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WO2024263805A1 - Micro-organismes génétiquement modifiés pour la détection de cellules malades - Google Patents

Micro-organismes génétiquement modifiés pour la détection de cellules malades Download PDF

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WO2024263805A1
WO2024263805A1 PCT/US2024/034854 US2024034854W WO2024263805A1 WO 2024263805 A1 WO2024263805 A1 WO 2024263805A1 US 2024034854 W US2024034854 W US 2024034854W WO 2024263805 A1 WO2024263805 A1 WO 2024263805A1
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genetically engineered
engineered microorganism
protein
gene
rna
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Jeffrey WAGNER
Cameron HABIB
Barry Polisky
Sydney Leigh SOLOMON
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Microbial Machines Inc
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Microbial Machines Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/34—Vector systems having a special element relevant for transcription being a transcription initiation element
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the present application contains an electronic Sequence Listing in XML file format named “MMS_004WO_SL.xml,” created on June 20, 2024, and having a size of 93.4 kilobytes, the contents of which are incorporated by reference herein in their entirety.
  • TECHNICAL FIELD The present technology generally relates to genetically engineered microorganisms, and in particular, to the use of genetically engineered microorganisms for detection of diseased cells.
  • GI gastrointestinal
  • 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
  • precancerous syndromes precancerous syndromes
  • 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.
  • CT computed tomography
  • focal, irregular, or asymmetrical gastrointestinal wall thickening on a CT scan can indicate a malignancy.
  • Segmental or diffuse gastrointestinal wall Fortem Ref. No. MMS.004WO thickening can indicate an ischemic, inflammatory, or infectious disease.
  • endoscopy, colonoscopy, etc. are invasive procedures and uncomfortable for the patient, particularly for patients that are at high risk for cancers of the GI tract, who may need these procedures more frequently.
  • FIG. 1 is a schematic illustration of a DNA construct encoding gene products having multiple forms, in accordance with embodiments of the present technology.
  • FIGS.2A–2D are schematic illustrations of a method for detecting diseased GI tissue using genetically engineered microorganisms, in accordance with embodiments of the present technology.
  • FIGS.3A–3C are schematic illustrations of a method for detecting diseased GI tissue using genetically engineered microorganisms, in accordance with embodiments of the present technology.
  • FIG. 4 is a schematic illustration of a strain for DNA transfer to mammalian cells, in accordance with embodiments of the present technology.
  • E. coli Nissle 1917 were engineered to have the invasin and listeriolysin O genes integrated on the bacterial chromosome under a bacterial promoter, and an episome containing the fluorescent protein iRFP670 under a mammalian promoter.
  • FIG.5A is a graph illustrating flow cytometry analysis of iRFP670 expression demonstrating DNA transfer to HEK293T cells by the E.
  • FIGS. 5B and 5C are microscopy images of HEK293T cells treated with a negative control strain (FIG. 5B) and the E. coli Nissle 1917 strain (FIG. 5C). iRFP670 expression was observed only in cells treated with the E. coli Nissle 1917 strain.
  • FIG. 6 is a schematic illustration of a strain for DNA transfer to mammalian cells. E. coli Nissle 1917 were engineered to have the invasin and listeriolysin O genes integrated on the bacterial chromosome under a bacterial promoter, and an episome containing the Renilla luciferase protein split by an intron under a mammalian promoter. Fortem Ref.
  • FIG. 7 is a graph illustrating luciferase expression in bacteria having the split Renilla luciferase gene (“RLuc-Int”) versus the wild type Renilla luciferase gene (“Rluc”), under either a bacterial promoter (“Bact”) or a mammalian promoter (“Mam”). Strains containing the Rluc-Int gene were unable to express the functional protein. The strain containing the wild type Rluc gene under the mammalian promoter showed some expression of the functional protein, suggesting leaky activity of the mammalian promoter in bacteria.
