelium of the ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct) includes: (i) administering to target epithelial cells (such as target cells of the GI tract) of a subject in need thereof, a genetically engineered microorganism of any of the embodiments disclosed herein comprising an exogenous gene encoding an Gluc or a fragment thereof; (ii) obtaining a biological sample from the subject selected from blood, serum, plasma, urine, or saliva; and (iii) measuring the Gluc or a fragment thereof in the biological sample obtained from the subject. In some embodiments, the detection is performed using a luminescence assay. In some embodiments, when the Gluc or a fragment thereof is detected in the biological sample, additional testing (e.g., colonoscopy) is indicated. In some embodiments, the expression of Gluc or a fragment thereof is detected based on luminescence, during colonoscopy. In such embodiments, coelenterazine may be administered to the subject before or during colonoscopy. In some embodiments, the expression of Gluc or a fragment thereof may be detected in a biopsy sample using a technique selected from RT-PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH. In some embodiments, the expression of Gluc is used for localizing diseased GI tissue and/or diseased epithelium of the ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct). In some embodiments, the secretable biomarker is human carcinoembryonic antigen (CEA). CEA is naturally expressed in some normal adult tissues, and in tumors expressing the same self-antigen. In some embodiments, the secretable biomarker is colon cancer secreted protein 2 (CCSP2). CCSP2 expression is generally absent in normal body tissues, but is induced in colon adenomas and colon cancers. In some embodiments, the secretable biomarker is cathepsin B. Cathepsin B belongs to a family of lysosomal cysteine proteases and plays an important role in intracellular proteolysis. It is found both in serum and urine. Attorney Docket No. MMS.003WO In some embodiments, the secretable biomarker is secretable by epithelial cells of GI tissue. Accordingly, in such embodiments, the secretable biomarker can be synthesized and sorted in the secretory pathway in mammalian cells that express the secretable biomarker. In some embodiments, the secretable biomarker includes an ER signal sequence, optionally located at or near the N-terminus. In some embodiments, the ER signal directs the ribosomes that are synthesizing the secretable biomarker to the rough ER. In some embodiments, the secretable biomarker includes a native signal peptide. Additionally or alternatively, in some embodiments, the secretable biomarker includes a heterologous signal sequence. In some embodiments, the heterologous signal sequence is selected from the signal sequence of interleukin-2, CD5, the Immunoglobulin Kappa light chain, trypsinogen, serum albumin, or prolactin. In some embodiments, the Gaussia luciferase (Gluc) comprises a native signal sequence. Additionally or alternatively, the Gaussia luciferase (Gluc) comprises a heterologous signal sequence. In some embodiments, the βhCG comprises a native signal sequence. Additionally or alternatively, the βhCG comprises a heterologous signal sequence. In some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one secretable biomarker to the target cells (e.g., diseased epithelial cells), the target cells express the secretable biomarker, and the target cells are detected based on presence of the secretable biomarker. Optionally, the payload gene(s) can include one or more introns such that the secretable biomarker is expressed in a detectable form only in the target cells, as described herein. In some embodiments, the secretable biomarker is detected in a biological sample collected from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva) using an in vitro assay. In some embodiments, the secretable biomarker is measured by using an enzymatic assay (e.g., where the secretable biomarker has enzymatic activity) or an immunoassay. In some embodiments, the secretable biomarker can be measured in the biological sample using an assay selected from agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, CD, chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, fluorimetry, flow cytometry, electrophoretic assay, enzymatic assay, ELISA, gas chromatography, gravimetry, HPLC, immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, or a combination of any two or more thereof. In some embodiments, when the secretable biomarker is detected in the biological sample, additional testing (e.g., colonoscopy) is indicated. In some embodiments, Attorney Docket No. MMS.003WO the expression of the secretable biomarker is detected in a biopsy sample using a technique selected from RT-PCR, immunohistochemistry, FISH (including RNA-FISH), or CISH. In some embodiments, the expression of the secretable biomarker is used for localizing diseased epithelial or GI tissue and/or epithelium of the ducts that are connected with GI tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct). In some embodiments, the detection marker is an MRI contrast agent. The MRI contrast agent can be a protein or peptide that causes the accumulation of a magnetic responsive atom, such as transition metal ions (e.g., Cu2+, Fe2+/Fe3+, Co2+, and Mn2+), or lanthanide metal ions (e.g., Eu3+, Gd3+, Ho3+, and Dy3+). For example, the MRI contrast agent can cause sequestration or chelation of metal ions (e.g., Fe3+) or can catalyze a biochemical reaction that leads to change in accumulation of ions (e.g., cleavage of a caged synthetic Gd3+ compound), and thereby allow the detection of the target cells via MRI. The MRI contrast agent can be selected from ferritin, transferrin receptor-1 (TfR1), Tyrosinase (TYR), beta-galactosidase, manganese-binding protein MntR, creatine kinase (CK), Magnetospirillum magnetotacticum magA, divalent metal transporter DMT1, protamine-1 (hPRM1), urea transporter (UT-B), and ferritin receptor Timd2 (T-cell immunoglobulin and mucin domain containing protein 2), sodium iodide symporter, E. coli dihydrofolate reductase, or norepinephrine transporter, or active variants thereof. Accordingly, in some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one MRI contrast agent to the target cells (e.g., diseased epithelial cells), the target cells express the MRI contrast agent, and the target cells are detected based on presence of the MRI contrast agent. Optionally, the payload gene(s) can include one or more introns such that the MRI contrast agent is expressed in a detectable form (e.g., a form that causes the accumulation of magnetic responsive atoms) only in the target cells, as described herein. In some embodiments, the detection of the target cells is performed using an MRI procedure. In some embodiments, the MRI procedure is noninvasive. In some embodiments, a substrate of the MRI contrast agent is administered during and/or before the MRI procedure. In some embodiments, the substrate of the MRI contrast agent is a source of magnetic responsive atoms, which can be accumulated by the MRI contrast agent within or on the surface of the target cells. For example, a caged synthetic Gd3+ compound comprising a galactoside may be administered when the MRI contrast agent is beta-galactosidase. In some embodiments, the administration of the substrate of the MRI contrast agent may be started prior Attorney Docket No. MMS.003WO to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection. In some embodiments, the detection marker is a PET reporter. The PET reporter can be a protein or peptide that causes the accumulation of a PET probe (e.g., a positron emitting radioisotope) in or on the surface of diseased epithelial cells. In some embodiments, the PET probe causes the accumulation of the PET probe through binding to a receptor, antibody, an enzyme, or a cellular transport mechanism. Illustrative PET probes include [18F]FHBG, [18F]FEAU, [124I]FIAU, [18F or 11C]BCNA, [11C] β-galactosyl triazoles, [18F]L- FMAU, [18F]FESP, [11C]Raclopride, [11C]N-methylspiperone, [18F]FES, 68Ga-DOTATOC, [18F]fluoropropyl-trimethoprim, Na124I, or a 225Ac-DOTA chelate. In some embodiments, the PET reporter is selected from thymidine kinase, deoxycytidine kinase, Dopamine 2 Receptor, estrogen receptor α surface protein binding domain, somatostatin receptor subtype 2, carcinoembryonic antigen, a sodium iodide symporter, a single-chain antibody specific to 1,4,7,10-tetraazacyclododecane-1, 4,7,10-tetraacetic acid (DOTA), or E. coli dihydrofolate reductase, or a variant thereof. Accordingly, in some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one PET reporter to the target cells (e.g., diseased epithelial cells), the target cells express the PET reporter, and the target cells are detected based on presence of the PET reporter. Optionally, the payload gene(s) can include one or more introns such that the PET reporter is expressed in a detectable form (e.g., a form that causes the accumulation of a PET probe) only in the target cells, as described herein. In some embodiments, the detection of the target cells is performed using a PET imaging procedure. In some embodiments, one or more PET probes are administered during and/or before the PET imaging procedure. In some embodiments, the administration of the PET probe may be started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection. In some embodiments, the detection marker is a SPECT reporter. The SPECT reporter can be a protein or peptide that causes the accumulation of a SPECT probe (e.g., a gamma-ray emitting radioisotope) in or on the surface of the target cells. In some embodiments, the SPECT reporter causes the accumulation of the SPECT probe through binding to a receptor, antibody, an enzyme, or a cellular transport mechanism. Illustrative Attorney Docket No. MMS.003WO SPECT probes include Sodium pertechnetate ( [99mTc]NaTcO4), Na123I, Na125I, Na131I, [123I]- NKJ64, [¹²⁵I]-NKJ64, [131I]-(R)-N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine, [123I]- NKJ64, [¹²⁵I]-(R)-N-methyl-3-(2-iodophenoxy)-3-phenylpropanamine, [131I]-(R)-N-methyl-3- (2-iodophenoxy)-3-phenylpropanamine, [123I]β-CIT (2β-carbomethoxy-3β-(4- iodophenyl)tropane), [125I]β-CIT (2β-carbomethoxy-3β-(4-iodophenyl)tropane), [131I]β-CIT [123I]-2β-carbomethoxy-3β-(4-iodophenyl)tropane, [125I]-2β-carbomethoxy-3β-(4- iodophenyl)tropane, [131I]-2β-carbomethoxy-3β-(4-iodophenyl)tropane, [123I]-2’- iodospiperone, [125I]-2’-iodospiperone, [131I]-2’-iodospiperone, [123I]epidepride, [125I]epidepride, [131I]epidepride, [123I]-5-iodo-7-N-[(1-ethyl-2- pyrrolidinyl)methyl]carboxamido-2,3-dihydrobenzofuran, [125I]-5-iodo-7-N-[(1-ethyl-2- pyrrolidinyl)methyl]carboxamido-2,3-dihydrobenzofuran, or [131I]-5-iodo-7-N-[(1-ethyl-2- pyrrolidinyl)methyl]carboxamido-2,3-dihydrobenzofuran. In some embodiments, the SPECT reporter is selected from sodium ion symporter, norepinephrine transporter, sodium iodide symporter, dopamine receptor, or dopamine transporter. Accordingly, in some embodiments, the genetically engineered microorganism delivers one or more payload genes encoding at least one SPECT reporter to the target cells (e.g., diseased epithelial cells), the target cells express the SPECT reporter, and the target cells are detected based on presence of the SPECT reporter. Optionally, the payload gene(s) can include one or more introns such that the SPECT reporter is expressed in a detectable form (e.g., a form that causes the accumulation of a SPECT probe) only in the target cells, as described herein. In some embodiments, the detection of the target cells is performed using a SPECT imaging procedure. In some embodiments, one or more SPECT probes are administered during and/or before the SPECT imaging procedure. In some embodiments, the administration of the SPECT probe may be started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection. In some embodiments, the detection marker is a photoacoustic reporter. Accordingly, in some embodiments, the genetically engineered microorganism delivers a one or more nucleic acid(s) encoding at least one photoacoustic reporter to target cells (e.g., diseased epithelial cells). In some embodiments, the detection of the target cells is performed using an endoscopic procedure, or colonoscopic procedure. Illustrative photoacoustic reporters include any fluorescent proteins disclosed herein. Attorney Docket No. MMS.003WO Representative examples of detection markers and their associated polynucleotide sequences are provided in Table 5 below. Table 5: Detection Markers
Figure imgf000057_0001
In some embodiments, the genetically engineered microorganism produces a detection marker having a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 9. In some embodiments, the genetically engineered microorganism includes a detection marker of SEQ ID NO: 9. Additional details and examples of detection markers are disclosed in International Publication Nos. WO 2021/247480 and WO 2022/261290, the disclosures of which are incorporated by reference herein in their entirety. 2. RNA-Based Detection In some embodiments, the detection marker is or includes at least one RNA molecule. For example, the at least one RNA molecule can include a messenger RNA (mRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a catalytic RNA (e.g., a ribozyme), a non- coding RNA, a riboswitch, or a combination thereof. In some embodiments, the RNA molecule is a naturally occurring RNA molecule or a derivative thereof. The naturally occurring RNA molecule can be any RNA molecule that is not endogenously produced by the cells of the subject (e.g., by target cells and/or non-target cells), such as an RNA molecule that is endogenously produced by a different type of organism than the subject (e.g., a non- mammalian organism). For example, the RNA molecule can include a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to a naturally occurring RNA molecule. Optionally, the RNA molecule can be an artificial RNA molecule (also known as a synthetic RNA molecule). Artificial RNA molecules can be generated in various ways, such as by modifying the sequence of a naturally occurring Attorney Docket No. MMS.003WO RNA molecule (e.g., to increase stability, spliceability) or via de novo design. In some embodiments, a library of artificial RNA molecules is generated and screened for the desired activity. In some embodiments, the RNA molecule serves as the sole detection marker for determining the presence or absence of target cells in the subject. In other embodiments, however, the RNA molecule can be used in combination with another detection marker for determining the presence or absence of target cells in the subject, such as a protein or peptide encoded by the RNA molecule, or a different protein or peptide. In some embodiments, the RNA molecule that serves as the detection marker is a mRNA encoding a protein or peptide. The protein or peptide can be any protein or peptide that is not endogenously produced by the cells of the subject (e.g., by target cells and/or by non-target cells within the subject). For instance, the protein or peptide can be a naturally occurring protein or peptide that is endogenously produced by a different type of organism than the subject (e.g., a non-mammalian organism), or can be a derivative of a naturally occurring protein or peptide. Optionally, the protein or peptide can be a synthetic protein or peptide. The protein or peptide produced by the mRNA can optionally also serve as a detection marker, in addition or alternatively to the mRNA itself. For example, the protein or peptide can be an enzyme, a hormone, an antigen, a fluorescent molecule, a bioluminescent molecule, etc., that is capable of being detected via any of the detection techniques provided herein. In some embodiments, the mRNA encodes for one or more of the following: a fluorescent marker, a bioluminescent marker, a CT contrast agent, an MRI contrast agent, a Positron Emission Tomography (PET) reporter, a Single Photon Emission Computed Tomography (SPECT) reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination thereof. In some embodiments, the mRNA encodes for a fluorescent protein selected from GFP, RFP, YFP, Sirius, Sandercyanin, shBFP-N158S/L173I, Azurite, EBFP2, mKalama1, mTagBFP2, TagBFP, shBFP, ECFP, Cerulean, mCerulean3, SCFP3A, CyPet, mTurquoise, mTurquoise2, TagCFP, mTFP1, monomeric Midoriishi-Cyan, Aquamarine, TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, Monomeric Azami Green, mWasabi, Clover, mNeonGreen, NowGFP, mClover3, TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, lanRFP-ΔS83, mPapaya1, mCyRFP1, Monomeric Kusabira-Orange, Attorney Docket No. MMS.003WO mOrange, mOrange2, mKOκ, mKO2, TagRFP, TagRFP-T, RRvT, mRuby, mRuby2, mTangerine, mApple, mStrawberry, FusionRed, mCherry, mNectarine, mRuby3, mScarlet, mScarlet-I, mKate2, HcRed-Tandem, mPlum, mRaspberry, mNeptune, NirFP, TagRFP657, TagRFP675, mCardinal, mStable, mMaroon1, mGarnet2, cyOFP, iFP1.4, iRFP713 (iRFP), iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mIFP, TDsmURFP, miRFP703, miRFP709, or miRFP670. In some embodiments, the mRNA encodes for a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713, iRFP720, or iSplit. In some embodiments, the near-infrared fluorescent protein is iRFP670, which requires biliverdin to fluoresce. In embodiments where the genetically engineered microorganism does not make biliverdin, iRFP670 fluorescence provides evidence that the iRFP670 was expressed in mammalian cells. In some embodiments, the mRNA encodes for a bioluminescent protein selected from a Ca+2 regulated photoprotein (e.g., aequorin, symplectin, Mitrocoma photoprotein, Clytia photoprotein, and Obelia photoprotein), North American firefly luciferase, Japanese firefly luciferase, Italian firefly luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, railroad worm luciferase, Renilla luciferase, Gaussia luciferase, Cypridina luciferase, Metridina luciferase, Metrida luciferase, OLuc protein, red firefly luciferase, bacterial luciferase, or active variants thereof. Additional details and examples of proteins and peptides that can serve as detection markers and may be encoded by the mRNA are provided in Section III.B.1 above. In other embodiments, however, the mRNA may encode for a protein or peptide that does not serve as a detection marker, or may not encode any protein or peptide. In some embodiments, the RNA molecule is a catalytic RNA molecule that catalyzes a specific biochemical reaction, such as a ribozyme. Examples of ribozymes include, but are not limited to, hammerhead ribozyme, hepatitis delta virus (HDV) ribozyme, HDV-like ribozyme, hairpin ribozyme, Varkud satellite (VS) ribozyme, glmS riboswitch, twister ribozyme, twister sister ribozyme, hatchet ribozyme, pistol ribozyme, Hovlinc ribozyme, Long Interpersed Nuclear Element-1 (LINE-1) ribozyme, beta-globin co-transcriptional cleavage ribozyme (CoTC ribozyme), Vg1 ribozyme, and protein-responsive ribozymes. In some embodiments, the RNA molecule includes one or more features to improve in vivo stability. For example, the RNA molecule can be a circular RNA, which may be more stable than linear RNAs. As another example, the RNA molecule can have a secondary Attorney Docket No. MMS.003WO structure that is associated with enhanced longevity in vivo, such as one or more hairpins, long range interactions, G-quadruplexes, or pseudoknots, or combinations thereof. Hairpins, also known as stem-loop structures, can include a hybridized stem and a single stranded loop, and can optionally contain mismatches and/or bulges. Pseudoknots can include nested stem-loop structures, with half of one stem intercalated between the two halves of another stem. G- quadruplexes can be composed of guanine-rich sequences and can be formed from G-quartets composed of four guanine nucleotides that interact with each other through Hoogsteen hydrogen bonding. In some embodiments, the RNA molecule can include a 5’ hairpin structure and/or 3’ poly A tail to increase stability. As yet another example, the RNA molecule can bind to a stabilization factor, which can be a protein, peptide, nucleic acid, or combination thereof. In a further example, the RNA molecule is capable of self-replicating and/or self-amplifying. For instance, the payload gene encoding the RNA molecule can be present in a cassette that directs RNA self-amplification of the cassette in target cells. In some embodiments, the cassette includes an RNA viral genome replication apparatus, such an RNA- dependent RNA polymerase (RdRP). In some embodiments, the RdRP is an alphavirus RdRP. In some embodiments, the alphavirus RdRP includes nsp1, nsp2, nsp3, and/or nsp4. The RNA molecules described herein can have any suitable length, such as a length within a range from 100 nt to 20 knt, 100 nt to 10 knt, 100 nt to 5 knt, 100 nt to 1 knt, 100 nt to 500 nt, 100 nt to 200 nt, 200 nt to 20 knt, 200 nt to 10 knt, 200 nt to 5 knt, 200 nt to 1 knt, 200 nt to 500 nt, 500 nt to 20 knt, 500 nt to 10 knt, 500 nt to 5 knt, 500 nt to 1 knt, 1 knt to 20 knt, 1 knt to 10 knt, 1 knt to 5 knt, 5 knt to 20 knt, 5 knt to 10 knt, or 10 knt to 20 knt. For example, an RNA molecule can have a length of at least 50 nt, 100 nt, 500 nt, 1 knt, 2 knt, 5 knt, or 10 knt; and/or a length of no more than 20 knt, 15 knt, 10 knt, 5 knt, 2 knt, 1 knt, or 500 nt. In some embodiments, the RNA molecule has at least two forms, including a first form when expressed by a target cell and a second, different from when expressed by a non-target cell (e.g., the genetically engineered microorganism). For example, the RNA molecule can be encoded by a DNA sequence including one or more introns (e.g., spliceosomal introns) that are spliced out in the RNA molecule to produce spliced RNA and/or different RNA splice variants, as described herein. In some embodiments, the first form of the RNA molecule that is produced by the target cell (e.g., a spliced RNA or first splice variant) is an mRNA encoding a functional and/or full-length protein or peptide, and the second form of the RNA molecule that is produced by the non-target cell (e.g., an unspliced RNA or second splice Attorney Docket No. MMS.003WO variant) is an mRNA encoding a nonfunctional and/or truncated protein or peptide. In some embodiments, the first form of the RNA molecule that is produced by the target cell is a functional form of a catalytic RNA, and the second form of the RNA molecule that is produced by the nontarget cell is a nonfunctional form of the catalytic RNA. Optionally, the first form of the RNA molecule that is produced by the target cell can have improved stability compared to the second form of the RNA molecule that is produced by the nontarget cell. In some embodiments, the RNA molecule is detected using RT-PCR, multiplex RT-PCR, qPCR, RT-qPCR, dPCR, ddPCR, semi-quantitative PCR, semi-quantitative RT- PCR, gel electrophoresis, RNA sequencing, FISH (e.g., RNA-FISH), CISH, or suitable combinations thereof. For example, the RNA molecule can be detected in a biological sample from the subject using a whole RNA extraction and qPCR sampling technique. The biological sample can include, for example, feces, blood, serum, plasma, mucus, urine, and/or saliva. In some embodiments, the RNA molecule is present within cells in the biological sample. The total RNA can be extracted from the cells in the biological sample by disrupting the biological sample (e.g., via vortexing, centrifugation, buffers, surfactants) and/or lysing the cells (e.g., via a lysis buffer). The RNA can then be converted to cDNA using suitable primers, such as random hexamers to reverse transcribe all RNA, an oligo dT primer to selectively reverse transcribe mammalian RNA only, or with primers specific to the gene encoded by the RNA (e.g., the payload gene). Optionally, an enrichment step can be performed before cDNA conversion using techniques such as bead-based pull down for either mammalian RNA (e.g., using oligo dT probes) or specific RNA (e.g., using gene-specific probes). The cDNA can then be quantified using qPCR. In some embodiments, qPCR is performed using primers that are capable of hybridizing and amplifying cDNA produced from a first form of the RNA molecule produced by target cells (e.g., a spliced RNA molecule, a first splice variant), but are not capable of hybridizing and amplifying cDNA produced from a second form of the RNA molecule produced by non-target cells (e.g., an unspliced RNA molecule, a second splice variant). For example, the first form of the RNA can include a first exon directly ligated to a second exon, while the second form of the RNA molecule can include an intron interposed between the first and second exons. In such embodiments, the qPCR primer can be designed to hybridize to the portion of the corresponding cDNA at the interface between the first and second exons, e.g., the 5’ region of the primer hybridizes to the first exon in the cDNA and the 3’ region of the primer hybridizes to the second exon, or vice-versa. The number of nucleotides in the 5’ region Attorney Docket No. MMS.003WO and the 3’ region can be selected so that if the intron is present in the cDNA, either the 5’ region or the 3’ region of the primer will fail to hybridize to the cDNA. In some embodiments, the 5’ region of the primer includes at least one, two, three, four, five, six, seven, eight, nine, or 10 nucleotides that hybridize to the first exon; and/or the 3’ region of the primer includes at least one, two, three, four, five, six, seven, eight, nine, or 10 nucleotides that hybridize to the second exon. Optionally, in embodiments where the RNA molecule is a catalytic RNA (e.g., a ribozyme), the catalytic RNA can be detected using an enzymatic assay. For instance, the catalytic RNA can be extracted from a biological sample and/or enriched as described above. The presence of the catalytic RNA can then be measured, e.g., by combining the catalytic RNA with a substrate and quantifying the amount of RNA based on the amount of converted substrate. Alternatively or in combination, the in vivo activity of the catalytic RNA molecule can create products (e.g., nucleic acid fragments) that can be detected in a biological sample from the subject using RT-PCR, multiplex RT-PCR, qPCR, RT-qPCR, dPCR, ddPCR, semi- quantitative PCR, semi-quantitative RT-PCR, gel electrophoresis, RNA sequencing, and/or any of the other techniques described herein. C. Additional Sequence Elements In some embodiments, the genetically engineered microorganisms of the present disclosure deliver a nucleic acid to target cells (e.g., diseased and/or abnormal cells). The nucleic acid can be a DNA molecule (e.g., a plasmid DNA, also referred to herein as a payload plasmid). In some embodiments, the payload plasmid is present in multiple copies (ranging from about 1 to about 300 copies, from about 20 to about 50 copies, from about 2 to about 10 copies, or from about 5 to about 10 copies) per cell, or is a single copy plasmid. Copy number may depend on the particular genetic characteristics of the plasmid. In some embodiments, the DNA molecule (e.g., payload plasmid) harbors one or more payload genes encoding the gene products described herein (e.g., therapeutic agents, such as prodrug converting enzymes, and/or detection markers). The DNA molecule can also include one or more sequence elements operably linked to the payload genes that control the expression of the gene products. The sequence element(s) may control and regulate the transcription, transcript stability, translation, protein stability, cellular localization, and/or secretion of the gene products. In some embodiments, the sequence element(s) may prevent expression of the gene product by the genetically engineered microorganism. Alternatively, the Attorney Docket No. MMS.003WO sequence element(s) may allow expression (e.g., transcription and/or translation) of the gene product by the genetically engineered microorganism. In some embodiments, the gene products are expressed in one form by the target cell and in another form by the genetically engineered microorganism, as described herein. In some embodiments, the one or more payload genes are operably linked to a mammalian promoter. In some embodiments, the one or more payload genes include at least one microbial repressor binding site to inhibit bacterial transcription. In some embodiments, the one or more payload genes include microbial transcription terminators. In some embodiments, the one or more payload genes include at least one intron, as described herein. In some embodiments, the one or more payload genes include codon usage optimized for mammalian expression. Without being bound by theory, it is believed that certain optional sequence elements present in the payload gene (e.g., mammalian promoters, microbial repressor binding sites (e.g., operators), internal ribosome entry sites) allow production of the gene product in mammalian cells, while reducing or preventing the expression of the gene product in the genetically engineered microorganism. Moreover, as described herein, the use of introns that are not spliced by genetically engineered microorganism allows for different forms of the gene product to differentiate between expression by the mammalian cells versus background expression by the genetically engineered microorganism (e.g., due to leaky expression, background activity of mammalian promoters in the microorganism). Therefore, in some embodiments, the presence of a particular form of the gene product provides a true readout of the presence of the target cells (e.g., diseased epithelial cells), with little or no background expression in the genetically engineered microorganism. Accordingly, in some embodiments, the one or more payload genes may be operably linked to a mammalian promoter. In some embodiments, the mammalian promoter directs expression in the specific target cell type, such as GI tract epithelial cell-specific expression. Illustrative examples of suitable mammalian promoters that direct GI tract epithelial cell-specific expression are MUC2 gene promoter, T3b gene promoter, intestinal fatty acid binding protein gene promoter, lysozyme gene promoter, and villin gene promoter. In some embodiments, the mammalian promoter directs an inducible GI tract epithelial cell- specific expression. An illustrative example of a suitable inducible mammalian promoter includes a cytochrome P450 promoter element that is transcriptionally up-regulated in response to a lipophilic xenobiotic such as β-napthoflavone. In some embodiments, the inducible mammalian promoter may be regulated by tetracycline, cumate, or an estrogen. In some Attorney Docket No. MMS.003WO embodiments, the inducible mammalian promoter may be a Tet-On or Tet-Off promoter. Accordingly, in some embodiments, the one or more payload genes may be inducible and/or repressible, and optionally controlled by delivering the inducer or repressor to the subject. The microbial repressor binding site(s), which are optionally present in the one or more payload genes, may repress the expression of the payload genes in bacteria, while exerting little or no repressive effect in mammalian cells. In some embodiments, the repressor sequence may be selected from one or more lac operator(s), one or more ara operator(s), one or more trp operator(s), one or more SOS operator(s), one or more integration host factor (IHF) binding sites, one or more histone-like protein HU binding sites, or a combination of two or more thereof. The microbial transcription termination site(s) can cause premature termination of the transcription of the one or more payload genes in the genetically engineered microorganism, without causing premature termination of the transcription of the one or more payload genes in mammalian cells. In some embodiments, the one or more payload genes include a rho-independent microbial transcription termination site. In some embodiments, the one or more payload genes include a 5’ untranslated region, and the 5’ untranslated region includes a rho-independent microbial transcription termination site. In some embodiments, the rho-independent microbial transcription termination site includes a short hairpin followed by a run of 4–8 Ts (e.g., TTTTTT and TTTTT). Illustrative rho-independent microbial transcription termination sites are T7 terminator, rrnB terminator, and T0 terminator. In some embodiments, the DNA molecule (e.g., payload plasmid) includes at least one binding site for a DNA binding protein. In some embodiments, the at least one binding site for a DNA binding protein forms an array of multiple adjacent binding sites for the DNA binding protein. In some embodiments, the DNA binding protein includes one or more nuclear localization signals (NLS). In these embodiments, the DNA binding protein binds the DNA molecule (e.g., a plasmid) and promotes the nuclear translocation of the DNA molecule (e.g., a plasmid) via the one or more NLS. In some embodiments, the NLS is SV40 T antigen NLS sequence (KKKRKV). In some embodiments, the DNA binding protein is NFκB. In some embodiments, the genetically engineered microorganism includes a gene encoding the DNA binding protein having the one or more NLS. Without being bound by theory, it is believed that the DNA binding protein comprising one or more NLS binds the at least one binding site for the DNA binding protein on the plasmid and promotes nuclear translocation of the plasmid via the action of the one or more NLS. Thus, in these embodiments, the target cells express the Attorney Docket No. MMS.003WO payload genes from the DNA molecule delivered by the microorganism. In some embodiments, the gene encoding the DNA binding protein is genomically integrated, is present on a payload plasmid harboring one or more payload genes, or is present on another plasmid. In some embodiments, the genetically engineered microorganisms of the present disclosure deliver an RNA molecule (e.g., an mRNA molecule) encoding the gene products described herein (e.g., therapeutic agents, such as prodrug converting enzymes, and/or detection markers). Accordingly, in such embodiments, the payload gene(s) encoding the gene product may be operably linked to a microbial promoter (e.g., a proD promoter). In some embodiments, the genetically engineered microorganism delivers an mRNA molecule encoding the gene products to the cytoplasm of the target cells, which are capable of translating the mRNA to produce the encoded gene products. In some embodiments, the payload gene(s) encoding the gene products includes an internal ribosome entry site(s) (IRES). In these embodiments, the IRES can promote translation of the mRNA molecule delivered by the genetically engineered microorganism in the target cell. In some embodiments, the mRNA sequence that is delivered includes an element that imparts stability on the mRNA molecule. Non-limiting examples of the elements that impart stability on the mRNA molecule include 5’ hairpin structures and 3’poly A tails. In some embodiments, a genetic change in the genetically engineered microorganism can improve stability of the mRNA molecule. In some embodiments, the genetic change is a deletion of an RNase gene. In some