  • FIG.8A is a graph illustrating Renilla luciferase expression levels in HEK293T cells treated with various bacterial strains.
  • FIG.8B is a graph illustrating Renilla luciferase expression levels in HEK293T cells treated with bacterial strains at various multiplicities of infection.
  • FIG.8C is a graph illustrating Renilla luciferase expression levels in HEK293T cells over time.
  • FIGS. 9A–9C are schematic illustrations of primers designed to differentiate between spliced Renilla luciferase RNA without an intron (mammalian origin) versus unspliced Renilla luciferase RNA still containing the intron (bacterial origin).
  • FIG. 9A–9C are schematic illustrations of primers designed to differentiate between spliced Renilla luciferase RNA without an intron (mammalian origin) versus unspliced Renilla luciferase RNA still containing the intron (bacterial origin).
  • FIG. 9A–9C are schematic illustrations of primers designed to differentiate between spliced Renilla
  • FIG. 10A is a schematic illustration of different primers together with the Renilla luciferase gene split with an intron.
  • FIG. 10B shows the relative DNA levels obtained with different primer sets when tested on plasmids containing the Renilla luciferase gene split with an intron versus plasmids containing the Renilla luciferase gene without the intron.
  • FIG. 11A is a graph illustrating expression of Renilla luciferase protein in HCT116 cells (expressing or lacking ⁇ 1 integrin) that were treated with the E. coli Nissle 1917 Rluc-Int strain. Protein expression was dependent on the presence of ⁇ 1 integrin.
  • FIG.11B is a graph illustrating expression of spliced Renilla luciferase RNA in HCT116 cells (expressing or lacking ⁇ 1 integrin) that were treated with the E. coli Nissle 1917 Rluc-Int strain. RNA expression was dependent on the presence of ⁇ 1 integrin.
  • FIG. 12A is a graph illustrating expression of Renilla luciferase protein in HEK293T cells that were treated with the E. coli Nissle 1917 Rluc-Int strain (expressing or lacking invasin). Protein expression was dependent on the presence of invasin. Fortem Ref. No.
  • FIG.12B is a graph illustrating expression of spliced Renilla luciferase RNA in HEK293T cells that were treated with the E. coli Nissle 1917 Rluc-Int strain (expressing or lacking invasin). RNA expression was dependent on the presence of invasin.
  • FIG. 13A is a graph illustrating expression of Renilla luciferase protein in HEK293T cells that were treated with the E. coli Nissle 1917 Rluc-Int strain (expressing or lacking LLO). Protein expression was dependent on the presence of LLO.
  • FIG.13B is a graph illustrating expression of spliced Renilla luciferase RNA in HEK293T cells that were treated with the E. coli Nissle 1917 Rluc-Int strain (expressing or lacking LLO). RNA expression was dependent on the presence of LLO.
  • FIG. 14A is a schematic illustration of a study protocol for DNA-based detection of diseased cells.
  • FIGS. 14B and 14C illustrate detection of a DNA payload in fecal samples in mice with and without induced tumors.
  • FIG.15 schematically illustrates a non-inducible bacterial lysis system (top) and an inducible bacterial lysis system (bottom).
  • FIG. 16 is a graph illustrating the efficacy of in vitro transfer of a Renilla luciferase payload plasmid to mammalian cells using different bacterial strains.
  • FIG. 17 is a graph illustrating the in vitro invasion rate of different bacterial strains into target cells.
  • FIG.18 is a graph showing pH-inducible gene expression over time in bacteria including an mNeonGreen fluorescent marker under the control of a pASR acid-sensitive promoter.
  • FIG. 19A schematically illustrates bacterial strains for assessing pH-induced expression of listeriolysin O in vitro.
  • FIG. 19B is an image showing a Western blot to evaluate listeriolysin O expression in the bacterial strains of FIG.19A under different pH conditions.
  • FIG. 19A schematically illustrates bacterial strains for assessing pH-induced expression of listeriolysin O in vitro.