embodiments, the RNase gene is RNase III (rnc). In some embodiments, when the genetically engineered microorganism delivers one or more mRNA molecules encoding the therapeutic agent and/or detection marker to the target cell (e.g., diseased epithelial cells), the one or more payload gene(s) encoding the RNAs are integrated in the genome of the genetically engineered microorganism. In some embodiments, the one or more payload gene(s) are present on a plasmid. Accordingly, in these embodiments, the payload gene(s) encoding the therapeutic agent (e.g., prodrug converting enzyme) and/or detection marker may be operably linked to a microbial promoter. Illustrative examples of suitable microbial promoters include a natural promoter of any chromosomal gene, plasmid gene, or bacteriophage gene that functions in the genetically engineered microorganism (e.g., E. coli). In some embodiments, the microbial promoter is a synthetic promoter derived from a promoter consensus sequence. In some embodiments, the microbial promoter is a constitutive promoter. In some embodiments, the microbial promoter is an inducible promoter. Illustrative examples of suitable inducible Attorney Docket No. MMS.003WO microbial promoter include the araBAD promoter and the lac promoter. Accordingly, in some embodiments, the payload gene(s) encoding the therapeutic agent and/or detection marker may be inducible and/or repressible, and optionally controlled by delivering the inducer and/or repressor to the subject. In some embodiments, the promoter is a T7 or T3 promoter, and the genetically engineered microorganism expresses a T7 or T3 RNA polymerase gene to support expression of the payload gene(s). For example, in some embodiments, the genetically engineered microorganism expresses a T7 RNA polymerase (T7RNAP) encoded by a T7RNAP gene, and harbors one or more payload genes encoding the gene products described herein (e.g., therapeutic agents and/or detection markers) under the control of a T7 promoter. In some embodiments, the T7RNAP is integrated on the bacterial chromosome. In some embodiments, the T7RNAP is present on a plasmid. In some embodiments, the T7RNAP is controlled by an inducible promoter (e.g., an araBAD or lacUV5 promoter). In such embodiments, the bacteria can express mRNA encoding the therapeutic agent and/or the detection marker. In some embodiments, the genetically engineered microorganism delivers mRNA encoding the therapeutic agent and/or the detection marker to the target cell (e.g., diseased epithelial cells). The mRNA encoding the therapeutic agent and/or the detection marker that is delivered to the target cell can include an internal ribosome entry site. In some embodiments, the genetically engineered microorganism delivers the therapeutic agent and/or the detection marker to the target cell. An internal ribosome entry site (IRES) is an RNA element that allows for translation initiation in a cap-independent manner. In some embodiments, the IRES may be selected from an IRES from an encephalomyocarditis virus (EMCV), an IRES from a hepatitis C virus (HCV), or an IRES from a cricket paralysis virus (CrPV). In some embodiments, the IRES(s) present in the payload gene(s) encoding the therapeutic agent and/or detection marker allow for the production of the therapeutic agent and/or detection marker in mammalian cells using an mRNA produced in the genetically engineered microorganism. One or more intron(s), which are optionally present in the payload gene(s) encoding the therapeutic agent and/or detection marker, can prevent the functional expression of the therapeutic agent and/or detection marker in bacteria, while allowing expression of payload gene(s) encoding the therapeutic agent and/or detection marker in mammalian cells, irrespective of whether the mRNA encoding the therapeutic agent and/or detection marker may be transcribed in the genetically engineered microorganism or a mammalian cell. In some Attorney Docket No. MMS.003WO embodiments, the intron may be a spliceosomal intron. In some embodiments, the intron creates a frameshift or a premature stop codon in an unspliced mRNA encoding the therapeutic agent and/or detection marker. Therefore, in some embodiments, the genetically engineered microorganism provides a true readout of the presence of target cells (e.g., diseased cells), without background expression of the detection marker in the genetically engineered microorganism. In some embodiments, the genetically engineered microorganism delivers to the target cells an RNA molecule that is capable of self-replicating and/or self-amplifying. In some embodiments, the payload gene(s) encoding the therapeutic agent and/or the detection marker are present in a cassette that directs RNA self-amplification of the cassette in the target cells. In some embodiments, the cassette comprises an RNA viral genome replication apparatus. In some embodiments, the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). In some embodiments, the RdRP is an alphavirus RdRP. In some embodiments, the alphavirus RdRP includes nsp1, nsp2, nsp3, and/or nsp4. In some embodiments, the payload gene(s) encoding the therapeutic agent and/or the detection marker includes codon usage optimized for mammalian expression. In some embodiments, the one or more payload genes optionally further include a sequence element selected from Kozak sequences, 2A peptide sequences, mammalian transcription termination sequences, polyadenylation sequences (pA), leader sequences for protein secretion, or a combination of any two or more thereof. The Kozak sequence is a nucleic acid motif that functions as the protein translation initiation site in most eukaryotic mRNA transcripts. The Kozak sequence present in the one or more payload genes can improve correct translation initiation. In some embodiments, the Kozak sequence has the following nucleotide sequence: 5’-(GCC)GCCRCCAUGG-3’. The 2A peptides, when present, can function by preventing the synthesis of a peptide bond between the glycine and proline residues found at the end of the 2A peptides, and that the 2A peptides allow production of equimolar levels of multiple proteins from the same mRNA. The 2A peptides become attached to C-terminus upstream protein, while the downstream protein starts with a proline. In some embodiments, the 2A peptide is selected from E2A ((GSG)QCTNYALLKLAGDVESNPGP), F2A ((GSG)VKQTLNFDLLKLAGDVESNP GP), P2A ((GSG)ATNFSLLKQAGDVEENPGP), or Attorney Docket No. MMS.003WO T2A ((GSG)EGRGSLLTCGDVEE NPGP). In some embodiments, the GSG sequence (which is included in the parentheses) may be optionally present. The polyadenylation sequences (pA) cause addition of a polyA tail to mRNA, which can be important for the nuclear export, translation, and stability of mRNA. The mammalian transcription termination sequences can terminate transcription and promote the addition of polyA tail. In some embodiments, the one or more payload genes include a sequence element that is both a mammalian transcription termination sequence and a polyadenylation sequence, such as a SV40 terminator, hGH terminator, BGH terminator, or rbGlob terminator. In some embodiments, the one or more payload genes optionally include leader sequences for protein secretion. In some embodiments, the one or more payload genes optionally include upstream sequences for display of the corresponding protein or peptide on a mammalian cell surface. In some embodiments, the genetically engineered microorganisms of the present disclosure deliver a protein or peptide that is a therapeutic agent (e.g., a prodrug converting enzyme) and/or a detection marker. Accordingly, in these embodiments, the payload gene(s) encoding the therapeutic agent (e.g., prodrug converting enzyme) and/or detection marker may be operably linked to a microbial promoter (e.g., a proD promoter). Illustrative examples of suitable microbial promoters include a natural promoter of any chromosomal gene, plasmid gene, or bacteriophage gene that functions in the genetically engineered microorganism (e.g., E. coli). In some embodiments, the microbial promoter is a synthetic promoter derived from a promoter consensus sequence. In some embodiments, the microbial promoter is a constitutive promoter. In some embodiments, the microbial promoter is an inducible promoter. Illustrative examples of suitable inducible microbial promoters include the araBAD promoter and the lac promoter. Accordingly, in some embodiments, the payload gene(s) encoding the therapeutic agent and/or detection marker may be inducible and/or repressible, and optionally controlled by delivering the inducer and/or repressor to the subject. The payload gene(s) can be present on a plasmid, can be integrated into a genomic site, or suitable combinations thereof. Representative examples of DNA constructs including a therapeutic agent, detection marker, and additional sequence elements are provided in Table 6 below. Attorney Docket No. MMS.003WO Table 6: DNA Constructs
Figure imgf000069_0001
Attorney Docket No. MMS.003WO
Figure imgf000070_0001
In some embodiments, the genetically engineered microorganism includes a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the genetically engineered microorganism includes the sequence of SEQ ID NO: 10. IV. Compositions and Methods of Use In some embodiments, about 103 to about 1011 viable genetically engineered microorganisms are administered to a subject, depending on the species of the subject (e.g., a human or non-human subject), as well as the disease or condition that is being diagnosed and/or treated. In some embodiments, about 105 to about 109 viable genetically engineered microorganisms of the present disclosure are administered to a subject. The genetically engineered microorganisms of the present disclosure may be administered between 1 and about 50 times, e.g., prior to detection of an expressed marker. The genetically engineered microorganisms may be administered from 1 to about 21 times, or from 1 to about 14 times, or from 1 to about 7 times, e.g., prior to the marker detection. The genetically engineered microorganisms may be administered starting between about 1 hour to about 2 months prior to marker detection. The administration of the genetically engineered microorganisms may be started prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days. The genetically engineered microorganisms of the present disclosure may be administered by any route as long as they are capable of invading their target cells upon Attorney Docket No. MMS.003WO administration and capable of delivery of their payload. The payload that the genetically engineered microorganisms of the present disclosure deliver can include a nucleic acid molecule (e.g., plasmid DNA harboring one or more payload genes, introns, and/or additional sequence elements; or an mRNA harboring one or more payload genes, introns, and/or additional sequence elements), and/or can include a protein or peptide, as already described. In some embodiments, the genetically engineered microorganisms of the present technology are administered by an oral and/or rectal route. The genetically engineered microorganisms of the present disclosure can be administered along with a pharmaceutically acceptable carrier and/or diluent. As used herein, “pharmaceutically acceptable” may refer to compositions that do not substantially produce adverse reactions, e.g., toxic, allergic, or immunological reactions, when administered to a subject. The particular pharmaceutically acceptable carrier and/or diluent employed can be varied as desired. Examples of diluents include a phosphate buffered saline, buffer for buffering against gastric acid in the stomach, such as citrate buffer (pH 7.0) containing sucrose, bicarbonate buffer (pH 7.0) alone, or bicarbonate buffer (pH 7.0) containing ascorbic acid, lactose, and optionally aspartame. Examples of carriers include proteins (e.g., as found in skim milk), sugars (e.g., sucrose), or polyvinylpyrrolidone. The carriers can be used at any suitable concentration, such as a concentration within a range from 0.1% to 30% (w/v), or within a range from 1% to 10% (w/v). The pharmaceutically acceptable carriers and/or diluents which may be used for delivery may depend on specific routes of administration. Any such carrier or diluent can be used for administration of the genetically engineered microorganisms of the present disclosure, so long as the genetically engineered microorganisms of the present disclosure are still capable of invading a target cell and delivering the payload that they carry to the target cells. In vitro or in vivo tests for invasiveness can be performed to determine appropriate diluents and carriers. The genetically engineered microorganisms described herein can be administered as part of a pharmaceutical composition. A pharmaceutical composition may be any combination of an active ingredient (e.g., the genetically engineered microorganisms) with a carrier, inert or active, that makes the composition suitable for diagnostic and/or therapeutic use. The pharmaceutical compositions of the present disclosure can be formulated for oral and/or rectal administration. Lyophilized forms are also included, so long as the genetically engineered microorganisms are invasive and capable of delivering their payload upon contact Attorney Docket No. MMS.003WO with a target cell or upon administration to the subject. Techniques and formulations generally may be found in Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients (e.g., carriers and/or diluents), such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose), fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc or silica), disintegrants (e.g., potato starch or sodium starch glycolate), and/or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and/or preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring agents, and/or sweetening agents as appropriate. The pharmaceutical compositions provided herein may be administered rectally in the forms of suppositories, pessaries, pastes, powders, creams, ointments, solutions, emulsions, suspensions, gels, foams, sprays, or enemas. These dosage forms can be manufactured using conventional processes as described in Remington: The Science and Practice of Pharmacy, supra. Rectal suppositories are solid bodies for insertion into rectum, which are solid at ordinary temperatures but melt or soften at body temperature to release the genetically engineered microorganisms of the present disclosure inside the rectum. Pharmaceutically acceptable carriers utilized in rectal suppositories can include bases or vehicles, such as stiffening agents, which produce a melting point in the proximity of body temperature, when formulated with the pharmaceutical compositions provided herein; and antioxidants, including bisulfite and sodium metabisulfite. Suitable vehicles include, but are not limited to, cocoa butter (theobroma oil), glycerin-gelatin, carbowax (polyoxyethylene glycol), spermaceti, paraffin, white and yellow wax, appropriate mixtures of mono-, di- and triglycerides of fatty acids, or hydrogels (such as polyvinyl alcohol, hydroxyethyl methacrylate, polyacrylic acid, Attorney Docket No. MMS.003WO glycerinated gelatin). Combinations of the various vehicles may be used. Rectal suppositories may be prepared by the compressed method or molding. The typical weight of a rectal suppository is from about 2 g to about 3 g. In some embodiments, the genetically engineered microorganisms of the present disclosure are administered as a single composition, or are administered individually at the same or different times and via the same or different route (e.g., oral and rectal) of administration. In some embodiments, the genetically engineered microorganisms of the present disclosure are provided in a mixture or solution suitable for rectal instillation and that includes sodium thiosulfate, bismuth subgallate, vitamin E, and/or sodium cromolyn. In some embodiments, a pharmaceutical composition of the present disclosure includes, in a suppository form, butyrate, and glutathione monoester, glutathione diethylester or other glutathione ester derivatives. The suppository can optionally include sodium thiosulfate and/or vitamin E. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. In some embodiments, the genetically engineered microorganisms of the present disclosure are formulated as an enema formulation. The enema formulation can include a reducing agent (or any other agent having a similar mode of action). The enema formulation can optionally comprise polysorbate-80 (or any other suitable emulsifying agent), and/or any short chain fatty acid (e.g., a five, four, three, or two carbon fatty acid) as a colonic epithelial energy source, such as sodium butyrate (4 carbons), proprionate (3 carbons), acetate (2 carbons), etc., and/or any mast cell stabilizer, such as cromolyn sodium (GASTROCROM) or Nedocromil sodium (ALOCRIL). In some embodiments, the pharmaceutical composition includes from about 105 to about 109 viable genetically engineered microorganisms of the present disclosure. If the composition includes cromolyn sodium, the cromolyn sodium can be present in an amount from about 10 mg to about 200 mg, or from about 20 mg to about 100 mg, or from about 30 mg to about 70 mg. If the composition includes polysorbate-80, the polysorbate-80 can be provided at a concentration from about 1% (v/v) to about 10% (v/v). If the composition includes sodium butyrate, the sodium buyrate can be present in an amount of about 500 to about 1500 mg. In some embodiments, the composition suitable for administration as an enema is formulated to include genetically engineered microorganisms of the present disclosure, Attorney Docket No. MMS.003WO cromolyn sodium, and polysorbate-80. In some embodiments, the composition further includes alpha-lipoic acid and/or L-glutamine and/or N-acetyl cysteine and/or sodium butyrate (1.1 gm). The compositions may, if desired, be presented in a pack or dispenser device and/or a kit that may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. In some embodiments, the present technology provides compositions that are useful for treating and/or detecting a disease, such as a disease of the GI tract and/or associated tissue. For example, a pharmaceutical composition for treating and/or detecting a target cell indicative of a disease can include a genetically engineered microorganism as disclosed herein, and, optionally, a pharmaceutically acceptable carrier. The genetically engineered microorganism can be a non-pathogenic and/or auxotrophic microorganism. The genetically engineered microorganism can deliver a payload gene specifically to the target cell. The payload gene can be part of a larger nucleic acid molecule, such as a DNA molecule (e.g., plasmid DNA). The payload gene can encode at least one gene product, such as a therapeutic agent, a detection marker, or a combination thereof. The payload gene can be operably linked to a mammalian promoter for expression by the target cell, e.g., in embodiments where the genetically engineered microorganism is configured to deliver a DNA molecule including the payload gene to the target cell. For instance, the mammalian promoter can be active and/or specific for epithelial expression (e.g., GI tract epithelial cell-specific expression). Alternatively, the payload gene can be operably linked to a microbial promoter for expression by the genetically engineered microorganism, e.g., in embodiments where the genetically engineered microorganism is configured to deliver an RNA molecule including the payload gene to the target cell, or in embodiment where the genetically engineered microorganism is configured to deliver a protein encoded by the payload gene to the target cell. In some embodiments, the present disclosure provides genetically engineered microorganisms that are administered for the purpose of treating and/or identifying target cells (e.g., diseased and/or abnormal cells) in a subject. In some embodiments, the genetically engineered microorganisms have been genetically altered to invade diseased and/or abnormal cells and deliver a nucleic acid encoding a prodrug converting enzyme, enabling the abnormal cells to convert a non-toxic prodrug into a pharmacologically active drug, and thereby provide a targeted treatment to the diseased and/or abnormal cells. In some embodiments, the engineered microorganisms delivers the prodrug converting enzyme directly. In some Attorney Docket No. MMS.003WO embodiments, the engineered microorganisms additionally produce a detection marker or deliver a nucleic acid encoding the detection marker that allows detection of diseased and/or abnormal cells. In some embodiments, the genetically engineered microorganism includes a targeting gene encoding a targeting element that facilitates binding to and/or entry into the target cell. For example, the targeting element can be a surface protein that specifically interacts with a surface marker on the target cell. In some embodiments, the target cell is a diseased epithelial cell, and the surface protein specifically interacts with one or more cell membrane receptors which are not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, common bile duct, etc., but are exposed to the luminal side of diseased epithelial cells of the GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, common bile duct, etc., in a subject suffering from a disease. In some embodiments, the present technology provides a genetically engineered microorganism useful in the treating of cells having mislocalized and/or aberrantly expressed cell surface molecules in the GI tract, and optionally, for diagnosing, prognosing, and/or evaluating a disease or condition associated with cells having mislocalized and/or aberrantly expressed cell surface molecules in the GI tract. In some embodiments, the genetically engineered microorganism includes a lysis gene encoding a lysis element that allows the genetically engineered microorganism and/or a payload plasmid including the payload gene to enter the cytoplasm of the target cell. For example, the lysis element can lyse an endocytic vacuole to allow the genetically engineered microorganism and/or payload plasmid to escape the phagosome. The payload plasmid can subsequently enter the nucleus of the target cell. The genetically engineered microorganism can also include an auxotrophic mutation in an essential gene that causes lysis of the genetically engineered microorganism within the target cell to release the payload plasmid into the cytoplasm of the target cell. The genetically engineered microorganism can also include a functional copy of the essential gene to serve as a selection mechanism. In some embodiments, the targeting gene and lysis gene are integrated into the genome of the genetically engineered microorganism, while the payload gene and functional copy of the essential gene are located on an episomal plasmid of the genetically engineered microorganism. Attorney Docket No. MMS.003WO The genetically engineered microorganism can also include a payload gene encoding at least one therapeutic agent. For example, the therapeutic agent can be a prodrug converting enzyme that converts an inactive precursor to a therapeutically active product that kills the target cell. In some embodiments, the prodrug converting enzyme is an enzyme that converts a precursor of 5-FU (e.g., 5-FC) into 5-FU, such as a cytosine deaminase or a derivative thereof (e.g., a CD:UPRT fusion protein). As another example, the therapeutic agent can be a direct cytotoxic agent that directly kills the target cell. The genetically engineered microorganism can also include a payload gene encoding at least one detection marker. The detection marker can be a protein, peptide, or nucleic acid molecule (e.g., an RNA molecule). In some embodiments, the detection marker is a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), or an ion channel reporter (e.g., a cAMP activated cation channel), or a combination of any two or more these. The detection marker may be detectable in situ in the subject (e.g., using an endoscopic or colonoscopic procedure) and/or may be detectable in a biological sample from the subject (e.g., feces, blood, serum, plasma, mucus, urine, and/or saliva; and/or from a biopsy sample). In some embodiments, a single genetically engineered microorganism includes a first payload gene encoding at least one therapeutic agent, and a second payload gene encoding at least one detection marker. In some embodiments, a first genetically engineered microorganism includes a first payload gene encoding at least one therapeutic agent, and a second genetically engineered microorganism includes a second payload gene encoding at least one detection marker. In such embodiments, the first genetically engineered microorganism can be administered to the subject before, concurrently with, or after the second genetically engineered microorganism. In some embodiments, the genetically engineered microorganism is non- pathogenic, auxotrophic, and includes an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s). In some embodiments, the cell membrane receptor is not exposed on the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but is exposed on the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc. in a subject suffering from a disease. The surface protein can promote binding and invasion of the microorganism in the Attorney Docket No. MMS.003WO diseased epithelial cells. The microorganism can also include one or more payload gene(s) encoding at least one detection marker and/or therapeutic agent (e.g., prodrug converting enzyme) operably linked to a promoter to drive mammalian or bacterial RNA expression. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the mammalian promoter directs epithelial-specific expression, such as GI tract epithelial cell- specific expression. In some embodiments, the promoter is a bacterial promoter (or a bacteriophage promoter that functions in the genetically engineered microorganism), and the resulting mRNA is translatable by the genetically engineered microorganism and/or a mammalian cell. In some embodiments, the genetically engineered microorganism of the present disclosure delivers a protein to target cells (e.g., diseased epithelial cells). In these embodiments, the one or more payload genes encoding the therapeutic agent and/or detection marker is operably linked to a microbial promoter. In some embodiments, the one or more payload genes encoding the therapeutic agent and/or detection marker includes one or more microbial transcription terminators. In some embodiments, the one or more payload genes encoding the therapeutic agent and/or detection marker include one or more bacterial ribosome binding sites (e.g., Shine-Dalgarno sequences). In some embodiments, the microbial promoter is inducible and/or repressible. In some embodiments, the microbial promoter is constitutive. In some embodiments, the one or more payload genes encoding the therapeutic agent and/or detection marker are inserted on a plasmid. In some embodiments, the one or more payload genes encoding the therapeutic agent and/or detection marker are stably integrated on the chromosome. In some embodiments, the present technology provides a genetically engineered microorganism useful in the detection of mislocalized and/or aberrantly expressed cell surface molecules in the GI tract, and optionally, for prognosing, evaluating, and/or treating a disease or condition, where the genetically engineered microorganism is capable of delivering to the diseased cells an RNA molecule that is capable of self-replicating or self-amplifying. In some embodiments, the RNA molecule that is capable of self-replicating or self-amplifying produces an mRNA encoding one or more therapeutic agents and/or detection markers. In some embodiments, the RNA molecule that is capable of self-replicating or self-amplifying is produced by expression of a cassette that comprises an RNA viral genome replication apparatus and genes encoding one or more detection markers. In some embodiments, the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). In some Attorney Docket No. MMS.003WO embodiments, the RdRP is an alphavirus RdRP. In some embodiments, the alphavirus RdRP includes nsp1, nsp2, nsp3, and/or nsp4. In some embodiments, the one or more payload genes encoding the therapeutic agent and/or detection marker have codon usage optimized for mammalian expression. In some embodiments, the present technology provides methods for treating and/or detecting a target cell in a subject, such as a diseased and/or abnormal cell. For example, the target cell can be a diseased epithelial cell indicative of a disease of the GI tract and/or associated tissues, such as a precancerous lesion, a GI tract cancer, IBD (e.g., ulcerative colitis, Crohn’s disease), IBS, or Barrett’s esophagus. Illustrative precancerous lesions and GI tract cancers include squamous cell carcinoma of anus, low-grade squamous intraepithelial lesions (LSIL) of anus, high-grade squamous intraepithelial lesions (HSIL) of anus, colorectal cancer (e.g., colorectal adenocarcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer), colorectal polyposis (e.g., Peutz-Jeghers syndrome, juvenile polyposis syndrome, MUTYH-associated polyposis, familial adenomatous polyposis/Gardner’s syndrome, Cronkhite-Canada syndrome), carcinoid tumor, pseudomyxoma peritonei, duodenal adenocarcinoma, premalignant adenoma of small bowel, distal bile duct carcinoma, BilIN (e.g., BilIN-1, BilIN-2, BilIN-3, cholangiocarcinoma), PanIN (e.g., PanIN-1, PanIN-2, PanIN-3, pancreatic ductal adenocarcinoma), gastric carcinoma, signet ring cell carcinoma (SRCC), gastric lymphoma (MALT lymphoma), linitis plastic (Brinton’s disease), or squamous cell carcinoma of esophagus and adenocarcinoma. In some embodiments, the precancerous lesion is an adenoma. In some embodiments, the precancerous lesion includes a polyp such as a sessile polyp, a serrated polyp (e.g., hyperplastic polyps, sessile serrated adenomas/polyps, traditional serrated adenoma), a sessile serrated polyp, a flat polyp, a sub-pedunculated polyp, pedunculated polyp, and a combination thereof. In some embodiments, the polyp is a diminutive polyp. In some embodiments, the precancerous lesion is a BilIN selected from BilIN-1, BilIN-2, BilIN-3, or cholangiocarcinoma. In some embodiments, the precancerous lesion is a PanIN selected from PanIN-1, PanIN-2, PanIN-3, or pancreatic ductal adenocarcinoma. In some embodiments, the precancerous lesion has a size within a range from about 0.05 mm to about 30 mm. In some embodiments, the precancerous lesion has a size less than about 0.1 mm, less than about 0.25 mm, less than about 0.5 mm, less than about 1 mm, less than about 2 mm, less than about 5 mm, less than about 8 mm, less than about 10 mm, less than about 15 mm, less than about 20 mm, less than about 25 mm, or less than about 30 mm. Attorney Docket No. MMS.003WO In some embodiments, the cancer is a polyp, an adenoma, or a frank cancer. In some embodiments, the cancer is CRC, Lynch syndrome, familial adenomatous polyposis, hereditary non-polyposis colon cancer (HNPCC), or a sporadic cancer. In some embodiments, the cancer is a BilIN (e.g., BilIN-1, BilIN-2, BilIN-3, cholangiocarcinoma) or a PanIN (e.g., PanIN-1, PanIN-2, PanIN-3, pancreatic ductal adenocarcinoma). In some embodiments, the present technology provides a method for treating a subject, where the method includes administering a genetically engineered microorganism to the subject (e.g., to a GI tract of the subject). The genetically engineered microorganism can include a payload gene encoding a therapeutic agent, such as a prodrug converting enzyme or direct cytotoxic agent. The genetically engineered microorganism can also include a targeting gene encoding a targeting element (e.g., invasin) that binds to a surface marker on a target cell (e.g., a diseased and/or abnormal cell of the GI tract) and/or promotes internalization of the genetically engineered microorganism by the target cell. For instance, the surface marker can be a cell surface marker that is mislocalized in the target cell, such as an integrin. The genetically engineered microorganism can optionally include a lysis gene encoding a lysis element (e.g., a lysin) that lyses an endocytic vacuole of the target cell. The genetically engineered microorganism can optionally include an auxotrophic mutation, e.g., to facilitate lysis of the genetically engineered microorganism in the target cell. In other embodiments, the lysis gene and/or the auxotrophic mutation to facilitate lysis may be omitted. In some embodiments, the genetically engineered microorganism is administered multiple times during a course of treatment, such as at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more. In such embodiments, the genetically engineered microorganism may be administered once every 12 hours, once every 24 hours, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 week, once every month, once every 2 months, once every 3 months, once every 6 months, or once every 12 months. In other embodiments, however, the genetically engineered microorganism may be administered only a single time during the course of treatment. In embodiments where the therapeutic agent is a prodrug converting enzyme that converts a prodrug into an active drug, the method can further include administering a prodrug to the subject. For example, the prodrug can be 5-FC, the active drug can be 5-FU, and the prodrug converting enzyme can be or include a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof (e.g., a Attorney Docket No. MMS.003WO CD:UPRT fusion protein). The prodrug can be administered via any suitable route, such as an oral route, a rectal route, a sublingual route, an intravenous route, an intramuscular route, a subcutaneous route, a transdermal route, an intranasal route, or a vaginal route. The prodrug may be administered via the same route as the genetically engineered microorganism (e.g., an oral route, such as part of a single oral capsule or as two different oral capsules), or may be administered via a different route than the genetically engineered microorganism. The timing of the administration of the genetically engineered microorganism and the prodrug may be varied as desired. In some embodiments, the genetically engineered microorganism is administered concurrently with the prodrug. In some embodiments, the genetically engineered microorganism is administered before the prodrug, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the genetically engineered microorganism is administered to the subject. In some embodiments, the prodrug is administered before the genetically engineered microorganism, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the prodrug is administered to the subject. In some embodiments, the prodrug is administered multiple times during a course of treatment, such as at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more. In such embodiments, the prodrug may be administered once every 12 hours, once every 24 hours, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 2 weeks, once every 3 weeks, once every 4 week, once every month, once every 2 months, once every 3 months, once every 6 months, or once every 12 months. In other embodiments, however, the genetically engineered microorganism may be administered only a single time during the course of treatment. The administration frequency of the prodrug may be the same as the administration frequency of the genetically engineered microorganism, or the administration frequency of the prodrug may be different than the administration frequency of the genetically engineered microorganism. In some embodiments, the genetically engineered microorganism includes a second payload gene encoding a detection marker. The detection marker can be a protein, peptide, or a nucleic acid molecule, as described herein. For example, the detection marker can include one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI Attorney Docket No. MMS.003WO contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter. Alternatively or in combination, the method can include administering a second genetically engineered microorganism including the second payload gene encoding the detection marker. In such embodiments, the second genetically engineered microorganism can include any of the other sequence elements described herein, such as a targeting gene encoding a targeting element, a lysis gene encoding a lysis element, an auxotrophic mutation, and/or additional sequence elements. In some embodiments, the second genetically engineered microorganism is administered concurrently with the genetically engineered microorganism. In some embodiments, the second genetically engineered microorganism is administered before the genetically engineered microorganism, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the genetically engineered microorganism is administered to the subject. In some embodiments, the genetically engineered microorganism is administered before the second genetically engineered microorganism, e.g., at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the second genetically engineered microorganism is administered to the subject. In some embodiments, the method further includes detecting the detection marker (e.g., encoded by the second payload gene of the genetically engineered microorganism or the second genetically engineered microorganism). The method can include collecting a biological sample from the subject after administration of the genetically engineered microorganism and/or the second genetically engineered microorganism. The biological sample can include feces, blood, serum, plasma, mucus, urine, saliva, or a combination thereof. In some embodiments, the biological sample is collected using a noninvasive technique (e.g., without performing colonoscopy or endoscopy). The biological sample can be collected at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 5 days, at least about 7 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, or at least about 60 days after administration of the genetically engineered microorganism and/or the second genetically engineered microorganism to the subject. In some embodiments, multiple biological samples are collected at multiple time points, such as at least two, three, four, five, 10, 20, or more different time points. For example, Attorney Docket No. MMS.003WO biological samples can be collected once every hour, once every two hours, once every 12 hours, once every 24 hours, once every 48 hours, or once every week after administration of the genetically engineered microorganism and/or the second genetically engineered microorganism to the subject. The method can further include detecting whether at least one detection marker is present in the biological sample. For example, the detection marker can be or include a protein, a peptide, or a nucleic acid. In some embodiments, the detection marker is a gene product encoded by a payload gene that is transferred to the target cell by the genetically engineered microorganism and/or the second genetically engineered microorganism. In some embodiments, the biological sample includes a plurality of cells, including the target cell (e.g., diseased and/or abnormal cells) and non-target cells (e.g., the genetically engineered microorganism, healthy cells). In such embodiments, the method can include processing the biological sample to extract any detection marker present on the target cells, within the target cells, and/or secreted by the target cells. The method can further include performing an assay on the extracted detection marker to determine the presence and/or amount of the detection marker. The assay can include agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, CD, chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, flow cytometry, fluorimetry, electrophoretic assay, enzymatic assay, ELISA, gas chromatography, gravimetry, HPLC, immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, RT-PCR, multiplex RT-PCR, qPCR, RT-qPCR, dPCR, ddPCR, semi-quantitative PCR, semi-quantitative RT-PCR, gel electrophoresis, RNA sequencing, FISH (e.g., RNA-FISH), CISH, or suitable combinations thereof. For instance, in embodiments where the biological sample is a fecal sample, the fecal sample can contain diseased and healthy epithelial cells sloughed off from the GI tract, as well as the genetically engineered microorganism and/or the second genetically engineered microorganism. The fecal sample can be collected by defecation into a collection device, or can be collected during a procedure, such as an enema, a fecal swab, or an endoscopy. The fecal sample can be tested immediately, or can be stored in a buffer prior to testing, such as an aqueous buffer, a glycerol-based buffer, a polar solvent based-buffer, an osmotic balance buffer, or other buffer sufficient for preserving the fecal sample. The fecal sample can optionally be refrigerated, for example, at 4 °C, 0 °C, -20 °C, -80 °C, or lower. Attorney Docket No. MMS.003WO The fecal sample can be disrupted in the presence of a buffer and/or a surfactant to form a suspension. The buffer can be a biologically compatible buffer, such as Hanks’ balanced salt solution, Alsever’s solution, Earle’s balanced salt solution, Gey’s balanced salt solution, phosphate buffered saline, Puck’s balanced salt solution, Ringer’s balanced salt solution, Simm’s balanced salt solution, TRIS-buffered saline, or Tyrode’s balanced salt solution. The surfactant can be an ionic or non-ionic surfactant, such as Tween-20 or Triton- X-100. In embodiments where the detection marker is or includes an RNA molecule, the fecal sample can be disrupted in the presence of a ribonuclease inhibitor. The ribonuclease inhibitor can be solvent based, protein based, or another type of method to prevent RNA destruction, including, for example, Protector RNase Inhibitor (Roche), RNasin® (Promega), SUPERase- In™ (Thermo Fisher Scientific), RNaseOUT™ (Thermo Fisher Scientific), ANTI-RNase, Recombinant RNase Inhibitor, or a cloned RNase Inhibitor. In some embodiments, the fecal sample is disrupted by vortexing, shaking, stirring, rotating, or otherwise mechanically agitating the fecal sample with the buffer, surfactant, and/or ribonuclease inhibitor to form the suspension. The detection marker can be extracted from the suspension using techniques such as centrifugation, filtration, targeted probes that specifically bind the detection marker, antibodies, column-based techniques, bead-based techniques, solvent-based techniques, chromatographic techniques, or suitable combinations thereof. In embodiments where the detection marker is an intracellular marker, the cells present in the fecal sample can be lysed using a lysis buffer and/or mechanical lysis techniques, and the lysate can be processed via centrifugation, filtration, column-based techniques, bead-based techniques, solvent-based techniques, chromatographic techniques, etc., to extract the detection marker from the lysate. In embodiments where the detection marker is or includes an RNA molecule, the fecal sample can be treated with DNase to degrade DNA. The detection marker extracted from the biological sample can then be measured or otherwise analyzed using one or more of the techniques described herein. Presence of the detection marker in the biological sample can indicate that the target cell is present in the subject, which can be indicative of the subject having the disease or being at risk of developing the disease. Alternatively or in combination, the method can include other techniques for detecting the presence of the detection marker. For example, the detection marker can be detected in situ within the subject using techniques such as endoscopy, colonoscopy, MRI, CT Attorney Docket No. MMS.003WO imaging, PET imaging, or a combination thereof. enzyme. Cells that accumulate the detection marker (e.g., on the surface and/or within the intracellular space) can be identified as the target cells (e.g., diseased cells), while cells that do not accumulate the detection marker can be identified as non-target cells (e.g., normal cells). In embodiments where the detection marker is detectable upon interaction with a substrate, the method can optionally include administering the substrate the subject, such as a substrate of a bioluminescent protein, a substrate of an MRI contrast agent, a PET probe, a substrate of an enzyme reporter, a SPECT probe, or a combination of any two or more thereof. The substrate can be administered prior to marker detection by at least about 1 hour, at least about 6 hours, at least about 12 hours, at least about 24 hours, at least about 2 days, or at least about 3 days prior to marker detection. In some embodiments, the substrate may be administered after the administration of the genetically engineered microorganism and/or the second genetically engineered microorganism. The method can further include selecting the subject for a treatment if the detection marker is determined to be present, thus indicating that the subject has the disease or is at risk of developing the disease, and/or indicating that previous treatments (e.g., treatment with a genetically engineered microorganism including a payload gene encoding a therapeutic agent) did not sufficiently treat or otherwise mitigate the disease. In some embodiments, the treatment is or includes surgery, e.g., to remove diseased tissue. Alternatively or in combination, the treatment is or includes administration of a therapeutic agent. The therapeutic agent can be selected from a chemotherapeutic agent, a cytotoxic agent, an immune checkpoint inhibitor, an immunosuppressive agent, a sulfa drug, a corticosteroid, an antibiotic, or a combination of any two or more thereof. Alternatively or in combination, the treatment is or includes administering a third genetically engineered microorganism to the subject. The third genetically engineered microorganism can include a payload gene encoding a therapeutic agent, which may the same payload gene and therapeutic agent as the genetically engineered microorganism, or can be a different payload gene and therapeutic agent. The third genetically engineered microorganism can include any of the other sequence elements described herein, such as a targeting gene encoding a targeting element, a lysis gene encoding a lysis element, an auxotrophic mutation, and/or additional sequence elements. In some embodiments, the methods described herein are performed on a subject that is at low risk for cancer (e.g., CRC) and/or colorectal polyps. In some embodiments, the subject has no previous incidence of cancer and/or colorectal polyps. In some embodiments, the method is performed on a subject that is high or medium risk for cancer and/or colorectal Attorney Docket No. MMS.003WO polyps. In some embodiments, the subject has a prior incidence of cancer and/or colorectal polyps, and/or has a family history of cancer and/or colorectal polyps. In some embodiments, the subject has a genetic predisposition to cancer. In some embodiments, the subject has FAP, attenuated FAP, Lynch syndrome, oligopolyposis, Peutz-Jeghers syndrome, MUTYH- associated polyposis, juvenile polyposis syndrome, and/or Cowden syndrome. In some embodiments, the subject is at least 45 years of age, at least 50 years of age, at least 55 years of age, or at least 60 years of age. In some embodiments, the present technology provides compositions and methods that are useful for treating diseased GI tissue. In some embodiments, the present technology relates to a method for treating diseased epithelial tissue, where the method includes administering to the GI tract of a subject in need thereof, a genetically engineered microorganism engineered to direct expression of a prodrug converting enzyme specifically in diseased epithelial cells of the GI tract. In some embodiments, the diseased epithelial tissue is selected from GI tract epithelium and/or bile duct epithelium. In some embodiments, the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 5-FC into 5-FU. In some embodiments, the enzyme is selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450 (e.g., that acts on the prodrug as a substrate), a uracil phosphoribosyltransferase (yUPRT), and a combination thereof or a fusion protein comprising two or more thereof. In some embodiments, the enzyme is a fusion protein of a cytosine deaminase and a UPRT. In some embodiments, the cytosine deaminase and/or the UPRT is derived from yeast. In various aspects, the present technology relates to a method for treating a disease characterized by the presence of diseased GI tissue in a subject in need thereof. In some embodiments, the method includes administering to the epithelial tissue of a subject, an engineered microorganism that specifically invades diseased cells of the epithelium, and directs expression of a prodrug converting enzyme in the diseased cells. In some embodiments, the method further includes administering a prodrug. In some embodiments, the genetically engineered microorganism includes an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue. In some embodiments, the surface protein promotes binding and/or invasion of the microorganism in the diseased epithelial cells. The genetically engineered microorganism disclosed herein can include one or more gene(s) encoding a surface protein, where the surface protein specifically interacts with Attorney Docket No. MMS.003WO one or more cell membrane receptor(s), where the one or more cell membrane receptor(s) are not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc.; and where the one or more cell membrane receptor(s) are exposed to the luminal side of epithelial cells of diseased GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc. In some embodiments, the surface protein promotes the binding and invasion specifically of epithelial cells of diseased GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., by the genetically engineered microorganism. In some embodiments, the genetically engineered microorganism disclosed herein includes a second exogenous gene encoding a lysin that lyses the endocytotic vacuole, and thereby contributes to pore formation, breakage, or degradation of the phagosome. In some embodiments, the lysin is a cholesterol-dependent cytolysin. In some embodiments, the lysin is selected from the group consisting of listeriolysin O, ivanolysin O, streptolysin, perfringolysin, botulinolysin, leucocidin, and a mutant derivative thereof. In some embodiments, the lysin is listeriolysin O (SEQ ID NO: 3), or a mutant derivative thereof (without limitation, e.g., SEQ ID NO: 4). In various aspects, the present technology relates to a method for treating a disease characterized by the presence of diseased GI tissue in a subject in need thereof. The diseases that may be treated using the genetically engineered microorganisms and/or using the methods disclosed herein include precancerous lesions, GI tract cancers, ulcerative colitis, Crohn’s disease, Barrett ’s esophagus, irritable bowel syndrome and irritable bowel disease. GI tract cancers and precancerous syndromes include squamous cell carcinoma of anus, colorectal cancer (CRC) (including colorectal adenocarcinoma, familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer), colorectal polyposis (including Peutz-Jeghers syndrome, juvenile polyposis syndrome, MUTYH-associated polyposis, familial adenomatous polyposis/Gardner's syndrome, and Cronkhite-Canada syndrome), carcinoid, pseudomyxoma peritonei, duodenal adenocarcinoma, distal bile duct carcinomas, pancreatic ductal adenocarcinomas, gastric carcinoma, signet ring cell carcinoma (SRCC), gastric lymphoma (MALT lymphoma), linitis plastic (Brinton ’s disease), and squamous cell carcinoma of esophagus and adenocarcinoma. The diseased epithelial cells from subjects suffering from one or more of these indications may be treated using the genetically engineered microorganisms of the present technology. The genetically engineered microorganisms can specifically bind to diseased epithelial cells by specifically interacting with one or more cell membrane receptor(s) Attorney Docket No. MMS.003WO that are exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc. The genetically engineered microorganisms may not bind to normal (non-diseased) epithelial cells because the one or more cell membrane receptor(s) are not exposed to the luminal side of the normal epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc. In some embodiments, the genetically engineered microorganism delivers one or more nucleic acid(s) encoding at least one detection marker and/or prodrug converting enzyme to the diseased epithelial cells (target cells). In these embodiments, the diseased epithelial cells (target cells) can express the at least one detection marker and/or prodrug converting enzyme, allowing their detection or targeted delivery of the prodrug converting enzyme to them. For example, the diseased epithelial cells (target cells) can be identified as the cells that accumulate the at least one detection marker and/or prodrug converting enzyme inside them or on their surface, while the detection marker and/or prodrug converting enzyme is not present in or on the surface of the surrounding healthy cells (normal epithelial cells). Detection of the diseased epithelial cells may be carried out using a suitable technique such as colonoscopy, endoscopy, magnetic resonance imaging, CT scan, PET scan, SPECT scan, etc. A method for detecting and/or treating a target cell in a subject can include administering at least one genetically engineered microorganism of the present disclosure to the subject. In some embodiments, the genetically engineered microorganism is administered via an oral and/or rectal route. For example, the genetically engineered microorganism can be administered into a lumen of the GI tract of the subject. The genetically engineered microorganism can be administered as part of a pharmaceutical composition including a pharmaceutically acceptable excipient, as described herein. In some embodiments, the method optionally includes administration of a colon cleansing agent, such as a laxative. In some embodiments, the colon cleansing agent is administered prior to and/or after the administration of the microorganism. In various aspects, the present technology relates to a method for diagnosing and treating a disease characterized by the presence of diseased GI tissue in subject in need thereof. In some embodiments, the method comprises administering to the epithelial tissue of a subject, an engineered microorganism that specifically invades diseased cells of the epithelium, and directs expression of a prodrug converting enzyme and a detection marker in Attorney Docket No. MMS.003WO the diseased cells. In some embodiments, the method further includes administering a prodrug. In some embodiments, the prodrug is administered where diseased tissue is detected. In various embodiments, the methods include administering to the GI tract of a subject in need thereof, a genetically engineered microorganism that directs expression of a prodrug converting enzyme and a detection marker specifically in diseased cells. In some embodiments, the method further includes administering a prodrug. In some embodiments, the prodrug is administered to diseased tissue that is detected. The method can further involve detecting the expression of the detection marker to thereby detect the diseased epithelial cells. In some embodiments, the genetically engineered microorganism is non-pathogenic, auxotrophic, and includes an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s). The cell membrane receptor may not be exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but may be exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., in the subject suffering from a disease. Thus, the expression and/or localization of the one or more cell membrane receptor(s) can confer the specificity for diseased or abnormal cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., on the microorganism. The surface protein can thus promote binding and invasion of the microorganism in the diseased epithelial cells. The microorganism can also include one or more gene(s) encoding a prodrug converting enzyme and a detection marker operably linked to a promoter (e.g., a mammalian or bacterial promoter). Thereby, in some embodiments, the microorganism delivers a nucleic acid (e.g., a DNA or an mRNA molecule) for expression of the detection marker in diseased epithelial cells. In some embodiments, the diseased epithelial cells express a prodrug converting enzyme and a detection marker, thereby allowing their detection. In some embodiments, the method further includes administering a prodrug. In some embodiments, the prodrug is administered when diseased tissue is detected (e.g., when potential cancerous or malignant tissue is detected). In some embodiments, the promoter may be a mammalian promoter. In some embodiments, the mammalian promoter directs GI tract epithelial cell-specific expression. In some embodiments, the promoter is a bacterial promoter, and the resulting mRNA is translatable in the bacterial or mammalian cell. In various aspects, the present technology provides a method for diagnosis and/or prognosis of a disease or disorder in a subject, the method comprising (i) administering Attorney Docket No. MMS.003WO to the GI tract of a subject in need thereof, a genetically engineered microorganism disclosed herein; and (ii) detecting the expression of the detection marker, thereby detecting the diseased epithelial cells. As discussed above, the microorganism can include an exogenous gene encoding a surface protein, where the surface protein specifically interacts with one or more cell membrane receptor(s), which are not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but is exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., in the subject suffering from a disease. In some embodiments, the surface protein promotes binding and invasion of the microorganism in the diseased epithelial cells. The microorganism can also include one or more gene(s) encoding the detection marker operably linked to a promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the mammalian promoter that is active or specific for epithelial expression or GI tract epithelial cell-specific expression. In some embodiments, the mammalian promoter directs GI tract epithelial cell-specific expression. In these embodiments, the microorganism delivers a DNA molecule (e.g., a plasmid) to diseased epithelial cells. In some embodiments, the genetically engineered microorganism is administered via an oral or rectal route. In some embodiments, the method further includes administration of a colon cleansing agent comprising a laxative. In some embodiments, the colon cleansing agent including the laxative is administered prior to the administration of the microorganism. In some embodiments, the genetically engineered microorganism is non-pathogenic. In some embodiments, the genetically engineered microorganism is auxotrophic. In some embodiments, the genetically engineered microorganism is non-pathogenic and auxotrophic. In various aspects, the present technology provides a genetically engineered microorganism for use in a method of diagnosis and/or prognosis of a disease or disorder in a subject, the method comprising (i) administering to the gastrointestinal tract of a subject in need thereof, disclosed herein; and (ii) detecting the expression of the detection marker, thereby detecting the diseased epithelial cells. As discussed above, the microorganism can include an exogenous gene encoding a surface protein, where the surface protein specifically interacts with one or more cell membrane receptor(s), which are not exposed to the luminal side of epithelial cells of normal GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, etc., but is exposed to the luminal side of diseased epithelial cells of GI tissue and/or epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct, Attorney Docket No. MMS.003WO etc., in the subject suffering from a disease. In some embodiments, the surface protein promotes binding and invasion of the microorganism in the diseased epithelial cells. The microorganism can also include one or more gene(s) encoding the detection marker operably linked to a promoter. In some embodiments, the promoter is a mammalian promoter. In some embodiments, the mammalian promoter directs GI tract epithelial cell-specific expression. In some embodiments, the genetically engineered microorganism is administered via an oral or rectal route. In some embodiments, the method further includes administration of a colon cleansing agent comprising a laxative. In some embodiments, the colon cleansing agent including the laxative is administered prior to the administration of the microorganism. In some embodiments, the genetically engineered microorganism is non-pathogenic. In some embodiments, the genetically engineered microorganism is auxotrophic. In some embodiments, the genetically engineered microorganism is non-pathogenic and auxotrophic. Disclosed herein, in various aspects, are methods of selecting a subject suffering from or suspected to be suffering from a disease for a treatment, the method comprising: (i) administering to the gastrointestinal tract of the subject a genetically engineered microorganism of any one of the embodiments disclosed herein; (ii) detecting elevated expression of the detection marker compared to surrounding normal epithelial cells; and (iii) selecting the subject for treatment if expression of the detection marker is observed compared to surrounding normal epithelial cells. In some embodiments, the disease is selected from a precancerous lesion, cancer, ulcerative colitis, Crohn’s disease, Barrett’s esophagus, irritable bowel syndrome, and irritable bowel disease. In some embodiments, the treatment is surgery or administration of a therapeutic agent. In some embodiments, the surgery removes diseased tissue. In some embodiments, the therapeutic agent is selected from a chemotherapeutic agent, a cytotoxic agent, an immune checkpoint inhibitor, an immunosuppressive agent, a sulfa drug, a corticosteroid, an antibiotic, and a combination of any two or more thereof. Also disclosed herein, in various aspects, are methods of treating a cancer in a patient. These methods comprise: (i) administering to the GI tract of the subject a genetically engineered microorganism of the present technology; (ii) detecting the expression of the detection marker, thereby detecting the diseased epithelial cells; and (iii) administering a treatment if the expression of the detection marker is observed. In some embodiments, the treatment is surgery or administration of a therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of a chemotherapeutic agent, a cytotoxic Attorney Docket No. MMS.003WO agent, an immune checkpoint inhibitor, an immunosuppressive agent, a sulfa drug, a corticosteroid, an antibiotic, and a combination of any two or more thereof. V. Examples The present technology is further illustrated by the following non-limiting examples. Example 1: Delivery of DNA Encoding a Chemotherapeutic to Diseased Cells by Genetically Engineered Bacterial Strains Bacteria of the current disclosure can specifically detect diseased cells and deliver a prodrug converting enzyme to diseased cells, thereby enabling targeted drug delivery to diseased cells. Without being bound by theory, it is hypothesized that payload delivery to diseased cells proceeds through four distinct steps. As shown in FIG. 1C and FIG. 10A, the genetically engineered microorganisms of the current disclosure can bind to diseased epithelial cells through mislocalized receptors, and undergo internalization (FIG. 1C). Upon internalization, the bacteria may undergo lysis, inter alia, due to the dapA attenuation mutation, which causes a defect in cell wall synthesis (FIG.1C). Listeriolysin O (LLO, or Hly; SEQ ID NO: 3) can then be released and lyses the phagosome or is naturally exported from the E. coli strain. As shown in FIG.1C, the plasmid carrying the prodrug converting enzyme can undergo nuclear localization, which can occur naturally or be guided by binding of a protein that includes a nuclear localization signal. As shown in FIG.1C, the plasmid carrying the prodrug converting enzyme can drive the expression of the prodrug converting enzyme in the diseased epithelial cells of the GI tract and/or epithelium of the ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and/or common bile duct). Without wishing to be bound by theory, the diseased epithelial cells may exhibit mislocalized receptors and/or expression of novel mammalian membrane receptors. The novel receptors may be receptors that are normally not found in the cells and/or receptor formed by translocations and other genomic rearrangement. Disclosed herein are bacterial strains derived from Escherichia coli Nissle 1917 for DNA transfer to diseased epithelial cells. FIG.3 shows a schematic of a strain for DNA transfer. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG. 3 (top panel)). This strain carries a Attorney Docket No. MMS.003WO plasmid containing a payload gene under a eukaryotic promoter and selection based on a wild- type alr gene (FIG.3 (bottom panel)). The above strain was further modified as shown in FIG.4 for a targeted delivery of a chemotherapeutic to diseased epithelial cells. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv) and listeriolysin O (hlyA) (FIG. 4 (top panel)). The strain carries a plasmid having an intron- containing gene encoding a chimeric bifunctional yeast cytosine deaminase (yCD) and the yeast uracil phosphoribosyltransferase (yUPRT, fusion protein is yCD:UPRT) gene under a eukaryotic promoter. The plasmid also carries a promoter-less iRFP670 gene following an internal ribosome entry site (IRES) from an encephalomyocarditis virus (EMCV) (SEQ ID NO: 10, FIG.4 (bottom panel)). The yCD:UPRT gene, which contains an intron, was placed under the control of a eukaryotic promoter, to prevent the expression of yCD:UPRT enzyme in bacteria. To test this scheme, the bacteria from this strain represented in FIG. 4 were coincubated with HEK293T cells (an immortalized cancer cell line) for two hours followed by washing away of extracellular bacteria. Bacteria lacking invasin or listeriolysin O were used as controls. To determine the efficiency of gene transfer, the HEK293T cells were analyzed by flow cytometry. As shown in FIG.5A, iRFP670 expression could be detected when the strain comprising invasin and listeriolysin O was used. On the other hand, the bacteria lacking invasin or listeriolysin O showed only background levels of iRFP670 expression (FIG.5A). The HEK293T cells were further incubated in the presence of increasing concentrations of 5-fluorocytosine (5-FC) and their viability was measured. yCD:UPRT deaminates the nontoxic prodrug 5-fluorocytosine (5-FC) to the chemotherapeutic compound 5-fluorouracil (5-FU), which is used in the treatment of malignant tumors. As shown in FIG. 5B, HEK293T cells incubated with the strain comprising invasin and listeriolysin O (shown in FIG.4) were killed. On the other hand, the bacteria lacking invasin or listeriolysin O did not show significant lethality in the presence of 5-FC (FIG.5B). These results demonstrate, inter alia, an invasin- and LLO-dependent genetic transfer of a prodrug converting enzyme to diseased cells. Attorney Docket No. MMS.003WO Example 2: Delivery of DNA and mRNA to Diseased Cells by Genetically Engineered Bacterial Strains The above results suggest that the iRFP670 mRNA was translated in mammalian cells through action of the EMCV IRES, opening the door for RNA transfer. Therefore, mRNA transfer was directly tested. For that purpose another strain was designed. FIG. 6 shows a schematic of a strain for RNA transfer. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG.6 (top panel)). This strain carries a plasmid containing a payload (illustrated herein using the iRFP670 gene) under a prokaryotic promoter and selection based on a wild-type alr gene (FIG.6 (bottom panel)). The iRFP670 gene followed an internal ribosome entry site (IRES) from an encephalomyocarditis virus (EMCV) (SEQ ID NO: 10, FIG.6 (bottom panel)). DNA transfer and RNA transfer to diseased cells were studied. Without wishing to be bound by theory, the mechanism of DNA transfer is shown in FIG.1C. The bacteria of the strain represented in FIG.3 (with the iRFP670 gene as the payload gene) were coincubated with HEK293T cells (an immortalized cancer cell line) for one hour followed by washing away of extracellular bacteria. Bacteria lacking invasin or listeriolysin O were used as controls. To determine the efficiency of gene transfer, the HEK293T cells were analyzed by flow cytometry. As shown in FIG.7A, iRFP670 expression could be detected when the strain comprising invasin and listeriolysin O was used. On the other hand, the bacteria lacking invasin or listeriolysin O showed only background level of iRFP670 expression (FIG.7A). Without wishing to be bound by theory, the mechanism of RNA transfer is shown in FIG. 1D. As shown in FIG. 1D, the genetically engineered microorganisms of the current disclosure can bind to diseased epithelial cells through mislocalized receptors and undergo internalization (FIG.1D). Upon internalization, the bacteria may undergo lysis, inter alia, due to the dapA attenuation mutation, which causes a defect in cell wall synthesis (FIG. 1D). Listeriolysin O (LLO, or Hly; SEQ ID NO: 3) can then be released and lyses the phagosome or is naturally exported from the E. coli strain. The mRNA transcribed in the bacteria can then be transferred to the diseased cells, as shown in FIG. 1D, and translated in diseased cells under the control of the EMCV IRES, which thereby drives the expression of the prodrug converting enzyme in the diseased epithelial cells of GI tract and/or epithelium of the Attorney Docket No. MMS.003WO ducts that are connected with gastrointestinal tissue (e.g., epithelial tissue lining the bile duct, pancreatic duct, and common bile duct). To test this scheme in vitro, the bacteria of the strain represented in FIG.6 were coincubated with HEK293T cells (an immortalized cancer cell line) for two hours followed by washing away of extracellular bacteria. Bacteria lacking invasin or listeriolysin O were used as controls. To determine the efficiency of gene transfer, the HEK293T cells were analyzed by flow cytometry. As shown in FIG.7B, iRFP670 expression could be detected when the strain comprising invasin and listeriolysin O was used. On the other hand, the bacteria lacking invasin or listeriolysin O showed only background level of iRFP670 expression (FIG.7B). These results demonstrate, inter alia, an invasin- and LLO-dependent DNA or mRNA transfer to diseased cells. To improve the stability of mRNA in the bacteria, ribonuclease III (RNase III) in the bacteria was deleted. Thus, the resulting strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations, an rncΔ mutation, and chromosomal integration of genes encoding invasin (inv) and listeriolysin O (hlyA; LLO). To test RNA transfer using this strain, the invasin+ and listeriolysin O+ dapAΔ, alrΔ, dadXΔ rncΔ bacteria were coincubated with HEK293T for two hours followed by washing away of extracellular bacteria. Invasin+ and listeriolysin O+ dapAΔ, alrΔ, dadXΔ rnc+ bacteria, or invasin+ and listeriolysin O+ wild type bacteria harboring the plasmid shown were used as controls. Each strain carried the wild-type alr gene-selected plasmid harboring the iRFP670 gene under a prokaryotic promoter and an EMCV IRES (FIG.6 (bottom panel)). To determine the efficiency of gene transfer, the HEK293T cells were analyzed by flow cytometry to identify the portion of the HEK293T cells that expressed iRFP670 under the control of EMCV IRES. As shown in FIGS.8A and 8B, iRFP670 expression increased when the rncΔ strain was used (“-rnc”) compared to rnc+ bacteria (“+rnc”). These results demonstrate that RNA delivery by the strains disclosed herein may be improved by mutations that reduce activity of an RNase gene such as RNase III. Example 3: In Vivo Bacterial Targeting of Diseased Cells The data above show that genetic transfer depends on invasin. The following experiment was carried out to confirm that the initial binding to diseased tissue depends on presence of a receptor for bacteria on the cells. Briefly, β1 integrin in HCT116 cells was knocked out. To test invasin dependent binding in HCT116 cells lacking β1 integrin (having the chromosomal gene for production of Attorney Docket No. MMS.003WO β1 integrin knocked out) or β1 integrin+ HCT116 cells, invasin+ and dapAΔ, alrΔ, dadXΔ bacteria were coincubated with the cells for one hour followed by washing away of extracellular bacteria. dapAΔ, alrΔ, dadXΔ bacteria lacking invasin were used as a control. Each strain carried the wild-type alr gene-selected plasmid expressing mNeonGreen gene under a prokaryotic promoter. To determine the efficiency of binding, the HCT116 cells were analyzed by flow cytometry. As shown in FIG.10B, wild type HCT116 cells were positive for the presence of mNeonGreen only when bacteria expressing wild-type invasin (“invasin+”) were used. Only background levels of mNeonGreen positive cells were observed when bacteria expressing a truncated invasin incapable of binding to β1 integrin were used (“invasin-”) (FIG. 10B). As expected, as shown in FIG. 10C, HCT116 cells lacking β1 integrin showed background levels of mNeonGreen positive cells even when invasin+ bacteria were used. Wild type HCT116 cells were identified as mNeonGreen positive when invasin+ bacteria were used (FIG.10C). These results demonstrate that the initial binding to diseased tissue depends both on the presence of a receptor for bacteria on the diseased cells as well as a ligand for the diseased cells on the bacteria. The Apcfl/fl,VilCre mouse model was used to study mislocalization of receptors for the bacteria in diseased cells and in vivo genetic transfer to diseased cells by the strains disclosed herein. The tumor suppressor gene Apc in this strain is deleted in colonic villi when (Z) 4-hydroxytamoxifen is administered. FIG. 9A shows a schematic representation of the model. Upon injection of (Z) 4-hydroxytamoxifen, these mice develop tumors in the distal colon but not in the proximal colon (FIG. 9A (right panel)). Mislocalization of β1 integrin is detected in diseased tissue (FIG. 9B), which shows a representative confocal fluorescence microscopy image of colon tissue section showing healthy tissue and diseased tissue. The development of tumors in this model was studied by analyzing the β1 integrin localization at 3, 5, 7, 11, 13, and 21 days after the injection of 4-hydroxytamoxifen. As shown, progressive induction of colorectal cancer in Apcfl/fl,VilCre mice was seen as demonstrated by confocal fluorescence microscopy images of colon tissue sections taken 3 days (FIG.9C), 5 days (FIG. 9D), 7 days (FIG.9E), 11 days (FIG.9F), 13 days (FIG.9G), and 3 weeks (FIG.9H) after the injection of 4-hydroxytamoxifen. To test binding to diseased cells in vivo, tumors were induced in the Apcfl/fl,VilCre mice and three weeks later, bacteria of the following strains were co- administered to the mice by enema: Attorney Docket No. MMS.003WO • Experimental bacteria: invasin+ and alrΔ, dadXΔ bacteria harboring a wild-type alr gene-selected plasmid expressing a GFP gene under a prokaryotic promoter, and • Control bacteria: invasin- and alrΔ, dadXΔ bacteria harboring a wild-type alr gene- selected plasmid expressing an RFP gene under a prokaryotic promoter. Three hours later, the colon was extracted from the mice and analyzed using epifluorescence microscopy (FIG.11A). As shown in FIG.11B, GFP expressing bacteria were observed bound to diseased tissue (tumors) but not normal tissue. There was no detectable RFP expressing bacteria bound to diseased or normal tissue. These results demonstrate, inter alia, the binding of diseased cells but not normal cells by invasin+ bacteria. To explore discrimination of diseased tissue in mice having early cancerous lesions, the following experiment was performed: tumors were induced in the Apcfl/fl,VilCre mice and 3 to 7 days later, bacteria of the following strains were co-administered to the mice by enema: • Experimental bacteria: invasin+ and alrΔ, dadXΔ bacteria harboring a wild-type alr gene-selected plasmid expressing a GFP gene under a prokaryotic promoter, and • Control bacteria: invasin- and alrΔ, dadXΔ bacteria harboring a wild-type alr gene- selected plasmid expressing an RFP gene under a prokaryotic promoter. Three hours later, the colon was extracted from the mice and analyzed using epifluorescence microscopy (FIG.12A). As shown in FIG.12B, GFP expressing bacteria were observed binding to diseased tissue as early as 5 days after induction of tumor induction by Apc deletion. GFP expressing bacteria were not observed in normal tissue. There was no detectable RFP expressing bacteria in diseased or normal tissue. The experiment was re-performed after swapping fluorescent markers. Specifically, tumors were induced in the Apcfl/fl,VilCre mice and 3 to7 days later, bacteria of the following strains were co-administered to the mice by enema: • Experimental bacteria: invasin+ and alrΔ, dadXΔ bacteria harboring a wild-type alr gene-selected plasmid expressing an RFP gene under a prokaryotic promoter, and • Control bacteria: invasin- and alrΔ, dadXΔ bacteria harboring a wild-type alr gene- selected plasmid expressing a GFP gene under a prokaryotic promoter. Three hours later, colon was extracted from the mice and analyzed using epifluorescence microscopy (FIG.13A). As shown in FIG.13B, RFP expressing bacteria were observed binding to diseased tissue as early as 5 days after induction of tumor induction by Attorney Docket No. MMS.003WO Apc deletion. RFP expressing bacteria were not observed in normal tissue. There was no detectable GFP expressing bacteria in diseased or normal tissue. These results demonstrate, inter alia, the discrimination of precancerous lesions from healthy tissue as early as five days after induction of carcinogenesis. To detect internalization of bacteria in the mice, the following experiment was performed: tumors were induced in the Apcfl/fl,VilCre mice and two weeks later, a surgical intervention to increase bacterial concentration in the colon was performed. Briefly, the cecum and colon were pulled out after opening the skin and peritoneum, and the proximal colon was tied with 5-0 Vet. CRYL (string) to prevent bacterial diffusion (FIG.14A, left panel; FIG.14A, right panel). The cecum and colon were returned to the abdomen, and the skin and peritoneum were closed (FIG. 14A, middle panel). The invasin+ alrΔ, dadXΔ bacteria harboring a wild- type alr gene-selected plasmid expressing GFP and a kanamycin resistance marker gene under a prokaryotic promoter were administered to the mice by enema, and the anus was closed with 5-0 Vet. CRYL (string) and glue was placed on the anus.3 hours later, the mice were sacrificed, and the colon was extracted (FIG. 14A, right panel)) and colon tissue was subjected to fluorescence microscopy. The associated bacteria in diseased cells and normal cells were detected by Image J software and counted. Table 7 below shows the number of bacteria in tumor and non-tumor samples: Table 7: Number of Bacteria Detected
Figure imgf000097_0001
The number of bacteria was plotted. As shown in FIG. 14B, no bacteria were associated with normal tissue. In contrast, diseased tissue showed the presence of hundreds of bacteria (FIG. 14B). FIG. 14C shows fluorescence microscopy from different mice showing bacteria that were associated with tumor tissue (right images) but not in non-tumor tissue (left images). These results demonstrate, inter alia, that the bacteria disclosed herein showed low levels of false negative detection of diseased tissue. Attorney Docket No. MMS.003WO Example 4: In Vitro and In Vivo Invasion of Diseased Cells by Genetically Engineered Bacteria To further study internalization of bacteria in diseased cell in vitro, the following experiment was performed (FIG.15A): invasin+ and listeriolysin O+ dapAΔ, alrΔ, dadXΔ bacteria were coincubated with HCT116 for 1 hour. Listeriolysin O+ dapAΔ, alrΔ, dadXΔ bacteria lacking invasin were used as controls. After 1 hour, extracellular bacteria were washed away and the cells were further incubated for 0, 2, 4 or 40 hours. Gentamicin was added to the cells to kill any non-invaded bacteria. After incubation for 1 hour, the HCT116 cells were lysed using a detergent, and serial dilutions of the lysate was plated (FIG.15A). As shown in FIG. 15B, thousands of invasin+ bacteria could be detected in the lysate. In contrast, no bacteria lacking invasin were detected in the lysate. As shown in FIG. 15B, the number of invaded bacteria decreased with time, which could be due to their lysis. These results demonstrate, inter alia, invasin-dependent invasion of the bacteria disclosed herein in diseased cells. To demonstrate the internalization of bacteria into diseased tissue in vivo, tumors were induced in Apcfl/fl,VilCre mice. Two weeks later, the surgery noted above to seal the colon was performed on the mice, followed by administration of an enema with invasin+ alrΔ, dadXΔ bacteria harboring a wild-type alr gene-selected plasmid expressing GFP and a kanamycin resistance marker gene under a prokaryotic promoter. After 3 hours, the proximal colon tissue (non-tumor tissue) and distal colon tissue (having tumors) specimens were extracted and viewed by fluorescence and phase contrast microscopy. As shown in FIG.16A, the bacteria were seen associated with tumor tissue but not non-tumor tissue. The colon tissue was added into a 1.5 mL tube with 200 µL PBS and vortexed twice for 10 seconds each (sample # 1). Serial dilutions of the supernatants were spotted on an LB plate supplemented with diaminopimelic acid. As shown in FIG. 16B, bacteria associated with but not internalized within tumor tissue could grow on LB plates supplemented with diaminopimelic acid and kanamycin. The washed colon tissue was incubated with gentamycin to kill any remaining extracellular bacteria, followed by washing away of the gentamycin and a treatment with 0.3% Triton-X (#2), then centrifuged at 400g for 1 min to remove tissue fragments. The supernatant was sample #3. The tissue pellet was resuspended in PBS (sample #4). The rest of the supernatant was centrifuged 21000g for 5 minutes, and the pellet was resuspended in PBS (sample #5). Serial dilutions of the samples #2–#5 were spotted on a LB plate supplemented with diaminopimelic acid and kanamycin. As shown in FIG.16C, each of sample # 2 to sample # 5 could grow on LB plates supplemented with diaminopimelic acid and kanamycin. Each of Attorney Docket No. MMS.003WO sample # 1 to sample # 5 and the input bacteria were serially diluted and plated on LB plates supplemented with diaminopimelic acid. The number of colonies were counted. Table 8 below shows the number of bacteria in each of the samples: Table 8: Number of Bacteria Detected
Figure imgf000099_0001
These results demonstrate, inter alia, that the engineered bacteria are selectively endocytosed by diseased cells. Example 5: Self-Amplifying RNA for Amplification of the Gene Encoding a Prodrug Converting Enzyme in the Diseased Cells A schematic representation of amplification of a payload in target cells using self-amplifying RNA is shown in FIG. 17. A schematic representation of the improvement of the signal from RNA transfer of payload using self-amplifying RNA is illustrated in FIG.18. The cell on the left side in FIG.18 shows RNA transfer without amplification. The cell on the right side in FIG.18 shows the amplification of RNA in target cells using self-amplifying RNA. This strategy will first be tested using an iRFP670 reporter. A construct for amplification of mRNA encoding iRFP670 or GFP will be made. The construct will include, under the control of a prokaryotic promoter, an IRES, the functional components of an RNA- dependent RNA polymerase (RdRP) or viral genome replication apparatus, which includes nsp1, nsp2, nsp3 and nsp4, an iRFP670 or GFP sequence (indicated in FIG.17 as a “payload sequence”), and a poly(A) tail. As shown, upon translation of the nsp1-4 genes present on the (+) strand of the construct, an RdRP will be produced. The RdRP is expected to produce a (-) strand using the (+) strand as a template (FIG. 17). The RdRP is then expected to produce another copy (+) strand using the (-) strand as a template (FIG.17). It is expected that the RdRP will catalyze multiple rounds of production of the (-) strand and (+) strand. Transcription of the (-) strand will produce an mRNA encoding the iRFP670 or GFP, which is translated to the Attorney Docket No. MMS.003WO iRFP670 or GFP. It is expected that the construct for amplification of mRNA disclosed herein will show higher levels of expression of iRFP670 or GFP. Therefore, these data will suggest that a construct for amplification of mRNA, which includes RdRP, will enhance the detection of diseased cells. A construct for amplification of mRNA encoding yCD:UPRT or other potential payloads to aid in treatment or detection of diseased lesions will be made. The construct will include, under the control of a prokaryotic promoter, the functional components of an RNA- dependent RNA polymerase (RdRP) or viral genome replication apparatus, which includes nsp1, nsp2, nsp3 and nsp4, a yCD:UPRT sequence (indicated in FIG. 17 as a “payload sequence”), and a poly(A) tail. As shown, upon translation of the nsp1-4 genes present on the (+) strand of the construct, an RdRP will be produced. The RdRP is expected to produce a (-) strand using the (+) strand as a template (FIG. 17). The RdRP is then expected to produce another copy (+) strand using the (-) strand as a template (FIG.17). It is expected that the RdRP will catalyze multiple rounds of production of the (-) strand and (+) strand. Transcription of the (-) strand will produce an mRNA encoding the yCD:UPRT, which is translated to the yCD:UPRT. It is expected that the construct for amplification of mRNA disclosed herein will show higher levels of expression of yCD:UPRT. Therefore, these data will suggest that a construct for amplification of mRNA, which includes RdRP, will enhance the targeted expression of a prodrug converting enzyme disclosed herein in diseased cells. Example 6: β1 Integrin Mislocalization to the Apical Membrane Early in Adenoma Formation This example describes studies to investigate the identification of mislocalized β1 integrin receptors in dysplastic colon cells early in the process of adenoma formation. The incidence of colorectal cancer (CRC) is rising, particularly in young adults. Early detection of CRC can be a key factor in improving patient outcomes. The current standard-of-care for CRC prevention, colonoscopy, is invasive and requires significant pre- procedure preparation, a physician visit, and an endoscopy team, leading to low compliance rates. Moreover, the procedure has a significant miss rate for small, precancerous lesions. Recent developments in non-invasive testing have given rise to several fecal-based diagnostic assays, however, these screening tests cannot sensitively detect small precancerous lesions. The non-invasive detection of these lesions is a major unmet medical need. Attorney Docket No. MMS.003WO Without wishing to be bound by theory, it is hypothesized that CRC cells are depolarized such that receptors normally found only on the basolateral membranes of the cell are mislocalized to the apical membrane. These mislocalized receptors are attractive targets for differentiating normal and dysplastic cells in the gastrointestinal tract as they can freely interact with bacteria resident in the intestinal lumen. However, it is unknown how early cell surface receptors become mislocalized in the process in the formation of dysplastic lesions. Accordingly, studies to investigate receptor mislocalization were performed using a mouse model of adenoma formation. ApcL/L ;Villin-CreER mice were used as a mouse model of adenoma formation, as described in Example 3 above. Briefly, 100 μM 4-hydroxytamoxifen was freshly prepared in 1x PBS and 2x 50 μL injections were placed into the distal colon of mice. Colon tissue was harvested at various time points after the injection of 4-hydroxytamoxifen. Harvested tissue was fixed in 10% formalin, paraffin embedded, and sectioned into 4–5 μm sections. Antigen retrieval was performed using sodium citrate buffer and a pressurized Decloaking Chamber (Biocare Medical, NxGen). The following primary antibodies and dilutions were used for immunofluorescence: mouse monoclonal anti β-catenin (1:100, BD Biosciences, 610164) and rabbit monoclonal anti β-1 integrin (1:100, CST, 34971). Alexa Fluor secondary antibodies, anti-rabbit 488 and anti-mouse 568, were used for visualization. Dapi (Biolegend 422801) was used for nuclear staining. The tissue was mounted using Invitrogen Prolong Gold (P36930) mounting medium. Images were acquired using either a 20x (Nikon Plan Apo), 40x objective (Nikon Plan Apo), or 60x oil immersion objective (Nikon Plan Apc VC) using a Nikon 90i upright microscope equipped with a Hamamatsu Orca-ER CCD camera and an APC line 1200 light source. Utilizing a mouse model of adenoma formation that allows for specific temporal and special staging of adenomas in the mouse colon, dysplasia was induced and stained for the localization of the cell surface receptor β1 integrin, in addition to β-catenin, which becomes mislocalized when Apc is knocked out. It was found that by 5 days after the induction of dysplasia, the mislocalization of β1 integrin to the apical membrane of the epithelial cells was clearly observed; these cells also contained mislocalized β-catenin (FIG. 9D). This phenomenon persisted as the formation of the adenoma became more advanced (FIGS. 9C– 9F). These results indicate that β1 integrin mislocalization occurs early in the dysplasia development process. Attorney Docket No. MMS.003WO Example 7: Identification of Dysplastic Tissue with Genetically Engineered Microorganisms This example describes studies to investigate the binding of β1 integrin by invasin expressed on E. coli Nissle 1917 (EcN) with high specificity for dysplastic tissue. The status of EcN, a probiotic bacterial strain, as a natural resident of the human gut, as well as the relatively advanced genetic tools for working with strains of E. coli, make EcN an attractive candidate for genetic engineering to identify mislocalized receptors associated with pre- cancerous lesions such as adenomas. The bacterial protein invasin from Yersina pseudotuberculosis is a binding partner of β1 integrin. EcN was isolated from a capsule of Mutaflor® and confirmed by established PCR methods and by whole genome sequencing. The cryptic plasmids of Mutaflor, pMUT1 and pMUT2, were cured from the strain by counterselection. Briefly, each plasmid was isolated and engineered to contain an ampicillin resistance marker and the sucrose counter selection marker sacB. Engineered plasmids were transformed into the wild-type EcN strain and selected by ampicillin. Strains were passaged in ampicillin for multiple days followed by selection on LB-agar plates containing 10% sucrose. Isolated colonies were selected and loss of the plasmid was confirmed by miniprep and PCR. In order to develop the present β1 integrin targeting strain, the cryptic plasmids pMUT1 and pMUT2 were removed from EcN and the alanine racemase genes alr and dadX were knocked out to render the strain auxotrophic for the essential cell wall component, D- alanine. Gene knockouts of alr and dadX were carried out using established lambda red recombineering methods. All strains were cured of antibiotic markers to generate fully antibiotic sensitive strains. Strains lacking episomes were cultured in lysogeny broth (LB) containing 1 mM D-alanine, whereas strains containing episomes expressing the alr gene were cultured in LB lacking D-alanine. The inv gene encoding for invasin was then integrated at the lambda phage attachment site. In addition, an episomal plasmid containing the alr gene and encoding the fluorescent protein mNeonGreen was transformed into the strain to generate a fluorescent EcN strain lacking any antibiotic resistance markers. As the invasin protein is not optimally active in E. coli grown at 37 °C, strains were grown at 30 °C unless otherwise indicated. A control strain of EcN expressing a truncated invasin incapable of binding β1 integrin (invasintrunc) and the flurorescent protein mScarlet was also prepared. HCT116 cells (ATCC® CCL-247) or HCT116 cells with the gene ITGB1 encoding β1 integrin knocked out (Abcam ab273724) were used for in vitro invasion assays. Attorney Docket No. MMS.003WO Cells were cultured in DMEM media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were seeded on 96-well tissue culture treated plates at densities ranging from 50–90% confluence depending on the experiment. For the in vitro invasion assays, bacterial cultures grown overnight at 30 °C were diluted 1:33 into LB and grown 3 hours to mid log phase. Bacteria were then washed and resuspended to target densities in DMEM containing 10% FBS with no antibiotics. HCT116 cells at approximately 80% confluence were washed once with PBS, followed by addition of 100 µL of media containing bacteria at the starting multiplicity of infection (MOI) noted. Plates were incubated for two hours at 37 °C with 5% CO2 and then washed in prewarmed PBS to remove unbound bacteria, followed by the addition of DMEM with 10% FBS and 40 µg/mL gentamycin to kill extracellular bacteria. Following 1 hour of incubation, cells were detached from plates using TrypLE (Thermo-Fischer) and then analyzed by flow cytometry. For in vivo invasion assays, mice received orthotopic injections for tumor induction as described in Example 6 above 5 or 7 days prior to bacterial enema. Bacterial cultures (invasin (mNeongreen); invasintrunc (mScarlet)) were grown in LB overnight. Bacteria were diluted 1:33 and grown to mind-log phase for 2 hours with shaking at 30 °C. The OD600 was measured, adjusted to 0.5 in SMEM (Thermo Fisher) and combined. Mice were anesthetized with isoflurane, colon flushed to remove any fecal material, and given the bacterial enema. Anuses were then sealed using Vetbond tissue adhesive (3M) for 3.5 hours. Mice were then sacrificed and the colons were flushed 4x 20 mL with 1x PBS. The colons were lateralized and placed luminal side down on microscope slides. Images were acquired using a 20x objective (Nikon) on a Nikon Eclipse Ti-S/L inverted microscope affixed with an Andor Zyla scMOS camera and a Lumencor Light Engine light source. To confirm that the engineered EcN strain could successfully and specifically bind β1 integrin, bacteria expressing mNeonGreen and either invasin or a truncated invasin incapable of binding β1 integrin (invasintrunc) were seeded on either wild-type HCT116 cells, or HCT116 cells with the β1 integrin knocked out at a range of multiplicities of infection. Cells were then washed to remove unbound bacteria, followed by detachment of the cells and analysis by flow cytometry. The results clearly demonstrated binding to cells in an invasin-β1 integrin dependent manner (FIG.19). Attorney Docket No. MMS.003WO Example 8: Endocytosis of Genetically Engineered Microorganisms into Cells Displaying β1 Integrin This example describes studies to investigate endocytosis of EcN expressing invasin by dysplastic cells via in vitro and in vivo gentamycin protection assays. For in vitro assays, two 96-well tissue culture treated plates containing DMEM with 10% FBS and 20 µg/mL of gentamycin (DMEMc) were seeded with HCT116 cells and allowed to grow to confluence in a static 37°C water jacket incubator at 5% CO2. Bacterial cultures were inoculated in LB and allowed to grow with shaking at 30 °C for approximately 3 hours. The OD600 of all cultures were taken and adjusted to 0.2 in DMEM and then plated for CFU on LB-agar with appropriate additive to determine MOI. Serial dilutions on the adjusted bacterial cultures were performed at 1:2 in DMEM for a total of 6 dilutions total. The media from the tissue culture plates was aspirated and the plates washed once with pre-warmed PBS. 100 µL of pre-warmed DMEM without antibiotic was added to each well, and then 100 µL of corresponding bacterial dilution in DMEM was subsequently added to that, with the two plates being inoculated identically. The plates were statically incubated for 1 hour at 37 °C and 5% CO2. Following the incubation, the media was aspirated off and each well washed twice with pre-warmed PBS. To one of the two plates, 50 µL of TrypLE was added and allowed to incubate for 5 minutes at 37 °C and 5% CO2, after which, 180 µL of DMEMc was added and each well thoroughly resuspended by pipetting to detach adherent cells. The number of adherent HCT116 cells was measured using a control well analyzed by Countess while the remainder of the samples were analyzed using an Attune NxT Flow Cytometer using the appropriate channels. To the remaining of the two plates, 200 µL of DMEMc with gentamycin adjusted to 50 µg/µL was added and allowed to incubate for 1 hour at 37 °C and 5% CO2. Following the incubation, wells were washed once with pre-warmed PBS and 100 µL of PBS with 0.1% Triton X-100 added to selectively lyse mammalian cells. The plate was incubated for 15 minutes at room temperature with periodic agitation by shaking. Each well was thoroughly resuspended by pipetting and a sample of each well was collected and serially diluted 1:10 in LB. Each dilution was plated in triplicate on LB-agar and allowed to incubate overnight at 37 °C. The following day, the colonies were counted and CFU calculated for each sample. For in vivo assays, mice with adenomas induced in the distal colon for either one week or two weeks were given enemas containing EcN strains either expressing invasin or invasintrunc. In addition, strains harbored an episome containing a kanamycin resistance gene to Attorney Docket No. MMS.003WO differentiate the introduced EcN strains from native gut microbiota. The anus was sealed by z- suture techniques after enema injection. After 2 hours of enema injection, the mice were sacrificed followed by removal of the colon and washing with PBS. The colon was divided into proximal (non-tumor lesion) and distal sections (tumor lesion) and incubated in the presence of collagenase (2 mg/mL) and gentamycin (100 µg/mL) for 30 minutes at 37 ºC After washing out collagenase and gentamycin with PBS, tissue was dissociated with TrypLETM Express for 30 minutes at 37 ºC on the shaker, then broken down into single cells using 16 G and 18 G needles. Samples were filtrated with 100 µM filter and then stained with an antibody to the epithelial marker Epcam for 20 minutes on ice. 7-AAD dye was used to exclude dead cells prior to the sample sorting. Only the Epcam+ fraction (epithelial cells) was sorted into 1.5 mL tube by flow cytometry. The supernatant was removed after the centrifuge and the palette was incubated with Triton X-100 (0.25%) for lysing cells at 37 ºC for 15 minutes. Lysate was plated on media containing kanamycin to select for the introduced EcN strain and incubated overnight at 37 ºC prior to counting the colony forming units. Gentamycin protection assays were performed to determine if invasin expressing EcN was endocytosed after binding to β1 integrin. After allowing bacteria to invade HCT116 cells, cells were incubated for one hour with media containing gentamycin, before cells were lysed and plated to count the number of surviving bacteria. EcN expressing invasin showed a clear, MOI dependent survival rate as compared to EcN expressing invasintrunc (FIG. 20). This technique was then adapted to an in vivo system. As before, mice with adenomas induced in the distal colon for either one week or two weeks were given enemas containing EcN strains either expressing invasin or invasintrunc. In addition, strains harbored an episome containing a kanamycin resistance gene to differentiate the introduced EcN strains from native gut microbiota. After 2 hours, the mice were sacrificed and the colons were removed and washed. The colons were then divided into proximal and distal sections and dissociated in the presence of gentamycin. The cells from the dissociated tissue were then stained with an antibody to the epithelial cell marker Epcam and sorted by flow cytometry to generate a population of only epithelial cells. These sorted cells were then lysed and the lysate was plated on media containing kanamycin to select for the introduced EcN strain. The data indicated that EcN expressing invasin were consistently endocytosed into dysplastic cells as opposed to normal cells (FIG. 21). These results indicate that EcN expressing invasin are selectively endocytosed into dysplastic tissue. Attorney Docket No. MMS.003WO Example 9: In Vitro Delivery of a Prodrug Converting Enzyme Using Genetically Engineered Microorganisms This example describes the construction of an engineered EcN strain capable of identifying mislocalized receptors along the gut epithelium and delivering a translational fusion of yeast cytosine deaminase and uracil phosphoribosyltransferase (yCD:UPRT). yCD:UPRT has high activity for the conversion of the antifungal 5-FC to cytotoxic 5-FU. This fusion was added to a high copy episome expressed under the ProD constitutive prokaryotic promoter. FIG.22 shows a schematic of a strain for protein transfer. The strain has dapAΔ, alrΔ, dadXΔ auxotrophic mutations and chromosomal integration of genes encoding invasin (inv, SEQ ID NOs: 1 and 2) and listeriolysin O (hlyA; LLO, SEQ ID NO: 3) (FIG. 22 (top panel)). This strain carries a plasmid containing a payload gene (e.g., yCD:UPRT, SEQ ID Nos: 6 and 7) under a prokaryotic promoter and selection based on a wild-type alr gene (FIG. 22 (bottom panel)). The studies in this example used an engineered EcN strain as depicted in FIG.22, except that the dapAΔ auxotrophic mutation and listeriolysin O gene were omitted. To test conversion of 5-FC into 5-FU by the engineered strain, bacteria were grown overnight at 30 °C, diluted 1:30 in LB, and grown 2 hours to mid log phase. The OD600 was measured and the amount of bacteria for an effective OD600 of 4.5 was calculated. Bacteria were washed and resuspended in 155 mM 5-FC.75 µL samples were removed at t = 0, 0.5, 1, 2, 3, 4, 5, 6, 7, and 24 hours and frozen at -80 °C. Samples were thawed, pelleted, and the supernatant was removed and diluted 10x in methanol. Samples were transferred to a 96 well plate and 1 µL from each sample was run on an Agilent 1260 Infinity II Bio-inert super critical fluid (SFC) system affixed to an Agilent 6545XT Advance LC/Q-TOF mass spectrometer using an ethanol/diethylamine/carbon dioxide gradient over a silica column. Concentration was determined as area under the curve. To test in vitro cell killing by 5-FC, bacterial cultures grown overnight at 30 °C were diluted 1:50 into LB and grown 3 hours to mid log phase. Bacteria were then washed and resuspended to target MOI of ~50 in DMEM with 10% FBS and no antibiotics. HCT116 cells either containing or lacking β1 integrin at approximately 50% confluence were washed once with prewarmed PBS, followed by addition of 100 µL of media containing bacteria and allowed to incubate 2 hours at 37 °C and 5% CO2. At this point, gentamycin was added to a final concentration of 40 µg/mL and the plates were washed in prewarmed PBS to remove unbound bacteria. After washing, the cells received 200 µL of DMEM with 10% FBS and 40 µg/mL gentamycin and were incubated overnight at 37 °C and 5% CO2. After this incubation, cells Attorney Docket No. MMS.003WO were washed again with prewarmed PBS followed by addition of 200 µL of DMEM with 10% FBS and 40 µg/mL gentamycin and the concentration of 5-FC indicated. Plates were then incubated for 3 days at 37 °C and 5% CO2. For analysis, plates were stained with near-IR Live/Dead Stain and, where appropriate, anti-β1 integrin antibody conjugated to Alexa Fluor 647. Cells were then detached with TrypLE and the number of live cells with and without β1 integrin quantified by flow cytometry. To determine if the yCD:UPRT fusion was active, engineered bacteria were incubated with 5-FC and the supernatant was removed at various time points for analysis by mass spectrometry. Bacteria containing the yCD:UPRT fusion showed significantly higher conversion of 5-FC to 5-FU over a 24-hour time period (FIG. 23). A small amount of conversion from 5-FC to 5-FU in the strain lacking the yCD:UPRT fusion was likely due to the native EcN cytosine deaminase. To determine whether the engineered EcN converting 5-FC to 5-FU would lead to cell death, bacteria were seeded on HCT116 cells and allowed to bind to cells. Cells were then washed to remove unbound bacteria and then incubated overnight in media containing gentamycin to kill extracellular bacteria. After about 24 hours, cells were washed again and the indicated concentration of 5-FC was added to the media followed by three days of incubation. The number of surviving cells was then measured using a Live/Dead Stain and flow cytometry. Engineered EcN expressing invasin and the yCD:UPRT fusion showed a clear dose response to 5-FC (FIG.24). A similar experiment on β1 integrin knockout cells showed no response to 5-FC, consistent with the invasin-β1 integrin interaction being necessary for EcN mediated killing (FIG.32). yCD:UPRT conversion of 5-FC to 5-FU may lead to not only killing of cell expressing the enzyme, but also nearby cells. This bystander effect may be desirable for treatments where the enzyme may not be delivered to every diseased cell. In order to determine if the present bacterial cells exhibited this bystander effect, a mixture of 1:1 WT HCT116: β1 integrin knockout HCT116 was seeded onto plates and the 5-FC dose response experiment was performed as before. In addition to staining for living cells, the presence of β1 integrin on cells was also stained, allowing the levels of each population to be measured by flow cytometry. Results indicated killing of both WT and β1 integrin knockout HCT116 cells at similar levels, indicating a clear bystander effect (FIG.25). Attorney Docket No. MMS.003WO These results indicate that invasin-expressing EcN can successfully deliver the prodrug converting enzyme yCD:UPRT to β1 integrin-expressing mammalian cells, and that the enzyme successfully converted 5-FC to 5-FU resulting in cell killing. Example 10: In Vivo Delivery of a Prodrug Converting Enzyme Using Genetically Engineered Microorganisms This example describes studies investigating in vivo activity of an engineered EcN strain capable of identifying mislocalized receptors along the gut epithelium and delivering yCD:UPRT. When combined with administration of the antifungal 5-FC, the protein fusion produced 5-FU at the pre-cancerous lesion, leading to a reduction in adenoma burden. Mice were injected as described in Example 6 above to initiate tumor formation, and treatment began on day 3 post injection. For each enema (twice daily), bacteria were grown overnight at 30 °C and diluted 1:30 in LB and grown for 2 hours to mid-log phase. The OD600 was measured and the effective OD600 of 4.5 was calculated. Bacteria were pelleted and resuspended in 30 µL of either 155 mM 5-FC (+/- yCD:UPRT) or 1x PBS (+/- invasin). Mice were anesthetized and colons were flushed to remove feces. After 15 minutes, mice were given bacterial enemas and the anuses were sutured shut. After 1 hour, the sutures were removed. Mice received 2x 375 µL intraperitoneal (IP) injections of 250 mg/kg 5-FC. Mice in the 5-FU treatment group received IP injections of 25 mg/kg 5-FU every other day over the course of the experiment. Mice were treated for 13 days. At day 11, tumor sizes were monitored via colonoscopy. On day 14, mice were euthanized and the colons were removed, flushed, and lateralized. Tumors were imaged on a Nikon SMZ18 dissection scope at 0.75x magnification. Colon tissue was preserved in 10% formalin for histology. Harvested tissue was fixed in 10% formalin, paraffin embedded, and sectioned into 4-5 µm. Antigen retrieval was performed using sodium citrate buffer and a pressurized Decloaking Chamber (Biocare Medical, NxGen). Antibodies and dilutions for immunohistochemistry (IHC) are as follows: rabbit monoclonal anti-Cleaved Caspase-3 (Asp175)(1:400, Cell Signaling Technologies (CST), 9661) and mouse monoclonal anti β-catenin (1:100, BD Biosciences, 610164). Biotin- conjugated secondary donkey anti-mouse or anti-rabbit antibodies were used (1:500, Jackson ImmunoResearch). Vectastain Elite ABC immunoperoxidase detection kit (Vector Laboratories, PK6100) followed by Signalstain DAB substrate kit (CST, 8049) were used for visualization. All IHC antibody dilutions were carried out in Signalstain Antibody Diluent (CST, 8112). For cleaved caspase-3 analysis, positive regions were counted in the base of 45 intact crypts in the proximal colon. The tissue was mounted using Invitrogen Prolong Gold Attorney Docket No. MMS.003WO (P36930) mounting medium. Images were acquired using either a 20x (Nikon Plan Apo), 40x objective (Nikon Plan Apo), or 60x oil immersion objective (Nikon Plan Apc VC) using a Nikon 90i upright microscope equipped with a Hamamatsu Orca-ER CCD camera and an APC line 1200 light source. In order to test the effectiveness of the engineered bacteria as a directed chemotherapy, dysplasia was induced in mice and the adenoma was allowed to develop for three days. Mice were given twice daily enemas of engineered bacteria resuspended in 5-FC, as well as twice daily IP injections of 250 mg/kg 5-FC (FIG.26). Control groups either received no treatment or IP injections of 25 mg/kg 5-FU every two days. On day 14 post injection, mice were sacrificed and the surface area of dysplastic tissue was measured. Mice receiving both the engineered EcN and 5-FC showed similar reduced lesion size both visually (FIG. 27) and quantitatively (FIG.28) as compared to 5-FU injection. Mice administered bacteria lacking the yCD:UPRT enzyme and mice administered 5-FC with no bacteria showed similar levels of dysplastic tissue to the untreated control. To test the advantage of targeting dysplastic tissue using the β1 integrin-invasin interaction, a similar experiment was performed with engineered bacteria expressing the yCD:UPRT enzyme and either invasin or invasintrunc. To minimize the risk of substrate conversion, mice were given bacterial enemas prepared in PBS in addition to twice daily IP injections of 250 mg/kg 5-FC. Mice treated with invasin expressing bacteria showed lower levels of dysplastic tissue when compared to mice treated with invasintrunc expressing bacteria, suggesting that specific targeting to dysplastic tissue by the β1 integrin-invasin interaction had a therapeutic advantage (FIG.29). In order to determine if the directed chemotherapy provided by the engineered EcN in conjunction with 5-FC would lead to less damage to normal tissue than 5-FU treatment, normal tissue was harvested from the mouse proximal colon and stained for the cleavage of caspase 3 as a marker for apoptosis. Visual inspection showed a clear difference in staining between 5-FU and untreated/bacterially treated mice (FIG. 30). Of note, the 5-FU treated cohort showed more apoptosis at the base of the colonic crypts, suggesting that the 5-FU was leading to the death of colonic stem cells. Quantification of these results indicated that 5-FU treatment was more damaging to normal gut tissue than engineered EcN in conjunction with 5-FC (FIG.31). Attorney Docket No. MMS.003WO In this example, a directed chemotherapy was developed to reduce levels of dysplastic tissue in a mouse model of CRC with less damage to healthy tissue as compared to a broad-spectrum chemotherapy. The system uses targeting to dysplastic tissue based on bacterial access to β1 integrin, in accordance with results in the previous examples showing that β1 integrin is mislocalized to the apical membrane of intestinal epithelial cells early in the dysplasia development process. Further, the results of the present example show that invasin- expressing bacteria bind and are internalized into cells in vivo, and that this effect can be used to convert 5-FC to 5-FU, leading to localized cell killing. The in vivo results suggest this approach may be a viable therapy to slow the development of adenomas in genetically predisposed populations, such as those suffered from familial adenomatous polyposis or Lynch syndrome. Significantly, the results seem to indicate that there is less damage done to the normal tissue of the gut with this directed chemotherapy intervention, as compared to the administration of the broad-spectrum chemotherapeutic 5-FU, currently the gold standard for chemotherapeutic treatment of CRC. These results demonstrate proof of concept for a potential treatment for early adenomas using engineered bacteria and prodrug combination therapy. Directions for future investigation include developing oral formulations of 5-FC and EcN contained in an enteric capsule for long term treatment, determining the optimal dosing and timing of doses in order to achieve the maximum therapeutic effect with minimal off target effects, and investigating the tolerance of long-term dosing with the bacteria and prodrug combination. Example 11: Delivery of a Direct Cytotoxic Agent Using Genetically Engineered Microorganisms This example describes the delivery of a direct cytotoxic agent by engineered bacteria. An engineered EcN strain is designed to deliver a mammalian cytotoxic protein as the payload, such as constitutively active caspase-3, which causes cell death. The strain can be generally similar to the strain design shown in FIG.22, except that the payload gene is a gene for the mammalian cytotoxic protein. Mice are injected as described in Example 6 above to initiate tumor formation, and treatment begins on day 3 post injection. For each enema (twice daily), bacteria are grown overnight at 30 °C and diluted 1:30 in LB and grown for 2 hours to mid-log phase. The OD600 is measured and the effective OD600 of 4.5 is calculated. Bacteria are pelleted and resuspended in 30 µL of 1x PBS. Mice are anesthetized and colons are flushed to remove feces. After 15 Attorney Docket No. MMS.003WO minutes, mice are given bacterial enemas and the anuses are sutured shut. After 1 hour, the sutures were removed. Mice are treated for 13 days. At day 11, tumor sizes are monitored via colonoscopy. On day 14, mice are euthanized and the colons were removed, flushed, and lateralized. Tumors were imaged on a Nikon SMZ18 dissection scope at 0.75x magnification. Mice treated with bacteria producing the cytotoxic protein are expected to have smaller tumors than mice treated with control bacteria that do not produce the cytotoxic protein and mice treated with control bacteria that express the cytotoxic protein and a truncated invasin. Additional Examples Additional examples of aspects of the present technology are described below as numbered clauses (1, 2, 3, etc.) for convenience. These are provided as examples and do not limit the present technology. 1. A method for treating a subject, the method comprising: administering a genetically engineered microorganism to a gastrointestinal (GI) tract of the subject, wherein the genetically engineered microorganism comprises: a targeting gene encoding a targeting element that binds to a surface marker on a target cell in the GI tract, and a payload gene encoding a therapeutic agent. 2. The method of Clause 1, wherein the therapeutic agent is a prodrug converting enzyme that converts a prodrug into an active drug, and wherein the method further comprises administering the prodrug to the subject. 3. The method of Clause 2, wherein the prodrug comprises a precursor of 5- fluorouracil (5-FU) and the active drug comprises 5-FU. 4. The method of Clause 3, wherein the precursor of 5-FU comprises 5- fluorocytosine (5-FC). 5. The method of Clause 4, wherein the prodrug converting enzyme comprises a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof. Attorney Docket No. MMS.003WO 6. The method of Clause 4 or 5, wherein the prodrug converting enzyme comprises a fusion protein of a cytosine deaminase and an uracil phosphoribosyltransferase (CD:UPRT). 7. The method of Clause 6, wherein the CD:UPRT comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 6. 8. The method of Clause 6 or 7, wherein the CD:UPRT comprises a sequence of SEQ ID NO: 6. 9. The method of any one of Clauses 6–8, wherein the payload gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO: 8. 10. The method of any one of Clauses 6–9, wherein the payload gene comprises a sequence of any one of SEQ ID NO: 7 or SEQ ID NO: 8. 11. The method of Clause 3, wherein the precursor of 5-FU comprises pentyl N- [1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4- yl]carbamate (capecitabine). 12. The method of Clause 11, wherein the prodrug converting enzyme comprises a carboxylesterase. 13. The method of Clause 3, wherein the precursor of 5-FU comprises 5-FU glucuronide. 14. The method of Clause 13, wherein the prodrug converting enzyme comprises a β-glucuronidase. 15. The method of Clause 2, wherein the prodrug converting enzyme comprises a purine nucleoside phosphorylase. Attorney Docket No. MMS.003WO 16. The method of Clause 15, wherein the prodrug comprises 6-methylpurine- deoxyriboside and the active drug comprises 6-methylpurine. 17. The method of Clause 15, wherein the prodrug comprises 9-beta-D- arabinofuranosyl-2-fluoroadenine monophosphate and the active drug comprises fluoroadenine. 18. The method of Clause 2, wherein the prodrug comprises 1-(2-deoxy-2-fluoro- b-D-arabinofuranosyl)uracil, and the active drug comprises 1-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl) 5-methyluracil monophosphate. 19. The method of Clause 18, wherein the prodrug converting enzyme comprises a thymidine kinase, a thymidine synthase, or a combination thereof. 20. The method of Clause 2, wherein the prodrug comprises ganciclovir, and the prodrug converting enzyme comprises a viral thymidine kinase. 21. The method of any one of Clauses 2–20, wherein the prodrug is administered via an oral route, a rectal route, a sublingual route, an intravenous route, an intramuscular route, a subcutaneous route, a transdermal route, an intranasal route, or a vaginal route. 22. The method of any one of Clauses 2–21, wherein the prodrug is administered concurrently with the genetically engineered microorganism. 23. The method of any one of Clauses 2–21, wherein the prodrug is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the genetically engineered microorganism is administered to the subject. 24. The method of Clause 1, wherein the therapeutic agent is a direct cytotoxic agent. Attorney Docket No. MMS.003WO 25. The method of Clause 24, wherein the direct cytotoxic agent comprises a microbial toxin or a caspase. 26. The method of Clause 24 or 25, wherein the direct cytotoxic agent comprises a cytotoxic protein or peptide. 27. The method of any one of Clauses 24–26, wherein the direct cytotoxic agent exhibits enhanced activity in the target cell compared to a nontarget cell. 28. The method of any one of Clauses 1–27, wherein the administration of the genetically engineered microorganism causes selective expression of the therapeutic agent in the target cell. 29. The method of Clause 28, wherein the selective expression of the therapeutic agent in the target cell results in killing of the target cell. 30. The method of any one of Clauses 1–29, wherein the target cell is a cell of a precancerous lesion. 31. The method of Clause 30, wherein the precancerous lesion is an adenoma. 32. The method of Clause 30 or 31, wherein the precancerous lesion has a size less than or equal to 0.1 mm, 0.25 mm, 0.5 mm, 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, 15 mm, 20 mm, 25 mm, or 30 mm. 33. The method of any one of Clauses 1–32, wherein the target cell is a diseased epithelial cell indicative of a cancer. 34. The method of Clause 33, wherein the cancer is one or more of the following: colorectal cancer, Lynch syndrome cancer, familial adenomatous polyposis, hereditary non- polyposis colon cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, or a sporadic cancer. Attorney Docket No. MMS.003WO 35. The method of any one of Clauses 1–34, wherein the payload gene comprises at least one intron. 36. The method of Clause 35, wherein the at least one intron comprises a spliceosomal intron. 37. The method of any one of Clauses 1–36, wherein the payload gene is operably linked to a promoter. 38. The method of Clause 37, wherein the promoter is a mammalian promoter. 39. The method of Clause 37, wherein the promoter is a microbial promoter. 40. The method of any one of Clauses 1–39, wherein the targeting element comprises one or more of the following: an invasin, an intimin, an adhesin, a flagellin, a component of a type III secretion system, a leptin, or an antibody. 41. The method of any one of Clauses 1–40, wherein the surface marker is mislocalized on the target cell. 42. The method of any one of Clauses 1–41, wherein the surface marker is present on a luminal side of the target cell and is not present on a luminal side of a non-target cell. 43. The method of any one of Clauses 1–42, wherein the targeting element comprises an invasin and the surface marker comprises an integrin. 44. The method of Clause 43, wherein the invasin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1, or comprises a sequence of SEQ ID NO: 1. 45. The method of Clause 43 or 44, wherein the targeting gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or a coding region thereof, or Attorney Docket No. MMS.003WO comprises a sequence of SEQ ID NO: 2 or the coding region thereof. 46. The method of any one of Clauses 1–45, wherein the targeting element promotes internalization of the genetically engineered microorganism by the target cell. 47. The method of any one of Clauses 1–45, wherein the genetically engineered microorganism remains external to the target cell. 48. The method of any one of Clauses 1–46, wherein the genetically engineered microorganism further comprises a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. 49. The method of Clause 48, wherein the lysis element comprises a lysin. 50. The method of Clause 49, wherein the lysin comprises listeriolysin O. 51. The method of Clause 50, wherein the lysin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4, or comprises a sequence of any one of SEQ ID NO: 3 or SEQ ID NO.4. 52. The method of any one of Clauses 1–51, wherein the genetically engineered microorganism comprises an auxotrophic mutation. 53. The method of Clause 52, wherein the auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell. 54. The method of Clause 52 or 53, wherein the auxotrophic mutation comprises a mutation in one or more of the following: dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF. Attorney Docket No. MMS.003WO 55. The method of any one of Clauses 52–54, wherein the auxotrophic mutation comprises a deletion of an essential gene, and the payload gene is located on a plasmid comprising a functional copy of the essential gene. 56. The method of Clause 55, wherein the plasmid comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or a coding region thereof, or the plasmid comprises a sequence of SEQ ID NO: 5 or the coding region thereof. 57. The method of any one of Clauses 1–56, wherein the genetically engineered microorganism is configured to deliver a DNA molecule comprising the payload gene to the target cell. 58. The method of Clause 57, wherein the DNA molecule comprises a plasmid. 59. The method of Clause 57 or 58, wherein the DNA molecule comprises at least one binding site for a DNA binding protein that comprises one or more nuclear localization signals. 60. The method of Clause 59, wherein the genetically engineered microorganism comprises a gene encoding the DNA binding protein. 61. The method of any one of Clauses 1–56, wherein the genetically engineered microorganism is configured to deliver an RNA molecule comprising the payload gene to the target cell. 62. The method of Clause 61, wherein the RNA molecule comprises an mRNA molecule. 63. The method of Clause 61 or 62, wherein the RNA molecule comprises a self- replicating or self-amplifying RNA molecule. Attorney Docket No. MMS.003WO 64. The method of Clause 63, wherein the genetically engineered microorganism comprises a cassette that directs RNA self-amplification of the cassette in the target cell, and the payload gene is present in the cassette. 65. The method of Clause 64, wherein the cassette comprises an RNA viral genome replication apparatus. 66. The method of Clause 65, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). 67. The method of Clause 66, wherein the RdRP comprises an alphavirus RdRP. 68. The method of Clause 67, wherein the alphavirus RdRP comprises one or more of nsp1, nsp2, nsp3, or nsp4. 69. The method of any one of Clauses 61–68, wherein the genetically engineered microorganism comprises a deletion of an RNase gene. 70. The method of Clause 69, wherein the RNase gene comprises RNase III. 71. The method of any one of Clauses 1–56, wherein the genetically engineered microorganism is configured to deliver a protein comprising the therapeutic agent to the target cell. 72. The method of Clause 71, wherein the payload gene is operably linked to a microbial promoter. 73. The method of Clause 72, wherein the microbial promoter is a constitutive promoter. 74. The method of any one of Clauses 71–73, wherein the genetically engineered microorganism does not include a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. Attorney Docket No. MMS.003WO 75. The method of any one of Clauses 71–74, wherein the genetically engineered microorganism does not include an auxotrophic mutation that facilitates lysis of the genetically engineered microorganism within the target cell. 76. The method of any one of Clauses 1–75, wherein the genetically engineered microorganism further comprises a second payload gene encoding a detection marker. 77. The method of any one of Clauses 1–76, further comprising administering a second genetically engineered microorganism to the subject, wherein the second genetically engineered microorganism comprises a second payload gene encoding a detection marker. 78. The method of Clause 77, wherein the second genetically engineered microorganism is administered before, concurrently with, or after administration of the genetically engineered microorganism to the subject. 79. The method of any one of Clauses 76–78, wherein the detection marker comprises a protein, a peptide, or a nucleic acid molecule. 80. The method of any one of Clauses 76–79, wherein the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter. 81. The method of any one of Clauses 76–80, further comprising: collecting a biological sample from the subject; and detecting whether the detection marker is present in the biological sample. 82. The method of Clause 81, wherein the biological sample comprises one or more of feces, blood, serum, plasma, mucus, urine, saliva, or a biopsy sample. 83. The method of any one of Clauses 76–82, further comprising performing an endoscopic or colonoscopic procedure to detect the detection marker in the subject. Attorney Docket No. MMS.003WO 84. The method of any one of Clauses 81–83, further comprising: upon detection of the detection marker, administering a third genetically engineered microorganism to the GI tract of the subject, wherein the third genetically engineered microorganism comprises the targeting gene and the payload gene. 85. The method of any one of Clauses 1–84, wherein the subject is low risk for colorectal cancer (CRC) or colorectal polyps. 86. The method of Clause 85, wherein the subject has no previous incidence of CRC or colorectal polyps. 87. The method of any one of Clauses 1–84, wherein the subject is high or medium risk for CRC or colorectal polyps. 88. The method of any one of Clauses 1–84, wherein the subject has prior incidence of CRC, prior incidence of colorectal polyps, a family history of CRC, or a combination thereof. 89. The method of any one of Clauses 1–84, wherein the subject has a genetic predisposition to CRC. 90. The method any one of Clauses 1–84, wherein the subject has familial adenomatous polyposis (FAP), attenuated FAP, Lynch syndrome, oligopolyposis, Peutz- Jeghers syndrome (PJS), MUTYH-associated polyposis (MAP), juvenile polyposis syndrome, or Cowden syndrome. 91. The method of any one of Clauses 1–90, wherein the subject is at least 45 years of age, at least 50 years of age, at least 55 years of age, or at least 60 years of age. 92. The method of any one of Clauses 1–91, wherein the target cell is indicative of a disease selected from a precancerous lesion, a GI tract cancer, an inflammatory bowel disease, irritable bowel syndrome, or Barrett’s esophagus. Attorney Docket No. MMS.003WO 93. The method of any one of Clauses 1–92, wherein the genetically engineered microorganism is Escherichia coli Nissle 1917 or a derivative thereof. 94. The method of any one of Clauses 1–93, wherein the genetically engineered microorganism is administered via an oral route or a rectal route. 95. The method of any one of Clauses 1–94, wherein about 103 to about 1011 genetically engineered microorganisms are administered to the subject. 96. A genetically engineered microorganism comprising: a targeting gene encoding a targeting element that binds to a surface marker on a target cell in a gastrointestinal (GI) tract; and a payload gene encoding a therapeutic agent. 97. The genetically engineered microorganism of Clause 96, wherein the therapeutic agent is a prodrug converting enzyme that converts a prodrug into an active drug. 98. The genetically engineered microorganism of Clause 97, wherein the prodrug comprises a precursor of 5-fluorouracil (5-FU) and the active drug comprises 5-FU. 99. The genetically engineered microorganism of Clause 98, wherein the precursor of 5-FU comprises 5-fluorocytosine (5-FC). 100. The genetically engineered microorganism of Clause 99, wherein the prodrug converting enzyme comprises a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof. 101. The genetically engineered microorganism of Clause 99 or 100, wherein the prodrug converting enzyme comprises a fusion protein of a cytosine deaminase and an uracil phosphoribosyltransferase (CD:UPRT). Attorney Docket No. MMS.003WO 102. The genetically engineered microorganism of Clause 101, wherein the CD:UPRT comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 6. 103. The genetically engineered microorganism of Clause 101 or 102, wherein the CD:UPRT comprises a sequence of SEQ ID NO: 6. 104. The genetically engineered microorganism of any one of Clauses 101–103, wherein the payload gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO: 8. 105. The genetically engineered microorganism of any one of Clauses 101–104, wherein the payload gene comprises a sequence of any one of SEQ ID NO: 7 or SEQ ID NO: 8. 106. The genetically engineered microorganism of Clause 98, wherein the precursor of 5-FU comprises pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2- yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine). 107. The genetically engineered microorganism of Clause 106, wherein the prodrug converting enzyme comprises a carboxylesterase. 108. The genetically engineered microorganism of Clause 98, wherein the precursor of 5-FU comprises 5-FU glucuronide. 109. The genetically engineered microorganism of Clause 108, wherein the prodrug converting enzyme comprises a β-glucuronidase. 110. The genetically engineered microorganism of Clause 97, wherein the prodrug converting enzyme comprises a purine nucleoside phosphorylase. Attorney Docket No. MMS.003WO 111. The genetically engineered microorganism of Clause 110, wherein the prodrug comprises 6-methylpurine-deoxyriboside and the active drug comprises 6-methylpurine. 112. The genetically engineered microorganism of Clause 110, wherein the prodrug comprises 9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate and the active drug comprises fluoroadenine. 113. The genetically engineered microorganism of Clause 97, wherein the prodrug comprises 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, and the active drug comprises 1- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5-methyluracil monophosphate. 114. The genetically engineered microorganism of Clause 113, wherein the prodrug converting enzyme comprises a thymidine kinase, a thymidine synthase, or a combination thereof. 115. The genetically engineered microorganism of Clause 97, wherein the prodrug comprises ganciclovir, and the prodrug converting enzyme comprises a viral thymidine kinase. 116. The genetically engineered microorganism of Clause 96, wherein the therapeutic agent is a direct cytotoxic agent. 117. The genetically engineered microorganism of Clause 116, wherein the direct cytotoxic agent comprises a microbial toxin or a caspase. 118. The genetically engineered microorganism of Clause 116 or 117, wherein the direct cytotoxic agent comprises a cytotoxic protein or peptide. 119. The genetically engineered microorganism of any one of Clauses 116–118, wherein the direct cytotoxic agent exhibits enhanced activity in the target cell compared to a nontarget cell. Attorney Docket No. MMS.003WO 120. The genetically engineered microorganism of any one of Clauses 96–119, wherein the target cell is a cell of a precancerous lesion. 121. The genetically engineered microorganism of Clause 120, wherein the precancerous lesion is an adenoma. 122. The genetically engineered microorganism of Clause 120 or 121, wherein the precancerous lesion has a size less than or equal to 0.1 mm, 0.25 mm, 0.5 mm, 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, 15 mm, 20 mm, 25 mm, or 30 mm. 123. The genetically engineered microorganism of any one of Clauses 96–122, wherein the target cell is a diseased epithelial cell indicative of a cancer. 124. The genetically engineered microorganism of Clause 123, wherein the cancer is one or more of the following: colorectal cancer, Lynch syndrome cancer, familial adenomatous polyposis, hereditary non-polyposis colon cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, or a sporadic cancer. 125. The genetically engineered microorganism of any one of Clauses 96–124, wherein the payload gene comprises at least one intron. 126. The genetically engineered microorganism of Clause 125, wherein the at least one intron comprises a spliceosomal intron. 127. The genetically engineered microorganism of any one of Clauses 96–126, wherein the payload gene is operably linked to a promoter. 128. The genetically engineered microorganism of Clause 127, wherein the promoter is a mammalian promoter. 129. The genetically engineered microorganism of Clause 127, wherein the promoter is a microbial promoter. Attorney Docket No. MMS.003WO 130. The genetically engineered microorganism of any one of Clauses 96–129, wherein the targeting element comprises one or more of the following: an invasin, an intimin, an adhesin, a flagellin, a component of a type III secretion system, a leptin, or an antibody. 131. The genetically engineered microorganism of any one of Clauses 96–130, wherein the surface marker is mislocalized on the target cell. 132. The genetically engineered microorganism of any one of Clauses 96–131, wherein the surface marker is present on a luminal side of the target cell and is not present on a luminal side of a non-target cell. 133. The genetically engineered microorganism of any one of Clauses 96–132, wherein the targeting element comprises an invasin and the surface marker comprises an integrin. 134. The genetically engineered microorganism of Clause 133, wherein the invasin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1, or comprises a sequence of SEQ ID NO: 1. 135. The genetically engineered microorganism of Clause 133 or 134, wherein the targeting gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or a coding region thereof, or comprises a sequence of SEQ ID NO: 2 or the coding region thereof. 136. The genetically engineered microorganism of any one of Clauses 96–135, wherein the targeting element promotes internalization of the genetically engineered microorganism by the target cell. 137. The genetically engineered microorganism of any one of Clauses 96–135, wherein the genetically engineered microorganism remains external to the target cell. Attorney Docket No. MMS.003WO 138. The genetically engineered microorganism of any one of Clauses 96–136, wherein the genetically engineered microorganism further comprises a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. 139. The genetically engineered microorganism of Clause 138, wherein the lysis element comprises a lysin. 140. The genetically engineered microorganism of Clause 139, wherein the lysin comprises listeriolysin O. 141. The genetically engineered microorganism of Clause 140, wherein the lysin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4, or comprises a sequence of any one of SEQ ID NO: 3 or SEQ ID NO.4. 142. The genetically engineered microorganism of any one of Clauses 96–141, wherein the genetically engineered microorganism comprises an auxotrophic mutation. 143. The genetically engineered microorganism of Clause 142, wherein the auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell. 144. The genetically engineered microorganism of Clause 142 or 143, wherein the auxotrophic mutation comprises a mutation in one or more of the following: dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF. 145. The genetically engineered microorganism of any one of Clauses 142–144, wherein the auxotrophic mutation comprises a deletion of an essential gene, and the payload gene is located on a plasmid comprising a functional copy of the essential gene. 146. The genetically engineered microorganism of Clause 145, wherein the plasmid comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, Attorney Docket No. MMS.003WO 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or a coding region thereof, or the plasmid comprises a sequence of SEQ ID NO: 5 or the coding region thereof. 147. The genetically engineered microorganism of any one of Clauses 96–146, wherein the genetically engineered microorganism is configured to deliver a DNA molecule comprising the payload gene to the target cell. 148. The genetically engineered microorganism of Clause 147, wherein the DNA molecule comprises a plasmid. 149. The genetically engineered microorganism of Clause 147 or 148, wherein the DNA molecule comprises at least one binding site for a DNA binding protein that comprises one or more nuclear localization signals. 150. The genetically engineered microorganism of Clause 149, wherein the genetically engineered microorganism comprises a gene encoding the DNA binding protein. 151. The genetically engineered microorganism of any one of Clauses 96–146, wherein the genetically engineered microorganism is configured to deliver an RNA molecule comprising the payload gene to the target cell. 152. The genetically engineered microorganism of Clause 151, wherein the RNA molecule comprises an mRNA molecule. 153. The genetically engineered microorganism of Clause 151 or 152, wherein the RNA molecule comprises a self-replicating or self-amplifying RNA molecule. 154. The genetically engineered microorganism of Clause 153, wherein the genetically engineered microorganism comprises a cassette that directs RNA self- amplification of the cassette in the target cell, and the payload gene is present in the cassette. 155. The genetically engineered microorganism of Clause 154, wherein the cassette comprises an RNA viral genome replication apparatus. Attorney Docket No. MMS.003WO 156. The genetically engineered microorganism of Clause 155, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). 157. The genetically engineered microorganism of Clause 156, wherein the RdRP comprises an alphavirus RdRP. 158. The genetically engineered microorganism of Clause 157, wherein the alphavirus RdRP comprises one or more of nsp1, nsp2, nsp3, or nsp4. 159. The genetically engineered microorganism of any one of Clauses 151–158, wherein the genetically engineered microorganism comprises a deletion of an RNase gene. 160. The genetically engineered microorganism of Clause 159, wherein the RNase gene comprises RNase III. 161. The genetically engineered microorganism of any one of Clauses 96–160, wherein the genetically engineered microorganism is configured to deliver a protein comprising the therapeutic agent to the target cell. 162. The genetically engineered microorganism of Clause 161, wherein the payload gene is operably linked to a microbial promoter. 163. The genetically engineered microorganism of Clause 162, wherein the microbial promoter is a constitutive promoter. 164. The genetically engineered microorganism of any one of Clauses 161–163, wherein the genetically engineered microorganism does not include a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. 165. The genetically engineered microorganism of any one of Clauses 161–164, wherein the genetically engineered microorganism does not include an auxotrophic mutation that facilitates lysis of the genetically engineered microorganism within the target cell. Attorney Docket No. MMS.003WO 166. The genetically engineered microorganism of any one of Clauses 96–165, wherein the genetically engineered microorganism further comprises a second payload gene encoding a detection marker. 167. The genetically engineered microorganism of Clause 166, wherein the detection marker comprises a protein, a peptide, or a nucleic acid molecule. 168. The genetically engineered microorganism of Clause 166 or 167, wherein the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter. 169. The genetically engineered microorganism of any one of Clauses 96–168, wherein the genetically engineered microorganism is Escherichia coli Nissle 1917 or a derivative thereof. 170. A method for treating a disease characterized by the presence of diseased gastrointestinal (GI) tissue in a subject in need thereof, the method comprising: (a) administering to the epithelial tissue of a subject, a genetically engineered microorganism that specifically invades diseased cells of the epithelium, and directs expression of a prodrug converting enzyme in the diseased cells, and (b) administering a prodrug. 171. The method of Clause 170, wherein the genetically engineered microorganism comprises an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue; and wherein the surface protein promotes binding and/or invasion of the microorganism in the diseased epithelial cells. 172. The method of Clause 170 or 171, wherein the subject is low risk for colorectal cancer (CRC) or colorectal polyps. Attorney Docket No. MMS.003WO 173. The method of Clause 172, wherein the subject has no previous incidence of CRC or colorectal polyps. 174. The method of Clause 170 or 171, wherein the subject is high or medium risk for CRC or colorectal polyps. 175. The method of Clause 170 or 171, wherein the subject has prior incidence of CRC or colorectal polyps, and/or family history of CRC. 176. The method of Clause 170 or 171, wherein the subject has a genetic predisposition to colorectal cancer. 177. The method of Clause 170 or 171, wherein the subject has familial adenomatous polyposis (FAP), attenuated FAP, Lynch syndrome (hereditary non-polyposis colorectal cancer (HNPCC)), oligopolyposis, Peutz-Jeghers syndrome (PJS), MUTYH- associated polyposis (MAP), juvenile polyposis syndrome, or Cowden syndrome. 178. The method of any one of Clauses 170–177, wherein the subject is at least 45 years of age, at least 50 years of age, at least 55 years of age, or at least 60 years of age. 179. The method of any one of Clauses 170–178, wherein the prodrug converting enzyme is selected from: an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5- fluorouracil (5-FU), the enzyme optionally selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, a uracil phosphoribosyltransferase (UPRT), and a combination thereof or a fusion protein comprising two or more thereof; an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU, optionally the enzyme being a carboxylesterase; an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU, optionally the enzyme being a β-glucuronidase; an enzyme capable of catalyzing conversion of 6-Methylpurine-deoxyriboside Attorney Docket No. MMS.003WO (MePdR) into 6-Methylpurine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); an enzyme capable of catalyzing conversion of 9-beta-D-Arabinofuranosyl-2- fluoroadenine monophosphate(fludarabine monophosphate) into fluoroadenine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); and/or an enzyme capable of catalyzing conversion of 1-(2-Deoxy-2-fluoro-b-D- arabinofuranosyl)uracil into 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5- methyluracil monophosphate (FMAUMP), optionally the enzyme being a thymidine kinase (TK) and/or a thymidylate synthase (TS). 180. The method of Clause 179, wherein the prodrug is 5-fluorocytosine (5-FC), pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin- 4-yl]carbamate (capecitabine), 5-FU glucuronide, 6-Methylpurine-deoxyriboside (MePdR), fludarabine monophosphate, 1-(2-Deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, and a combination of any two or more thereof. 181. The method of any one of Clauses 170–180, wherein the genetically engineered microorganism is non-pathogenic. 182. The method of Clause 181, wherein the genetically engineered microorganism harbors at least one auxotrophic mutation. 183. The method of any one of Clauses 170–182, wherein the prodrug converting enzyme is expressed from a mammalian promoter. 184. The method of Clause 183, wherein the mammalian promoter directs GI tract epithelial cell-specific expression. 185. The method of Clause 183 or 184, wherein a nucleic acid from the genetically engineered microorganism enters the nucleus of at least one diseased cell. 186. The method of Clause 185, wherein the nucleic acid is a plasmid molecule. Attorney Docket No. MMS.003WO 187. The method of Clause 186, wherein the plasmid molecule comprises a gene encoding the prodrug converting enzyme. 188. The method of Clause 186 or 187, wherein the plasmid molecule comprises at least one binding site for a DNA binding protein. 189. The method of Clause 188, wherein the at least one binding site for the DNA binding protein is selected from SV40 enhancer, a transcription factor binding site, and a bacterial operator. 190. The method of Clause 189, wherein the DNA binding protein comprises one or more nuclear localization signal(s) (NLS). 191. The method of Clause 190, wherein the NLS is an SV40 T antigen NLS sequence (KKKRKV). 192. The method of any one of Clauses 188–191, wherein the microorganism comprises a gene encoding the DNA binding protein. 193. The method of Clause 191 or 192, wherein the DNA binding protein is NFκB. 194. The method of any one of Clauses 170–182, wherein the prodrug converting enzyme is expressed from a microbial promoter, and an mRNA encoding the prodrug converting enzyme is translated in the diseased cells. 195. The method of Clause 194, wherein the genetically engineered microorganism has a deletion of an RNase gene. 196. The method of Clause 195, wherein the RNase gene is RNase III (rnc). 197. The method of any one of Clauses 194–196, wherein the gene encoding the prodrug converting enzyme is present in a cassette that directs RNA self-amplification of the cassette in the diseased cells. Attorney Docket No. MMS.003WO 198. The method of Clause 197, wherein the cassette comprises an RNA viral genome replication apparatus. 199. The method of Clause 198, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). 200. The method of Clause 199, wherein the RdRP is an alphavirus RdRP comprising nsp1, nsp2, nsp3, and nsp4. 201. The method of any one of Clauses 194–200, wherein the mRNA encoding the prodrug converting enzyme further comprises an internal ribosome entry site (IRES). 202. The method of any one of Clauses 170–201, wherein the genetically engineered microorganism is administered via an oral or rectal route. 203. The method of any one of Clauses 170–202, wherein the gene encoding the prodrug converting enzyme comprises at least one intron. 204. The method of Clause 203, wherein the at least one intron is a spliceosomal intron. 205. The method of any one of Clauses 170–204, wherein the method further comprises detecting diseased cells in the GI tract. 206. The method of Clause 205, wherein the method is performed as an alternative to colonoscopy. 207. The method of Clause 205, wherein the genetically engineered microorganism is administered after colonoscopy or other technique confirming presence of diseased tissue. 208. The method of any one of Clauses 205–207, wherein the method is repeated no more frequently than about biweekly, about monthly, bimonthly, every six months, annually, or biannually. Attorney Docket No. MMS.003WO 209. The method of any one of Clauses 205–208, wherein the genetically engineered microorganism further comprises a gene encoding a detection marker. 210. The method of Clause 209, wherein the detection marker is selected from a fluorescent protein, a bioluminescent protein, a contrast agent for magnetic resonance imaging (MRI), a Positron Emission Tomography (PET) reporter, an enzyme reporter, a contrast agent for use in computerized tomography (CT), a Single Photon Emission Computed Tomography (SPECT) reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), and ion channel reporters (e.g., cAMP activated cation channel), and a combination of any two or more thereof. 211. The method of Clause 210, wherein the diseased GI tissue is detected using endoscopy, colonoscopy, MRI, CT scan, PET scan, SPECT, or a combination thereof. 212. The method of Clause 211, wherein the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713 (iRFP), iRFP720 and iSplit. 213. The method of Clause 212, wherein the near-infrared fluorescent protein is detected by colonoscopy. 214. The method of Clause 209, wherein the detection marker is a secretable biomarker that is excreted in a biological fluid selected from mucus, saliva, and urine. 215. The method of Clause 214, wherein the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two or more thereof. 216. The method of Clause 214 or 215, wherein the detecting comprises: (c1) obtaining a biological sample of the subject; and (c2) evaluating the sample for the presence or amount of the secretable biomarker. Attorney Docket No. MMS.003WO 217. The method of Clause 216, wherein the biological sample is selected from blood, plasma, serum, urine, feces, saliva, and mucus, or a combination of any two or more thereof. 218. The method of Clause 216 or 217, wherein the secretable biomarker is measured by agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, circular dichroism (CD), chemiluminescence, colorimetry, dipstick assay, lateral flow immunoassay, fluorimetry, electrophoretic assay, enzymatic assay, enzyme linked immunosorbant assay (ELISA), gas chromatography, gravimetry, high pressure liquid chromatography (HPLC), immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, or a combination of any two or more thereof. 219. The method of any one of Clauses 170–218, wherein the microorganism is selected from Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, or Escherichia coli. 220. The method of Clause 219, wherein the microorganism is Escherichia coli, and optionally Escherichia coli Nissle 1917 or a derivative thereof. 221. The method of Clause 220, wherein the Escherichia coli Nissle 1917 or the derivative thereof harbors a plasmid pMUT1 and/or a plasmid pMUT2, and/or derivatives thereof. 222. The method of Clause 221, wherein the Escherichia coli Nissle 1917 or the derivative thereof is cured of the plasmid pMUT1 and/or the plasmid pMUT2. 223. The method of any one of Clauses 171–222, wherein the surface protein specifically interacts with one or more cell membrane receptor(s) that are not exposed on the luminal side of normal epithelial cells of GI tissue. 224. The method of Clause 223, wherein the surface protein is an invasin, or a fragment thereof. Attorney Docket No. MMS.003WO 225. The method of Clause 224, wherein the invasin is selected from Yersinia enterocolitica invasin, Yersinia pseudotuberculosis invasin, Salmonella enterica PagN, Candida albicans Als3 and E. coli intimin. 226. The method of Clause 223, wherein the surface protein comprises a peptide or protein that specifically binds to the surface of cancerous cells and/or pre-cancerous cells. 227. The method of Clause 226, wherein the peptide or protein is selected from a leptin, an antibody, or a fragment thereof. 228. The method of Clause 227, wherein the antibody, or a fragment thereof is selected from a single-domain antibody (sdAb) and an scFv fragment. 229. The method of any one of Clauses 226–228, wherein the peptide or protein is displayed on a microbial surface protein. 230. The method of Clause 229, wherein the peptide or protein is expressed as a fusion protein with the microbial surface protein. 231. The method of Clause 230, wherein the microbial surface protein is selected from invasin, intimin, and adhesin. 232. The method of any one of Clauses 170–231, wherein the genetically engineered microorganism further comprises an exogenous gene encoding a lysin that is capable of lysing the endocytotic vacuole. 233. The method of Clause 232, wherein the lysin is listeriolysin O, or a mutant derivative thereof. 234. The method of Clause 232 or 233, wherein the gene encoding the surface protein and/or the gene encoding the lysin is integrated in the genome of the genetically engineered microorganism. Attorney Docket No. MMS.003WO 235. The method of Clause 234, wherein the gene encoding the surface protein, and the gene encoding the lysin are integrated at a single genomic site. 236. The method of Clause 235, wherein the single genomic site is selected from an integration site of a bacteriophage and an integration site of a plasmid. 237. The method of Clause 232 or 233, wherein the gene encoding the surface protein, and/or the gene encoding the lysin are inserted on a plasmid. 238. The method of any one of Clauses 170–237, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is integrated in a genome of the genetically engineered microorganism or inserted on a plasmid. 239. The method of Clause 238, wherein the genes encoding the detection marker and the prodrug converting enzyme are integrated at the single genomic site. 240. The method of Clause 238, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a plasmid. 241. The method of any one of Clauses 170–236, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a second plasmid. 242. The method of Clause 241, wherein the microorganism is Escherichia coli Nissle 1917 or a derivative thereof and the plasmid and/or the second plasmid is selected from the plasmid pMUT1, the plasmid pMUT2, and a derivative thereof. 243. The method of Clause 241 or 242, wherein the plasmid and/or the second plasmid comprises a selection mechanism. 244. The method of Clause 243, wherein the selection mechanism is an antibiotic resistance marker selected from kanamycin resistance gene and tetracycline resistance gene. Attorney Docket No. MMS.003WO 245. The method of Clause 243, wherein the selection mechanism is a marker causing complementation of a mutation in an essential gene and/or a nutritional marker. 246. The method of Clause 245, wherein the essential gene and/or a nutritional marker is selected from dapA, dapD, murA, alr, dadX, murI, dapE, thyA and a combination of any two or more thereof. 247. The method of Clause 246, wherein the essential gene and/or a nutritional marker is alr and/or dadX. 248. The method of any one of Clauses 170–247, wherein the genetically engineered microorganism has an inactivation of alr and dadX genes. 249. The method of Clause 248, wherein the inactivation of alr and dadX genes is caused by a point mutation, a frameshift mutation or a nonsense mutation. 250. The method of Clause 249, wherein the inactivation of alr and dadX genes is caused by a partial or complete deletion. 251. The method of any one of Clauses 248–250, wherein the plasmid and/or the second plasmid comprises an active alr and/or dadX gene complementing the inactivation of alr and dadX genes. 252. The method of any one of Clauses 170–251, wherein the disease is selected from a precancerous lesion, cancer, ulcerative colitis, Crohn’s disease, Barrett’s esophagus, irritable bowel syndrome and irritable bowel disease. 253. The method of Clause 252, wherein the precancerous lesion comprises a polyp selected from sessile polyp, serrated polyp (e.g., hyperplastic polyps, sessile serrated adenomas/polyps, and traditional serrated adenoma), sessile serrated polyp, flat polyp, sub- pedunculated polyp, pedunculated polyp, and a combination thereof. Attorney Docket No. MMS.003WO 254. The method of Clause 252, wherein the precancerous lesion comprises a biliary intraepithelial neoplasm (BilIN) selected from BilIN-1, BilIN-2, BilIN-3, and cholangiocarcinoma. 255. The method of Clause 252, wherein the precancerous lesion comprises a pancreatic intraepithelial neoplasm (PanIN) selected from PanIN -1, PanIN -2, PanIN -3 and pancreatic ductal adenocarcinoma (PDAC). 256. The method of any one of Clauses 252–255, wherein the precancerous lesion is a diminutive polyp. 257. The method of any one of Clauses 252–255, wherein the precancerous lesion has a size of from about 0.05 mm to about 30 mm. 258. The method of Clause 257, wherein the precancerous lesion has a size of less than about 0.1 mm, less than about 0.25 mm, less than about 0.5 mm, less than about 1 mm, less than about 2 mm, less than about 5 mm, less than about 8 mm, less than about 10 mm, less than about 15 mm, less than about 20 mm, less than about 25 mm, or less than about 30 mm. 259. The method of Clause 252, wherein the cancer comprises a polyp, an adenoma, or a frank cancer. 260. The method of Clause 252, wherein the cancer comprises Lynch syndrome cancer, familial adenomatous polyposis, hereditary non-polyposis colon cancer (HNPCC), pancreatic ductal adenocarcinoma (PDAC), cholangiocarcinoma, or a sporadic cancer. 261. A genetically engineered microorganism comprising an exogenous gene encoding a surface protein, wherein the surface protein specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue, and which is optionally epithelial tissue of the gastrointestinal tract, wherein the surface protein promotes binding and invasion of epithelial cells of diseased tissue, Attorney Docket No. MMS.003WO wherein the microorganism comprises a gene encoding a prodrug converting enzyme that is capable of converting a non-toxic prodrug to a pharmacologically active compound inside a diseased cell, the gene encoding a prodrug converting enzyme being operably linked to a promoter. 262. The genetically engineered microorganism of Clause 261, wherein the prodrug converting enzyme is selected from: an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5- fluorouracil (5-FU), the enzyme optionally selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, a uracil phosphoribosyltransferase (yUPRT), and a combination thereof or a fusion protein comprising two or more thereof that; an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU, optionally the enzyme being a carboxylesterase; an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU, optionally the enzyme being a β-glucuronidase; an enzyme capable of catalyzing conversion of 6-methylpurine-deoxyriboside (MePdR) into 6-methylpurine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); an enzyme capable of catalyzing conversion of 9-beta-D-arabinofuranosyl-2- fluoroadenine monophosphate(fludarabine monophosphate) into fluoroadenine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); and/or an enzyme capable of catalyzing conversion of 1-(2-deoxy-2-fluoro-b-D- arabinofuranosyl)uracil into 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5- methyluracil monophosphate (FMAUMP), optionally the enzyme being a thymidine kinase (TK) and/or a thymidylate synthase (TS). 263. The genetically engineered microorganism of Clause 261 or 262, wherein the genetically engineered microorganism is non-pathogenic. Attorney Docket No. MMS.003WO 264. The genetically engineered microorganism of any one of Clauses 261–263, wherein the genetically engineered microorganism harbors at least one auxotrophic mutation. 265. The genetically engineered microorganism of any one of Clauses 261–264, wherein the prodrug converting enzyme is expressed from a mammalian promoter. 266. The genetically engineered microorganism of Clause 265, wherein the mammalian promoter directs GI tract epithelial cell-specific expression. 267. The genetically engineered microorganism of Clause 265 or 266, wherein a nucleic acid from the genetically engineered microorganism enters the nucleus of at least one diseased cell. 268. The genetically engineered microorganism of Clause 267, wherein the nucleic acid is a plasmid molecule. 269. The genetically engineered microorganism of Clause 268, wherein the plasmid molecule comprises a gene encoding the prodrug converting enzyme. 270. The genetically engineered microorganism of Clause 268 or 269, wherein the plasmid molecule comprises at least one binding site for a DNA binding protein. 271. The genetically engineered microorganism of Clause 270, wherein the at least one binding site for the DNA binding protein is selected from SV40 enhancer, a transcription factor binding site, and a bacterial operator. 272. The genetically engineered microorganism of Clause 271, wherein the DNA binding protein comprises one or more nuclear localization signal(s) (NLS). 273. The genetically engineered microorganism of Clause 272, wherein the NLS is SV40 T antigen NLS sequence (KKKRKV). Attorney Docket No. MMS.003WO 274. The genetically engineered microorganism of any one of Clauses 270–273, wherein the microorganism comprises a gene encoding the DNA binding protein. 275. The genetically engineered microorganism of Clause 273 or 274, wherein the DNA binding protein is NFκB. 276. The genetically engineered microorganism of any one of Clauses 261–264, wherein the prodrug converting enzyme is expressed from a microbial promoter, and an mRNA encoding the prodrug converting enzyme is translated in the diseased cells. 277. The genetically engineered microorganism of Clause 276, wherein the genetically engineered microorganism has a deletion of an RNase gene. 278. The genetically engineered microorganism of Clause 277, wherein the RNase gene is RNase III (rnc). 279. The genetically engineered microorganism of any one of Clauses 276–278, wherein the gene encoding the prodrug converting enzyme is present in a cassette that directs RNA self-amplification of the cassette in the diseased cells. 280. The genetically engineered microorganism of Clause 279, wherein the cassette comprises an RNA viral genome replication apparatus. 281. The genetically engineered microorganism of Clause 280, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). 282. The genetically engineered microorganism of Clause 281, wherein the RdRP is an alphavirus RdRP comprising nsp1, nsp2, nsp3, and nsp4. 283. The genetically engineered microorganism of any one of Clauses 276–282, wherein the mRNA encoding the prodrug converting enzyme further comprises an internal ribosome entry site (IRES). Attorney Docket No. MMS.003WO 284. The genetically engineered microorganism of any one of Clauses 261–283, wherein the gene encoding the prodrug converting enzyme comprises at least one intron. 285. The genetically engineered microorganism of Clause 284, wherein the at least one intron is a spliceosomal intron. 286. The genetically engineered microorganism of any one of Clauses 261–285, wherein the engineered microorganism further comprises a gene encoding a detection marker. 287. The genetically engineered microorganism of Clause 286, wherein the detection marker is selected from a fluorescent protein, a bioluminescent protein, a contrast agent for magnetic resonance imaging (MRI), a Positron Emission Tomography (PET) reporter, an enzyme reporter, a contrast agent for use in computerized tomography (CT), a Single Photon Emission Computed Tomography (SPECT) reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), and ion channel reporters (e.g., cAMP activated cation channel), and a combination of any two or more thereof. 288. The genetically engineered microorganism of Clause 287, wherein the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713 (iRFP), iRFP720 and iSplit. 289. The genetically engineered microorganism of Clause 286, wherein the detection marker is a secretable biomarker that is excreted in a biological fluid selected from mucus, saliva and urine. 290. The genetically engineered microorganism of Clause 289, wherein the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two or more thereof. Attorney Docket No. MMS.003WO 291. The genetically engineered microorganism of any one of Clauses 261–290, wherein the microorganism is selected from Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, or Escherichia coli. 292. The genetically engineered microorganism of Clause 291, wherein the microorganism is Escherichia coli, and optionally Escherichia coli Nissle 1917 or a derivative thereof. 293. The genetically engineered microorganism of Clause 292, wherein the Escherichia coli Nissle 1917 or the derivative thereof harbors a plasmid pMUT1 and/or a plasmid pMUT2, and/or derivatives thereof. 294. The genetically engineered microorganism of Clause 292, wherein the Escherichia coli Nissle 1917 or the derivative thereof is cured of the plasmid pMUT1 and/or the plasmid pMUT2. 295. The genetically engineered microorganism of any one of Clauses 261–294, wherein the surface protein specifically interacts with one or more cell membrane receptor(s) that are not exposed on the luminal side of normal epithelial cells of GI tissue. 296. The genetically engineered microorganism of Clause 295, wherein the surface protein is an invasin, or a fragment thereof. 297. The genetically engineered microorganism of Clause 296, wherein the invasin is selected from Yersinia enterocolitica invasin, Yersinia pseudotuberculosis invasin, Salmonella enterica PagN, Candida albicans Als3, and E. coli intimin. 298. The genetically engineered microorganism of Clause 297, wherein the surface protein comprises a peptide or protein that specifically binds to the surface of cancerous cells and/or pre-cancerous cells. 299. The genetically engineered microorganism of Clause 298, wherein the peptide or protein is selected from a leptin, an antibody, or a fragment thereof. Attorney Docket No. MMS.003WO 300. The genetically engineered microorganism of Clause 299, wherein the antibody, or a fragment thereof is selected from a single-domain antibody (sdAb) and an scFv fragment. 301. The genetically engineered microorganism of any one of Clauses 298–300, wherein the peptide or protein is displayed on a microbial surface protein. 302. The genetically engineered microorganism of Clause 301, wherein the peptide or protein is expressed as a fusion protein with the microbial surface protein. 303. The genetically engineered microorganism of Clause 302, wherein the microbial surface protein is selected from invasin, intimin and adhesin. 304. The genetically engineered microorganism of any one of Clauses 261–303, wherein the microorganism further comprises an exogenous gene encoding a lysin that is capable of lysing the endocytotic vacuole. 305. The genetically engineered microorganism of Clause 304, wherein the lysin is listeriolysin O, or a mutant derivative thereof. 306. The genetically engineered microorganism of Clause 304 or 305, wherein the gene encoding the surface protein and/or the gene encoding the lysin is integrated in the genome of the microorganism. 307. The genetically engineered microorganism of Clause 306, wherein the gene encoding the surface protein, and the gene encoding the lysin are integrated at a single genomic site. 308. The genetically engineered microorganism of Clause 307, wherein the single genomic site is selected from an integration site of a bacteriophage and an integration site of a plasmid. Attorney Docket No. MMS.003WO 309. The genetically engineered microorganism of Clause 307 or 308, wherein the gene encoding the surface protein, and /or the gene encoding the lysin are inserted on a plasmid. 310. The genetically engineered microorganism of any one of Clauses 261–309, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is integrated in a genome of the microorganism or inserted on a plasmid. 311. The genetically engineered microorganism of Clause 310, wherein the genes encoding the detection marker and the prodrug converting enzyme are integrated at the single genomic site. 312. The genetically engineered microorganism of Clause 310, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a plasmid. 313. The genetically engineered microorganism of any one of Clauses 261–312, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a second plasmid. 314. The genetically engineered microorganism of Clause 313, wherein the microorganism is Escherichia coli Nissle 1917 or a derivative thereof and the plasmid and/or the second plasmid is selected from the plasmid pMUT1, the plasmid pMUT2, and a derivative thereof. 315. The genetically engineered microorganism of Clause 313 or 314, wherein the plasmid and/or the second plasmid comprises a selection mechanism. 316. The genetically engineered microorganism of Clause 315, wherein the selection mechanism is an antibiotic resistance marker selected from kanamycin resistance gene and tetracycline resistance gene. Attorney Docket No. MMS.003WO 317. The genetically engineered microorganism of Clause 315, wherein the selection mechanism is a marker causing complementation of a mutation in an essential gene and/or a nutritional marker. 318. The genetically engineered microorganism of Clause 316, wherein the essential gene and/or a nutritional marker is selected from dapA, dapD, murA, alr, dadX, murI, dapE, thyA and a combination of any two or more thereof. 319. The genetically engineered microorganism of Clause 318, wherein the essential gene and/or a nutritional marker is alr and/or dadX. 320. The genetically engineered microorganism of any one of Clauses 261–319, wherein the genetically engineered microorganism has an inactivation of alr and dadX genes. 321. The genetically engineered microorganism of Clause 320, wherein the inactivation of alr and dadX genes is caused by a point mutation, a frameshift mutation or a nonsense mutation. 322. The genetically engineered microorganism of Clause 320, wherein the inactivation of alr and dadX genes is caused by a partial or complete deletion. 323. The genetically engineered microorganism of any one of Clauses 318–322, wherein the plasmid and/or the second plasmid comprises an active alr and/or dadX gene complementing the inactivation of alr and dadX genes. 324. A pharmaceutical composition comprising the genetically engineered microorganism of any one of Clauses 96–169 or 261–323, and a pharmaceutically acceptable carrier. 325. A method for treating a subject comprising administering the genetically engineered microorganism of any one of Clauses 96–169 or 261–323 or the pharmaceutical composition of Clause 324 to the subject. Attorney Docket No. MMS.003WO Conclusion Although many of the embodiments are described above with respect to compositions and methods for treating and/or detecting diseases of the GI tract and/or associated tissues, the technology is applicable to other applications and/or other approaches, such as diagnosing and/or treating other types of diseases. Moreover, other embodiments in addition to those described herein are within the scope of the technology. Additionally, several other embodiments of the technology can have different configurations, components, or procedures than those described herein. A person of ordinary skill in the art, therefore, will accordingly understand that the technology can have other embodiments with additional elements, or the technology can have other embodiments without several of the features shown and described above with reference to FIGS.1A–32. The descriptions of embodiments of the technology are not intended to be exhaustive or to limit the technology to the precise form disclosed above. Where the context permits, singular or plural terms may also include the plural or singular term, respectively. Although specific embodiments of, and examples for, the technology are described above for illustrative purposes, various equivalent modifications are possible within the scope of the technology, as those skilled in the relevant art will recognize. For example, while steps are presented in a given order, alternative embodiments may perform steps in a different order. The various embodiments described herein may also be combined to provide further embodiments. As used herein, the terms “generally,” “substantially,” “about,” and similar terms are used as terms of approximation and not as terms of degree, and are intended to account for the inherent variations in measured or calculated values that would be recognized by those of ordinary skill in the art. The term “about,” in reference to a number, may be used herein to include numbers that fall within a range of 10%, 5%, or 1% in either direction (greater than or less than) the number unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Moreover, unless the word “or” is expressly limited to mean only a single item exclusive from the other items in reference to a list of two or more items, then the use of “or” in such a list is to be interpreted as including (a) any single item in the list, (b) all of the items in the list, or (c) any combination of the items in the list. As used herein, the phrase “and/or” as in “A and/or B” refers to A alone, B alone, and A and B. Additionally, the term “comprising” Attorney Docket No. MMS.003WO is used throughout to mean including at least the recited feature(s) such that any greater number of the same feature and/or additional types of other features are not precluded. As used herein, the term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, may refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. For purposes herein, percent identity and sequence similarity may be performed using the BLAST algorithm, which is described in Altschul et al. (J. Mol. Biol.215:403-410 (1990)). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. As used herein, the term “subject” may broadly refer to any animal, including but not limited to, human and non-human animals (e.g., dogs, cats, cows, horses, sheep, pigs, poultry, fish, crustaceans, etc.). To the extent any materials incorporated herein by reference conflict with the present disclosure, the present disclosure controls. It will also be appreciated that specific embodiments have been described herein for purposes of illustration, but that various modifications may be made without deviating from the technology. Further, while advantages associated with certain embodiments of the technology have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the technology. Accordingly, the disclosure and Attorney Docket No. MMS.003WO associated technology can encompass other embodiments not expressly shown or described herein.

Claims

Attorney Docket No. MMS.003WO CLAIMS I/We claim: 1. A method for treating a subject, the method comprising: administering a genetically engineered microorganism to a gastrointestinal (GI) tract of the subject, wherein the genetically engineered microorganism comprises: a targeting gene encoding a targeting element that binds to a surface marker on a target cell in the GI tract, and a payload gene encoding a therapeutic agent. 2. The method of claim 1, wherein the therapeutic agent is a prodrug converting enzyme that converts a prodrug into an active drug, and wherein the method further comprises administering the prodrug to the subject. 3. The method of claim 2, wherein the prodrug comprises a precursor of 5- fluorouracil (5-FU) and the active drug comprises 5-FU. 4. The method of claim 3, wherein the precursor of 5-FU comprises 5- fluorocytosine (5-FC). 5. The method of claim 4, wherein the prodrug converting enzyme comprises a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof. 6. The method of claim 4 or 5, wherein the prodrug converting enzyme comprises a fusion protein of a cytosine deaminase and an uracil phosphoribosyltransferase (CD:UPRT). 7. The method of claim 6, wherein the CD:UPRT comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 6. Attorney Docket No. MMS.003WO 8. The method of claim 6 or 7, wherein the CD:UPRT comprises a sequence of SEQ ID NO: 6. 9. The method of any one of claims 6–8, wherein the payload gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO: 8. 10. The method of any one of claims 6–9, wherein the payload gene comprises a sequence of any one of SEQ ID NO: 7 or SEQ ID NO: 8. 11. The method of claim 3, wherein the precursor of 5-FU comprises pentyl N-[1- [(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4- yl]carbamate (capecitabine). 12. The method of claim 11, wherein the prodrug converting enzyme comprises a carboxylesterase. 13. The method of claim 3, wherein the precursor of 5-FU comprises 5-FU glucuronide. 14. The method of claim 13, wherein the prodrug converting enzyme comprises a β-glucuronidase. 15. The method of claim 2, wherein the prodrug converting enzyme comprises a purine nucleoside phosphorylase. 16. The method of claim 15, wherein the prodrug comprises 6-methylpurine- deoxyriboside and the active drug comprises 6-methylpurine. 17. The method of claim 15, wherein the prodrug comprises 9-beta-D- arabinofuranosyl-2-fluoroadenine monophosphate and the active drug comprises fluoroadenine. Attorney Docket No. MMS.003WO 18. The method of claim 2, wherein the prodrug comprises 1-(2-deoxy-2-fluoro-b- D-arabinofuranosyl)uracil, and the active drug comprises 1-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl) 5-methyluracil monophosphate. 19. The method of claim 18, wherein the prodrug converting enzyme comprises a thymidine kinase, a thymidine synthase, or a combination thereof. 20. The method of claim 2, wherein the prodrug comprises ganciclovir, and the prodrug converting enzyme comprises a viral thymidine kinase. 21. The method of any one of claims 2–20, wherein the prodrug is administered via an oral route, a rectal route, a sublingual route, an intravenous route, an intramuscular route, a subcutaneous route, a transdermal route, an intranasal route, or a vaginal route. 22. The method of any one of claims 2–21, wherein the prodrug is administered concurrently with the genetically engineered microorganism. 23. The method of any one of claims 2–21, wherein the prodrug is administered at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 18 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after the genetically engineered microorganism is administered to the subject. 24. The method of claim 1, wherein the therapeutic agent is a direct cytotoxic agent. 25. The method of claim 24, wherein the direct cytotoxic agent comprises a microbial toxin or a caspase. 26. The method of claim 24 or 25, wherein the direct cytotoxic agent comprises a cytotoxic protein or peptide. 27. The method of any one of claims 24–26, wherein the direct cytotoxic agent exhibits enhanced activity in the target cell compared to a nontarget cell. Attorney Docket No. MMS.003WO 28. The method of any one of claims 1–27, wherein the administration of the genetically engineered microorganism causes selective expression of the therapeutic agent in the target cell. 29. The method of claim 28, wherein the selective expression of the therapeutic agent in the target cell results in killing of the target cell. 30. The method of any one of claims 1–29, wherein the target cell is a cell of a precancerous lesion. 31. The method of claim 30, wherein the precancerous lesion is an adenoma. 32. The method of claim 30 or 31, wherein the precancerous lesion has a size less than or equal to 0.1 mm, 0.25 mm, 0.5 mm, 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, 15 mm, 20 mm, 25 mm, or 30 mm. 33. The method of any one of claims 1–32, wherein the target cell is a diseased epithelial cell indicative of a cancer. 34. The method of claim 33, wherein the cancer is one or more of the following: colorectal cancer, Lynch syndrome cancer, familial adenomatous polyposis, hereditary non- polyposis colon cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, or a sporadic cancer. 35. The method of any one of claims 1–34, wherein the payload gene comprises at least one intron. 36. The method of claim 35, wherein the at least one intron comprises a spliceosomal intron. 37. The method of any one of claims 1–36, wherein the payload gene is operably linked to a promoter. Attorney Docket No. MMS.003WO 38. The method of claim 37, wherein the promoter is a mammalian promoter. 39. The method of claim 37, wherein the promoter is a microbial promoter. 40. The method of any one of claims 1–39, wherein the targeting element comprises one or more of the following: an invasin, an intimin, an adhesin, a flagellin, a component of a type III secretion system, a leptin, or an antibody. 41. The method of any one of claims 1–40, wherein the surface marker is mislocalized on the target cell. 42. The method of any one of claims 1–41, wherein the surface marker is present on a luminal side of the target cell and is not present on a luminal side of a non-target cell. 43. The method of any one of claims 1–42, wherein the targeting element comprises an invasin and the surface marker comprises an integrin. 44. The method of claim 43, wherein the invasin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1, or comprises a sequence of SEQ ID NO: 1. 45. The method of claim 43 or 44, wherein the targeting gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or a coding region thereof, or comprises a sequence of SEQ ID NO: 2 or the coding region thereof. 46. The method of any one of claims 1–45, wherein the targeting element promotes internalization of the genetically engineered microorganism by the target cell. 47. The method of any one of claims 1–45, wherein the genetically engineered microorganism remains external to the target cell. Attorney Docket No. MMS.003WO 48. The method of any one of claims 1–46, wherein the genetically engineered microorganism further comprises a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. 49. The method of claim 48, wherein the lysis element comprises a lysin. 50. The method of claim 49, wherein the lysin comprises listeriolysin O. 51. The method of claim 50, wherein the lysin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4, or comprises a sequence of any one of SEQ ID NO: 3 or SEQ ID NO.4. 52. The method of any one of claims 1–51, wherein the genetically engineered microorganism comprises an auxotrophic mutation. 53. The method of claim 52, wherein the auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell. 54. The method of claim 52 or 53, wherein the auxotrophic mutation comprises a mutation in one or more of the following: dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF. 55. The method of any one of claims 52–54, wherein the auxotrophic mutation comprises a deletion of an essential gene, and the payload gene is located on a plasmid comprising a functional copy of the essential gene. 56. The method of claim 55, wherein the plasmid comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or a coding region thereof, or the plasmid comprises a sequence of SEQ ID NO: 5 or the coding region thereof. Attorney Docket No. MMS.003WO 57. The method of any one of claims 1–56, wherein the genetically engineered microorganism is configured to deliver a DNA molecule comprising the payload gene to the target cell. 58. The method of claim 57, wherein the DNA molecule comprises a plasmid. 59. The method of claim 57 or 58, wherein the DNA molecule comprises at least one binding site for a DNA binding protein that comprises one or more nuclear localization signals. 60. The method of claim 59, wherein the genetically engineered microorganism comprises a gene encoding the DNA binding protein. 61. The method of any one of claims 1–56, wherein the genetically engineered microorganism is configured to deliver an RNA molecule comprising the payload gene to the target cell. 62. The method of claim 61, wherein the RNA molecule comprises an mRNA molecule. 63. The method of claim 61 or 62, wherein the RNA molecule comprises a self- replicating or self-amplifying RNA molecule. 64. The method of claim 63, wherein the genetically engineered microorganism comprises a cassette that directs RNA self-amplification of the cassette in the target cell, and the payload gene is present in the cassette. 65. The method of claim 64, wherein the cassette comprises an RNA viral genome replication apparatus. 66. The method of claim 65, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). Attorney Docket No. MMS.003WO 67. The method of claim 66, wherein the RdRP comprises an alphavirus RdRP. 68. The method of claim 67, wherein the alphavirus RdRP comprises one or more of nsp1, nsp2, nsp3, or nsp4. 69. The method of any one of claims 61–68, wherein the genetically engineered microorganism comprises a deletion of an RNase gene. 70. The method of claim 69, wherein the RNase gene comprises RNase III. 71. The method of any one of claims 1–56, wherein the genetically engineered microorganism is configured to deliver a protein comprising the therapeutic agent to the target cell. 72. The method of claim 71, wherein the payload gene is operably linked to a microbial promoter. 73. The method of claim 72, wherein the microbial promoter is a constitutive promoter. 74. The method of any one of claims 71–73, wherein the genetically engineered microorganism does not include a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. 75. The method of any one of claims 71–74, wherein the genetically engineered microorganism does not include an auxotrophic mutation that facilitates lysis of the genetically engineered microorganism within the target cell. 76. The method of any one of claims 1–75, wherein the genetically engineered microorganism further comprises a second payload gene encoding a detection marker. 77. The method of any one of claims 1–76, further comprising administering a second genetically engineered microorganism to the subject, wherein the second genetically Attorney Docket No. MMS.003WO engineered microorganism comprises a second payload gene encoding a detection marker. 78. The method of claim 77, wherein the second genetically engineered microorganism is administered before, concurrently with, or after administration of the genetically engineered microorganism to the subject. 79. The method of any one of claims 76–78, wherein the detection marker comprises a protein, a peptide, or a nucleic acid molecule. 80. The method of any one of claims 76–79, wherein the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter. 81. The method of any one of claims 76–80, further comprising: collecting a biological sample from the subject; and detecting whether the detection marker is present in the biological sample. 82. The method of claim 81, wherein the biological sample comprises one or more of feces, blood, serum, plasma, mucus, urine, saliva, or a biopsy sample. 83. The method of any one of claims 76–82, further comprising performing an endoscopic or colonoscopic procedure to detect the detection marker in the subject. 84. The method of any one of claims 81–83, further comprising: upon detection of the detection marker, administering a third genetically engineered microorganism to the GI tract of the subject, wherein the third genetically engineered microorganism comprises the targeting gene and the payload gene. 85. The method of any one of claims 1–84, wherein the subject is low risk for colorectal cancer (CRC) or colorectal polyps. Attorney Docket No. MMS.003WO 86. The method of claim 85, wherein the subject has no previous incidence of CRC or colorectal polyps. 87. The method of any one of claims 1–84, wherein the subject is high or medium risk for CRC or colorectal polyps. 88. The method of any one of claims 1–84, wherein the subject has prior incidence of CRC, prior incidence of colorectal polyps, a family history of CRC, or a combination thereof. 89. The method of any one of claims 1–84, wherein the subject has a genetic predisposition to CRC. 90. The method any one of claims 1–84, wherein the subject has familial adenomatous polyposis (FAP), attenuated FAP, Lynch syndrome, oligopolyposis, Peutz- Jeghers syndrome (PJS), MUTYH-associated polyposis (MAP), juvenile polyposis syndrome, or Cowden syndrome. 91. The method of any one of claims 1–90, wherein the subject is at least 45 years of age, at least 50 years of age, at least 55 years of age, or at least 60 years of age. 92. The method of any one of claims 1–91, wherein the target cell is indicative of a disease selected from a precancerous lesion, a GI tract cancer, an inflammatory bowel disease, irritable bowel syndrome, or Barrett’s esophagus. 93. The method of any one of claims 1–92, wherein the genetically engineered microorganism is Escherichia coli Nissle 1917 or a derivative thereof. 94. The method of any one of claims 1–93, wherein the genetically engineered microorganism is administered via an oral route or a rectal route. 95. The method of any one of claims 1–94, wherein about 103 to about 1011 genetically engineered microorganisms are administered to the subject. Attorney Docket No. MMS.003WO 96. A genetically engineered microorganism comprising: a targeting gene encoding a targeting element that binds to a surface marker on a target cell in a gastrointestinal (GI) tract; and a payload gene encoding a therapeutic agent. 97. The genetically engineered microorganism of claim 96, wherein the therapeutic agent is a prodrug converting enzyme that converts a prodrug into an active drug. 98. The genetically engineered microorganism of claim 97, wherein the prodrug comprises a precursor of 5-fluorouracil (5-FU) and the active drug comprises 5-FU. 99. The genetically engineered microorganism of claim 98, wherein the precursor of 5-FU comprises 5-fluorocytosine (5-FC). 100. The genetically engineered microorganism of claim 99, wherein the prodrug converting enzyme comprises a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, an uracil phosphoribosyltransferase, or a combination thereof. 101. The genetically engineered microorganism of claim 99 or 100, wherein the prodrug converting enzyme comprises a fusion protein of a cytosine deaminase and an uracil phosphoribosyltransferase (CD:UPRT). 102. The genetically engineered microorganism of claim 101, wherein the CD:UPRT comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 6. 103. The genetically engineered microorganism of claim 101 or 102, wherein the CD:UPRT comprises a sequence of SEQ ID NO: 6. 104. The genetically engineered microorganism of any one of claims 101–103, wherein the payload gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 7 or SEQ ID NO: 8. Attorney Docket No. MMS.003WO 105. The genetically engineered microorganism of any one of claims 101–104, wherein the payload gene comprises a sequence of any one of SEQ ID NO: 7 or SEQ ID NO: 8. 106. The genetically engineered microorganism of claim 98, wherein the precursor of 5-FU comprises pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5- fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine). 107. The genetically engineered microorganism of claim 106, wherein the prodrug converting enzyme comprises a carboxylesterase. 108. The genetically engineered microorganism of claim 98, wherein the precursor of 5-FU comprises 5-FU glucuronide. 109. The genetically engineered microorganism of claim 108, wherein the prodrug converting enzyme comprises a β-glucuronidase. 110. The genetically engineered microorganism of claim 97, wherein the prodrug converting enzyme comprises a purine nucleoside phosphorylase. 111. The genetically engineered microorganism of claim 110, wherein the prodrug comprises 6-methylpurine-deoxyriboside and the active drug comprises 6-methylpurine. 112. The genetically engineered microorganism of claim 110, wherein the prodrug comprises 9-beta-D-arabinofuranosyl-2-fluoroadenine monophosphate and the active drug comprises fluoroadenine. 113. The genetically engineered microorganism of claim 97, wherein the prodrug comprises 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, and the active drug comprises 1- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5-methyluracil monophosphate. Attorney Docket No. MMS.003WO 114. The genetically engineered microorganism of claim 113, wherein the prodrug converting enzyme comprises a thymidine kinase, a thymidine synthase, or a combination thereof. 115. The genetically engineered microorganism of claim 97, wherein the prodrug comprises ganciclovir, and the prodrug converting enzyme comprises a viral thymidine kinase. 116. The genetically engineered microorganism of claim 96, wherein the therapeutic agent is a direct cytotoxic agent. 117. The genetically engineered microorganism of claim 116, wherein the direct cytotoxic agent comprises a microbial toxin or a caspase. 118. The genetically engineered microorganism of claim 116 or 117, wherein the direct cytotoxic agent comprises a cytotoxic protein or peptide. 119. The genetically engineered microorganism of any one of claims 116–118, wherein the direct cytotoxic agent exhibits enhanced activity in the target cell compared to a nontarget cell. 120. The genetically engineered microorganism of any one of claims 96–119, wherein the target cell is a cell of a precancerous lesion. 121. The genetically engineered microorganism of claim 120, wherein the precancerous lesion is an adenoma. 122. The genetically engineered microorganism of claim 120 or 121, wherein the precancerous lesion has a size less than or equal to 0.1 mm, 0.25 mm, 0.5 mm, 1 mm, 2 mm, 5 mm, 8 mm, 10 mm, 15 mm, 20 mm, 25 mm, or 30 mm. 123. The genetically engineered microorganism of any one of claims 96–122, wherein the target cell is a diseased epithelial cell indicative of a cancer. Attorney Docket No. MMS.003WO 124. The genetically engineered microorganism of claim 123, wherein the cancer is one or more of the following: colorectal cancer, Lynch syndrome cancer, familial adenomatous polyposis, hereditary non-polyposis colon cancer, pancreatic ductal adenocarcinoma, cholangiocarcinoma, or a sporadic cancer. 125. The genetically engineered microorganism of any one of claims 96–124, wherein the payload gene comprises at least one intron. 126. The genetically engineered microorganism of claim 125, wherein the at least one intron comprises a spliceosomal intron. 127. The genetically engineered microorganism of any one of claims 96–126, wherein the payload gene is operably linked to a promoter. 128. The genetically engineered microorganism of claim 127, wherein the promoter is a mammalian promoter. 129. The genetically engineered microorganism of claim 127, wherein the promoter is a microbial promoter. 130. The genetically engineered microorganism of any one of claims 96–129, wherein the targeting element comprises one or more of the following: an invasin, an intimin, an adhesin, a flagellin, a component of a type III secretion system, a leptin, or an antibody. 131. The genetically engineered microorganism of any one of claims 96–130, wherein the surface marker is mislocalized on the target cell. 132. The genetically engineered microorganism of any one of claims 96–131, wherein the surface marker is present on a luminal side of the target cell and is not present on a luminal side of a non-target cell. Attorney Docket No. MMS.003WO 133. The genetically engineered microorganism of any one of claims 96–132, wherein the targeting element comprises an invasin and the surface marker comprises an integrin. 134. The genetically engineered microorganism of claim 133, wherein the invasin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 1, or comprises a sequence of SEQ ID NO: 1. 135. The genetically engineered microorganism of claim 133 or 134, wherein the targeting gene comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 2 or a coding region thereof, or comprises a sequence of SEQ ID NO: 2 or the coding region thereof. 136. The genetically engineered microorganism of any one of claims 96–135, wherein the targeting element promotes internalization of the genetically engineered microorganism by the target cell. 137. The genetically engineered microorganism of any one of claims 96–135, wherein the genetically engineered microorganism remains external to the target cell. 138. The genetically engineered microorganism of any one of claims 96–136, wherein the genetically engineered microorganism further comprises a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. 139. The genetically engineered microorganism of claim 138, wherein the lysis element comprises a lysin. 140. The genetically engineered microorganism of claim 139, wherein the lysin comprises listeriolysin O. 141. The genetically engineered microorganism of claim 140, wherein the lysin comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, Attorney Docket No. MMS.003WO 97%, 98%, 99%, 99.5%, or 100% sequence identity to any one of SEQ ID NO: 3 or SEQ ID NO: 4, or comprises a sequence of any one of SEQ ID NO: 3 or SEQ ID NO.4. 142. The genetically engineered microorganism of any one of claims 96–141, wherein the genetically engineered microorganism comprises an auxotrophic mutation. 143. The genetically engineered microorganism of claim 142, wherein the auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell. 144. The genetically engineered microorganism of claim 142 or 143, wherein the auxotrophic mutation comprises a mutation in one or more of the following: dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF. 145. The genetically engineered microorganism of any one of claims 142–144, wherein the auxotrophic mutation comprises a deletion of an essential gene, and the payload gene is located on a plasmid comprising a functional copy of the essential gene. 146. The genetically engineered microorganism of claim 145, wherein the plasmid comprises a sequence having at least 70%, 75%, 80%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to SEQ ID NO: 5 or a coding region thereof, or the plasmid comprises a sequence of SEQ ID NO: 5 or the coding region thereof. 147. The genetically engineered microorganism of any one of claims 96–146, wherein the genetically engineered microorganism is configured to deliver a DNA molecule comprising the payload gene to the target cell. 148. The genetically engineered microorganism of claim 147, wherein the DNA molecule comprises a plasmid. 149. The genetically engineered microorganism of claim 147 or 148, wherein the DNA molecule comprises at least one binding site for a DNA binding protein that comprises one or more nuclear localization signals. Attorney Docket No. MMS.003WO 150. The genetically engineered microorganism of claim 149, wherein the genetically engineered microorganism comprises a gene encoding the DNA binding protein. 151. The genetically engineered microorganism of any one of claims 96–146, wherein the genetically engineered microorganism is configured to deliver an RNA molecule comprising the payload gene to the target cell. 152. The genetically engineered microorganism of claim 151, wherein the RNA molecule comprises an mRNA molecule. 153. The genetically engineered microorganism of claim 151 or 152, wherein the RNA molecule comprises a self-replicating or self-amplifying RNA molecule. 154. The genetically engineered microorganism of claim 153, wherein the genetically engineered microorganism comprises a cassette that directs RNA self- amplification of the cassette in the target cell, and the payload gene is present in the cassette. 155. The genetically engineered microorganism of claim 154, wherein the cassette comprises an RNA viral genome replication apparatus. 156. The genetically engineered microorganism of claim 155, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). 157. The genetically engineered microorganism of claim 156, wherein the RdRP comprises an alphavirus RdRP. 158. The genetically engineered microorganism of claim 157, wherein the alphavirus RdRP comprises one or more of nsp1, nsp2, nsp3, or nsp4. 159. The genetically engineered microorganism of any one of claims 151–158, wherein the genetically engineered microorganism comprises a deletion of an RNase gene. Attorney Docket No. MMS.003WO 160. The genetically engineered microorganism of claim 159, wherein the RNase gene comprises RNase III. 161. The genetically engineered microorganism of any one of claims 96–160, wherein the genetically engineered microorganism is configured to deliver a protein comprising the therapeutic agent to the target cell. 162. The genetically engineered microorganism of claim 161, wherein the payload gene is operably linked to a microbial promoter. 163. The genetically engineered microorganism of claim 162, wherein the microbial promoter is a constitutive promoter. 164. The genetically engineered microorganism of any one of claims 161–163, wherein the genetically engineered microorganism does not include a lysis gene encoding a lysis element that lyses an endocytic vacuole of the target cell. 165. The genetically engineered microorganism of any one of claims 161–164, wherein the genetically engineered microorganism does not include an auxotrophic mutation that facilitates lysis of the genetically engineered microorganism within the target cell. 166. The genetically engineered microorganism of any one of claims 96–165, wherein the genetically engineered microorganism further comprises a second payload gene encoding a detection marker. 167. The genetically engineered microorganism of claim 166, wherein the detection marker comprises a protein, a peptide, or a nucleic acid molecule. 168. The genetically engineered microorganism of claim 166 or 167, wherein the detection marker comprises one or more of the following: a fluorescent marker, a bioluminescent marker, an MRI contrast agent, a CT contrast agent, a SPECT reporter, a PET reporter, an enzyme reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter, or an ion channel reporter. Attorney Docket No. MMS.003WO 169. The genetically engineered microorganism of any one of claims 96–168, wherein the genetically engineered microorganism is Escherichia coli Nissle 1917 or a derivative thereof. 170. A method for treating a disease characterized by the presence of diseased gastrointestinal (GI) tissue in a subject in need thereof, the method comprising: (a) administering to the epithelial tissue of a subject, a genetically engineered microorganism that specifically invades diseased cells of the epithelium, and directs expression of a prodrug converting enzyme in the diseased cells, and (b) administering a prodrug. 171. The method of claim 170, wherein the genetically engineered microorganism comprises an exogenous gene encoding a surface protein that specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue; and wherein the surface protein promotes binding and/or invasion of the microorganism in the diseased epithelial cells. 172. The method of claim 170 or 171, wherein the subject is low risk for colorectal cancer (CRC) or colorectal polyps. 173. The method of claim 172, wherein the subject has no previous incidence of CRC or colorectal polyps. 174. The method of claim 170 or 171, wherein the subject is high or medium risk for CRC or colorectal polyps. 175. The method of claim 170 or 171, wherein the subject has prior incidence of CRC or colorectal polyps, and/or family history of CRC. 176. The method of claim 170 or 171, wherein the subject has a genetic predisposition to colorectal cancer. Attorney Docket No. MMS.003WO 177. The method of claim 170 or 171, wherein the subject has familial adenomatous polyposis (FAP), attenuated FAP, Lynch syndrome (hereditary non-polyposis colorectal cancer (HNPCC)), oligopolyposis, Peutz-Jeghers syndrome (PJS), MUTYH- associated polyposis (MAP), juvenile polyposis syndrome, or Cowden syndrome. 178. The method of any one of claims 170–177, wherein the subject is at least 45 years of age, at least 50 years of age, at least 55 years of age, or at least 60 years of age. 179. The method of any one of claims 170–178, wherein the prodrug converting enzyme is selected from: an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5- fluorouracil (5-FU), the enzyme optionally selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, a uracil phosphoribosyltransferase (UPRT), and a combination thereof or a fusion protein comprising two or more thereof; an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU, optionally the enzyme being a carboxylesterase; an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU, optionally the enzyme being a β-glucuronidase; an enzyme capable of catalyzing conversion of 6-methylpurine-deoxyriboside (MePdR) into 6-methylpurine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); an enzyme capable of catalyzing conversion of 9-beta-D-arabinofuranosyl-2- fluoroadenine monophosphate(fludarabine monophosphate) into fluoroadenine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); and/or an enzyme capable of catalyzing conversion of 1-(2-Deoxy-2-fluoro-b-D- arabinofuranosyl)uracil into 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5- methyluracil monophosphate (FMAUMP), optionally the enzyme being a thymidine kinase (TK) and/or a thymidylate synthase (TS). Attorney Docket No. MMS.003WO 180. The method of claim 179, wherein the prodrug is 5-fluorocytosine (5-FC), pentyl N-[1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin- 4-yl]carbamate (capecitabine), 5-FU glucuronide, 6-methylpurine-deoxyriboside (MePdR), fludarabine monophosphate, 1-(2-deoxy-2-fluoro-b-D-arabinofuranosyl)uracil, and a combination of any two or more thereof. 181. The method of any one of claims 170–180, wherein the genetically engineered microorganism is non-pathogenic. 182. The method of claim 181, wherein the genetically engineered microorganism harbors at least one auxotrophic mutation. 183. The method of any one of claims 170–182, wherein the prodrug converting enzyme is expressed from a mammalian promoter. 184. The method of claim 183, wherein the mammalian promoter directs GI tract epithelial cell-specific expression. 185. The method of claim 183 or 184, wherein a nucleic acid from the genetically engineered microorganism enters the nucleus of at least one diseased cell. 186. The method of claim 185, wherein the nucleic acid is a plasmid molecule. 187. The method of claim 186, wherein the plasmid molecule comprises a gene encoding the prodrug converting enzyme. 188. The method of claim 186 or 187, wherein the plasmid molecule comprises at least one binding site for a DNA binding protein. 189. The method of claim 188, wherein the at least one binding site for the DNA binding protein is selected from SV40 enhancer, a transcription factor binding site, and a bacterial operator. Attorney Docket No. MMS.003WO 190. The method of claim 189, wherein the DNA binding protein comprises one or more nuclear localization signal(s) (NLS). 191. The method of claim 190, wherein the NLS is an SV40 T antigen NLS sequence (KKKRKV). 192. The method of any one of claims 188–191, wherein the microorganism comprises a gene encoding the DNA binding protein. 193. The method of claim 191 or 192, wherein the DNA binding protein is NFκB. 194. The method of any one of claims 170–182, wherein the prodrug converting enzyme is expressed from a microbial promoter, and an mRNA encoding the prodrug converting enzyme is translated in the diseased cells. 195. The method of claim 194, wherein the genetically engineered microorganism has a deletion of an RNase gene. 196. The method of claim 195, wherein the RNase gene is RNase III (rnc). 197. The method of any one of claims 194–196, wherein the gene encoding the prodrug converting enzyme is present in a cassette that directs RNA self-amplification of the cassette in the diseased cells. 198. The method of claim 197, wherein the cassette comprises an RNA viral genome replication apparatus. 199. The method of claim 198, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). 200. The method of claim 199, wherein the RdRP is an alphavirus RdRP comprising nsp1, nsp2, nsp3, and nsp4. Attorney Docket No. MMS.003WO 201. The method of any one of claims 194–200, wherein the mRNA encoding the prodrug converting enzyme further comprises an internal ribosome entry site (IRES). 202. The method of any one of claims 170–201, wherein the genetically engineered microorganism is administered via an oral or rectal route. 203. The method of any one of claims 170–202, wherein the gene encoding the prodrug converting enzyme comprises at least one intron. 204. The method of claim 203, wherein the at least one intron is a spliceosomal intron. 205. The method of any one of claims 170–204, wherein the method further comprises detecting diseased cells in the GI tract. 206. The method of claim 205, wherein the method is performed as an alternative to colonoscopy. 207. The method of claim 205, wherein the genetically engineered microorganism is administered after colonoscopy or other technique confirming presence of diseased tissue. 208. The method of any one of claims 205–207, wherein the method is repeated no more frequently than about biweekly, about monthly, bimonthly, every six months, annually, or biannually. 209. The method of any one of claims 205–208, wherein the genetically engineered microorganism further comprises a gene encoding a detection marker. 210. The method of claim 209, wherein the detection marker is selected from a fluorescent protein, a bioluminescent protein, a contrast agent for magnetic resonance imaging (MRI), a Positron Emission Tomography (PET) reporter, an enzyme reporter, a contrast agent for use in computerized tomography (CT), a Single Photon Emission Computed Tomography (SPECT) reporter, a photoacoustic reporter, an X-ray reporter, an Attorney Docket No. MMS.003WO ultrasound reporter (e.g., a bacterial gas vesicle), and ion channel reporters (e.g., cAMP activated cation channel), and a combination of any two or more thereof. 211. The method of claim 210, wherein the diseased GI tissue is detected using endoscopy, colonoscopy, MRI, CT scan, PET scan, SPECT, or a combination thereof. 212. The method of claim 211, wherein the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713 (iRFP), iRFP720 and iSplit. 213. The method of claim 212, wherein the near-infrared fluorescent protein is detected by colonoscopy. 214. The method of claim 209, wherein the detection marker is a secretable biomarker that is excreted in a biological fluid selected from mucus, saliva, and urine. 215. The method of claim 214, wherein the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two or more thereof. 216. The method of claim 214 or 215, wherein the detecting comprises: (c1) obtaining a biological sample of the subject; and (c2) evaluating the sample for the presence or amount of the secretable biomarker. 217. The method of claim 216, wherein the biological sample is selected from blood, plasma, serum, urine, feces, saliva, and mucus, or a combination of any two or more thereof. 218. The method of claim 216 or 217, wherein the secretable biomarker is measured by agglutination, amperometry, atomic absorption spectrometry, atomic emission spectrometry, circular dichroism (CD), chemiluminescence, colorimetry, dipstick assay, Attorney Docket No. MMS.003WO lateral flow immunoassay, fluorimetry, electrophoretic assay, enzymatic assay, enzyme linked immunosorbant assay (ELISA), gas chromatography, gravimetry, high pressure liquid chromatography (HPLC), immunoassay, immunofluorometric enzyme assay, mass spectrometry, nuclear magnetic resonance, radioimmunoassay, spectrometry, titrimetry, western blotting, or a combination of any two or more thereof. 219. The method of any one of claims 170–218, wherein the microorganism is selected from Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, or Escherichia coli. 220. The method of claim 219, wherein the microorganism is Escherichia coli, and optionally Escherichia coli Nissle 1917 or a derivative thereof. 221. The method of claim 220, wherein the Escherichia coli Nissle 1917 or the derivative thereof harbors a plasmid pMUT1 and/or a plasmid pMUT2, and/or derivatives thereof. 222. The method of claim 221, wherein the Escherichia coli Nissle 1917 or the derivative thereof is cured of the plasmid pMUT1 and/or the plasmid pMUT2. 223. The method of any one of claims 171–222, wherein the surface protein specifically interacts with one or more cell membrane receptor(s) that are not exposed on the luminal side of normal epithelial cells of GI tissue. 224. The method of claim 223, wherein the surface protein is an invasin, or a fragment thereof. 225. The method of claim 224, wherein the invasin is selected from Yersinia enterocolitica invasin, Yersinia pseudotuberculosis invasin, Salmonella enterica PagN, Candida albicans Als3 and E. coli intimin. 226. The method of claim 223, wherein the surface protein comprises a peptide or protein that specifically binds to the surface of cancerous cells and/or pre-cancerous cells. Attorney Docket No. MMS.003WO 227. The method of claim 226, wherein the peptide or protein is selected from a leptin, an antibody, or a fragment thereof. 228. The method of claim 227, wherein the antibody, or a fragment thereof is selected from a single-domain antibody (sdAb) and an scFv fragment. 229. The method of any one of claims 226–228, wherein the peptide or protein is displayed on a microbial surface protein. 230. The method of claim 229, wherein the peptide or protein is expressed as a fusion protein with the microbial surface protein. 231. The method of claim 230, wherein the microbial surface protein is selected from invasin, intimin, and adhesin. 232. The method of any one of claims 170–231, wherein the genetically engineered microorganism further comprises an exogenous gene encoding a lysin that is capable of lysing the endocytotic vacuole. 233. The method of claim 232, wherein the lysin is listeriolysin O, or a mutant derivative thereof. 234. The method of claim 232 or 233, wherein the gene encoding the surface protein and/or the gene encoding the lysin is integrated in the genome of the genetically engineered microorganism. 235. The method of claim 234, wherein the gene encoding the surface protein, and the gene encoding the lysin are integrated at a single genomic site. 236. The method of claim 235, wherein the single genomic site is selected from an integration site of a bacteriophage and an integration site of a plasmid. Attorney Docket No. MMS.003WO 237. The method of claim 232 or 233, wherein the gene encoding the surface protein, and/or the gene encoding the lysin are inserted on a plasmid. 238. The method of any one of claims 170–237, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is integrated in a genome of the genetically engineered microorganism or inserted on a plasmid. 239. The method of claim 238, wherein the genes encoding the detection marker and the prodrug converting enzyme are integrated at the single genomic site. 240. The method of claim 238, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a plasmid. 241. The method of any one of claims 170–236, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a second plasmid. 242. The method of claim 241, wherein the microorganism is Escherichia coli Nissle 1917 or a derivative thereof and the plasmid and/or the second plasmid is selected from the plasmid pMUT1, the plasmid pMUT2, and a derivative thereof. 243. The method of claim 241 or 242, wherein the plasmid and/or the second plasmid comprises a selection mechanism. 244. The method of claim 243, wherein the selection mechanism is an antibiotic resistance marker selected from kanamycin resistance gene and tetracycline resistance gene. 245. The method of claim 243, wherein the selection mechanism is a marker causing complementation of a mutation in an essential gene and/or a nutritional marker. 246. The method of claim 245, wherein the essential gene and/or a nutritional marker is selected from dapA, dapD, murA, alr, dadX, murI, dapE, thyA and a combination of any two or more thereof. Attorney Docket No. MMS.003WO 247. The method of claim 246, wherein the essential gene and/or a nutritional marker is alr and/or dadX. 248. The method of any one of claims 170–247, wherein the genetically engineered microorganism has an inactivation of alr and dadX genes. 249. The method of claim 248, wherein the inactivation of alr and dadX genes is caused by a point mutation, a frameshift mutation or a nonsense mutation. 250. The method of claim 249, wherein the inactivation of alr and dadX genes is caused by a partial or complete deletion. 251. The method of any one of claims 248–250, wherein the plasmid and/or the second plasmid comprises an active alr and/or dadX gene complementing the inactivation of alr and dadX genes. 252. The method of any one of claims 170–251, wherein the disease is selected from a precancerous lesion, cancer, ulcerative colitis, Crohn’s disease, Barrett’s esophagus, irritable bowel syndrome and irritable bowel disease. 253. The method of claim 252, wherein the precancerous lesion comprises a polyp selected from sessile polyp, serrated polyp (e.g., hyperplastic polyps, sessile serrated adenomas/polyps, and traditional serrated adenoma), sessile serrated polyp, flat polyp, sub- pedunculated polyp, pedunculated polyp, and a combination thereof. 254. The method of claim 252, wherein the precancerous lesion comprises a biliary intraepithelial neoplasm (BilIN) selected from BilIN-1, BilIN-2, BilIN-3, and cholangiocarcinoma. 255. The method of claim 252, wherein the precancerous lesion comprises a pancreatic intraepithelial neoplasm (PanIN) selected from PanIN -1, PanIN -2, PanIN -3 and pancreatic ductal adenocarcinoma (PDAC). Attorney Docket No. MMS.003WO 256. The method of any one of claims 252–255, wherein the precancerous lesion is a diminutive polyp. 257. The method of any one of claims 252–255, wherein the precancerous lesion has a size of from about 0.05 mm to about 30 mm. 258. The method of claim 257, wherein the precancerous lesion has a size of less than about 0.1 mm, less than about 0.25 mm, less than about 0.5 mm, less than about 1 mm, less than about 2 mm, less than about 5 mm, less than about 8 mm, less than about 10 mm, less than about 15 mm, less than about 20 mm, less than about 25 mm, or less than about 30 mm. 259. The method of claim 252, wherein the cancer comprises a polyp, an adenoma, or a frank cancer. 260. The method of claim 252, wherein the cancer comprises Lynch syndrome cancer, familial adenomatous polyposis, hereditary non-polyposis colon cancer (HNPCC), pancreatic ductal adenocarcinoma (PDAC), cholangiocarcinoma, or a sporadic cancer. 261. A genetically engineered microorganism comprising an exogenous gene encoding a surface protein, wherein the surface protein specifically interacts with one or more cell membrane receptor(s) that are specifically exposed on the luminal side of epithelial cells of diseased tissue, and which is optionally epithelial tissue of the gastrointestinal tract, wherein the surface protein promotes binding and invasion of epithelial cells of diseased tissue, wherein the microorganism comprises a gene encoding a prodrug converting enzyme that is capable of converting a non-toxic prodrug to a pharmacologically active compound inside a diseased cell, the gene encoding a prodrug converting enzyme being operably linked to a promoter. 262. The genetically engineered microorganism of claim 261, wherein the prodrug converting enzyme is selected from: an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5- Attorney Docket No. MMS.003WO fluorouracil (5-FU), the enzyme optionally selected from a cytosine deaminase, an aldehyde oxidase, a cytochrome P450, a uracil phosphoribosyltransferase (yUPRT), and a combination thereof or a fusion protein comprising two or more thereof that; an enzyme capable of catalyzing conversion of pentyl N-[1-[(2R,3R,4S,5R)-3,4- dihydroxy-5-methyloxolan-2-yl]-5-fluoro-2-oxopyrimidin-4-yl]carbamate (capecitabine) into 5-FU, optionally the enzyme being a carboxylesterase; an enzyme capable of catalyzing conversion of 5-FU glucoronide into 5-FU, optionally the enzyme being a β-glucuronidase; an enzyme capable of catalyzing conversion of 6-methylpurine-deoxyriboside (MePdR) into 6-methylpurine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); an enzyme capable of catalyzing conversion of 9-beta-D-arabinofuranosyl-2- fluoroadenine monophosphate(fludarabine monophosphate) into fluoroadenine, optionally the enzyme being a purine nucleoside phosphorylase (PNP); and/or an enzyme capable of catalyzing conversion of 1-(2-deoxy-2-fluoro-b-D- arabinofuranosyl)uracil into 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) 5- methyluracil monophosphate (FMAUMP), optionally the enzyme being a thymidine kinase (TK) and/or a thymidylate synthase (TS). 263. The genetically engineered microorganism of claim 261 or 262, wherein the genetically engineered microorganism is non-pathogenic. 264. The genetically engineered microorganism of any one of claims 261–263, wherein the genetically engineered microorganism harbors at least one auxotrophic mutation. 265. The genetically engineered microorganism of any one of claims 261–264, wherein the prodrug converting enzyme is expressed from a mammalian promoter. 266. The genetically engineered microorganism of claim 265, wherein the mammalian promoter directs GI tract epithelial cell-specific expression. Attorney Docket No. MMS.003WO 267. The genetically engineered microorganism of claim 265 or 266, wherein a nucleic acid from the genetically engineered microorganism enters the nucleus of at least one diseased cell. 268. The genetically engineered microorganism of claim 267, wherein the nucleic acid is a plasmid molecule. 269. The genetically engineered microorganism of claim 268, wherein the plasmid molecule comprises a gene encoding the prodrug converting enzyme. 270. The genetically engineered microorganism of claim 268 or 269, wherein the plasmid molecule comprises at least one binding site for a DNA binding protein. 271. The genetically engineered microorganism of claim 270, wherein the at least one binding site for the DNA binding protein is selected from SV40 enhancer, a transcription factor binding site, and a bacterial operator. 272. The genetically engineered microorganism of claim 271, wherein the DNA binding protein comprises one or more nuclear localization signal(s) (NLS). 273. The genetically engineered microorganism of claim 272, wherein the NLS is SV40 T antigen NLS sequence (KKKRKV). 274. The genetically engineered microorganism of any one of claims 270–273, wherein the microorganism comprises a gene encoding the DNA binding protein. 275. The genetically engineered microorganism of claim 273 or 274, wherein the DNA binding protein is NFκB. 276. The genetically engineered microorganism of any one of claims 261–264, wherein the prodrug converting enzyme is expressed from a microbial promoter, and an mRNA encoding the prodrug converting enzyme is translated in the diseased cells. Attorney Docket No. MMS.003WO 277. The genetically engineered microorganism of claim 276, wherein the genetically engineered microorganism has a deletion of an RNase gene. 278. The genetically engineered microorganism of claim 277, wherein the RNase gene is RNase III (rnc). 279. The genetically engineered microorganism of any one of claims 276–278, wherein the gene encoding the prodrug converting enzyme is present in a cassette that directs RNA self-amplification of the cassette in the diseased cells. 280. The genetically engineered microorganism of claim 279, wherein the cassette comprises an RNA viral genome replication apparatus. 281. The genetically engineered microorganism of claim 280, wherein the RNA viral genome replication apparatus encodes an RNA-dependent RNA polymerase (RdRP). 282. The genetically engineered microorganism of claim 281, wherein the RdRP is an alphavirus RdRP comprising nsp1, nsp2, nsp3, and nsp4. 283. The genetically engineered microorganism of any one of claims 276–282, wherein the mRNA encoding the prodrug converting enzyme further comprises an internal ribosome entry site (IRES). 284. The genetically engineered microorganism of any one of claims 261–283, wherein the gene encoding the prodrug converting enzyme comprises at least one intron. 285. The genetically engineered microorganism of claim 284, wherein the at least one intron is a spliceosomal intron. 286. The genetically engineered microorganism of any one of claims 261–285, wherein the engineered microorganism further comprises a gene encoding a detection marker. Attorney Docket No. MMS.003WO 287. The genetically engineered microorganism of claim 286, wherein the detection marker is selected from a fluorescent protein, a bioluminescent protein, a contrast agent for magnetic resonance imaging (MRI), a Positron Emission Tomography (PET) reporter, an enzyme reporter, a contrast agent for use in computerized tomography (CT), a Single Photon Emission Computed Tomography (SPECT) reporter, a photoacoustic reporter, an X-ray reporter, an ultrasound reporter (e.g., a bacterial gas vesicle), and ion channel reporters (e.g., cAMP activated cation channel), and a combination of any two or more thereof. 288. The genetically engineered microorganism of claim 287, wherein the detection marker is a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713 (iRFP), iRFP720 and iSplit. 289. The genetically engineered microorganism of claim 286, wherein the detection marker is a secretable biomarker that is excreted in a biological fluid selected from mucus, saliva and urine. 290. The genetically engineered microorganism of claim 289, wherein the secretable biomarker is selected from alkaline phosphatase, a human chorionic gonadotropin, a human carcinoembryonic antigen (CEA), colon cancer secreted protein 2, cathepsin B, a Gaussia luciferase (Gluc), a Metridia luciferase (MLuc), a subunit thereof, a fragment thereof, and a combination of any two or more thereof. 291. The genetically engineered microorganism of any one of claims 261–290, wherein the microorganism is selected from Lactobacillus, Bifidobacterium, Saccharomyces, Enterococcus, Streptococcus, Pediococcus, Leuconostoc, Bacillus, or Escherichia coli. 292. The genetically engineered microorganism of claim 291, wherein the microorganism is Escherichia coli, and optionally Escherichia coli Nissle 1917 or a derivative thereof. 293. The genetically engineered microorganism of claim 292, wherein the Escherichia coli Nissle 1917 or the derivative thereof harbors a plasmid pMUT1 and/or a plasmid pMUT2, and/or derivatives thereof. Attorney Docket No. MMS.003WO 294. The genetically engineered microorganism of claim 292, wherein the Escherichia coli Nissle 1917 or the derivative thereof is cured of the plasmid pMUT1 and/or the plasmid pMUT2. 295. The genetically engineered microorganism of any one of claims 261–294, wherein the surface protein specifically interacts with one or more cell membrane receptor(s) that are not exposed on the luminal side of normal epithelial cells of GI tissue. 296. The genetically engineered microorganism of claim 295, wherein the surface protein is an invasin, or a fragment thereof. 297. The genetically engineered microorganism of claim 296, wherein the invasin is selected from Yersinia enterocolitica invasin, Yersinia pseudotuberculosis invasin, Salmonella enterica PagN, Candida albicans Als3, and E. coli intimin. 298. The genetically engineered microorganism of claim 297, wherein the surface protein comprises a peptide or protein that specifically binds to the surface of cancerous cells and/or pre-cancerous cells. 299. The genetically engineered microorganism of claim 298, wherein the peptide or protein is selected from a leptin, an antibody, or a fragment thereof. 300. The genetically engineered microorganism of claim 299, wherein the antibody, or a fragment thereof is selected from a single-domain antibody (sdAb) and an scFv fragment. 301. The genetically engineered microorganism of any one of claims 298–300, wherein the peptide or protein is displayed on a microbial surface protein. 302. The genetically engineered microorganism of claim 301, wherein the peptide or protein is expressed as a fusion protein with the microbial surface protein. Attorney Docket No. MMS.003WO 303. The genetically engineered microorganism of claim 302, wherein the microbial surface protein is selected from invasin, intimin and adhesin. 304. The genetically engineered microorganism of any one of claims 261–303, wherein the microorganism further comprises an exogenous gene encoding a lysin that is capable of lysing the endocytotic vacuole. 305. The genetically engineered microorganism of claim 304, wherein the lysin is listeriolysin O, or a mutant derivative thereof. 306. The genetically engineered microorganism of claim 304 or 305, wherein the gene encoding the surface protein and/or the gene encoding the lysin is integrated in the genome of the microorganism. 307. The genetically engineered microorganism of claim 306, wherein the gene encoding the surface protein, and the gene encoding the lysin are integrated at a single genomic site. 308. The genetically engineered microorganism of claim 307, wherein the single genomic site is selected from an integration site of a bacteriophage and an integration site of a plasmid. 309. The genetically engineered microorganism of claim 307 or 308, wherein the gene encoding the surface protein, and /or the gene encoding the lysin are inserted on a plasmid. 310. The genetically engineered microorganism of any one of claims 261–309, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is integrated in a genome of the microorganism or inserted on a plasmid. 311. The genetically engineered microorganism of claim 310, wherein the genes encoding the detection marker and the prodrug converting enzyme are integrated at the single genomic site. Attorney Docket No. MMS.003WO 312. The genetically engineered microorganism of claim 310, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a plasmid. 313. The genetically engineered microorganism of any one of claims 261–312, wherein the gene encoding the detection marker and/or the prodrug converting enzyme is inserted on a second plasmid. 314. The genetically engineered microorganism of claim 313, wherein the microorganism is Escherichia coli Nissle 1917 or a derivative thereof and the plasmid and/or the second plasmid is selected from the plasmid pMUT1, the plasmid pMUT2, and a derivative thereof. 315. The genetically engineered microorganism of claim 313 or 314, wherein the plasmid and/or the second plasmid comprises a selection mechanism. 316. The genetically engineered microorganism of claim 315, wherein the selection mechanism is an antibiotic resistance marker selected from kanamycin resistance gene and tetracycline resistance gene. 317. The genetically engineered microorganism of claim 315, wherein the selection mechanism is a marker causing complementation of a mutation in an essential gene and/or a nutritional marker. 318. The genetically engineered microorganism of claim 316, wherein the essential gene and/or a nutritional marker is selected from dapA, dapD, murA, alr, dadX, murI, dapE, thyA and a combination of any two or more thereof. 319. The genetically engineered microorganism of claim 318, wherein the essential gene and/or a nutritional marker is alr and/or dadX. 320. The genetically engineered microorganism of any one of claims 261–319, wherein the genetically engineered microorganism has an inactivation of alr and dadX genes. Attorney Docket No. MMS.003WO 321. The genetically engineered microorganism of claim 320, wherein the inactivation of alr and dadX genes is caused by a point mutation, a frameshift mutation or a nonsense mutation. 322. The genetically engineered microorganism of claim 320, wherein the inactivation of alr and dadX genes is caused by a partial or complete deletion. 323. The genetically engineered microorganism of any one of claims 318–322, wherein the plasmid and/or the second plasmid comprises an active alr and/or dadX gene complementing the inactivation of alr and dadX genes. 324. A pharmaceutical composition comprising the genetically engineered microorganism of any one of claims 96–169 or 261–323, and a pharmaceutically acceptable carrier. 325. A method for treating a subject comprising administering the genetically engineered microorganism of any one of claims 96–169 or 261–323 or the pharmaceutical composition of claim 324 to the subject.
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