  • FIG. 19B is an image showing a Western blot to evaluate listeriolysin O expression in
  • FIG. 20 schematically illustrates a bacterial strain for assessing endosomal- induced expression of a mNeonGreen fluorescent marker.
  • FIG. 21A is a series of flow cytometry plots illustrating mNeonGreen expression over time after co-culture of engineered bacteria with HEK293T cells. Fortem Ref. No. MMS.004WO
  • FIG.21B is a graph showing mNeonGreen expression over time after co-culture of engineered bacteria with HEK293T cells.
  • FIG.22 schematically illustrates a bacterial strain including an inducible lysis system.
  • FIG.23 is a graph showing in vitro survival of engineered bacteria including an inducible lysis system based on LytE under various pH conditions.
  • FIG.24 is a graph showing in vitro survival of engineered bacteria including an inducible lysis system based on LytE or CPP-Lysozyme under various conditions.
  • FIG. 25 is a graph illustrating the efficacy of in vitro transfer of a Renilla luciferase payload plasmid to mammalian cells using bacteria including inducible or non- inducible lysis systems.
  • FIG. 26 is a graph illustrating the efficacy of in vitro transfer of a Renilla luciferase payload plasmid to mammalian cells using bacteria including a CPP-Lysozyme inducible lysis system.
  • FIG. 25 is a graph illustrating the efficacy of in vitro transfer of a Renilla luciferase payload plasmid to mammalian cells using bacteria including a CPP-Lysozyme inducible lysis system.
  • FIG. 27 is a series of graphs illustrating the efficacy of in vitro transfer of a Renilla luciferase payload plasmid to mammalian cells using bacteria including inducible or non-inducible lysis systems.
  • FIG. 28 schematically illustrates two different positions for an intron in a Renilla luciferase payload gene.
  • FIG.29 is a graph illustrating expression of a Renilla luciferase payload gene with different introns resulting from Lipofectamine transfection of HEK239T or HCT116 cells.
  • FIG.30 is a graph illustrating expression levels of a Renilla luciferase payload gene with different introns resulting from payload transfer from engineered bacteria to HEK293T cells.
  • FIG.31 is a graph illustrating expression levels of a Renilla luciferase payload gene with different introns resulting from payload transfer from engineered bacteria to HCT116 cells.
  • FIG.32 is a series of graphs illustrating invasion rate (left graph) and payload transfer efficiency (right graph) of engineered bacteria.
  • Fortem Ref. No. MMS.004WO DETAILED DESCRIPTION The present technology relates to genetically engineered microorganisms and associated compositions and methods of use.
  • a method for detecting a target cell in a subject is provided. The method can include administering a genetically engineered microorganism to the subject.
  • the genetically engineered microorganism can deliver a payload gene specifically to the target cell (e.g., a diseased and/or abnormal mammalian cell).
  • the payload gene can encode at least one gene product (e.g., an RNA molecule, protein, or peptide) that has a first form when expressed by the target cell and a second, different form when expressed by the genetically engineered microorganism.
  • the payload gene can include one or more introns that can be spliced out by the target cell via spliceosomal machinery present in eukaryotic cells, but not by the genetically engineered microorganism.
  • the first form of the at least one gene product can be a spliced RNA and/or a protein or peptide produced from the spliced RNA
  • the second form of the at least one gene product can be an unspliced RNA and/or a protein or peptide produced from the unspliced RNA.
  • the method further includes collecting a biological sample from the subject (e.g., a fecal sample), and detecting whether the first form of the at least one gene product is present in the biological sample.
  • GI gastrointestinal
  • 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.
  • the present disclosure addresses these and other challenges by providing genetically engineered microorganisms that allow for detection of diseased cells in a minimally invasive or noninvasive manner.
  • the genetically engineered microorganisms described herein can detect the onset of disease (e.g., precancerous lesions) at an earlier stage compared to conventional techniques, which can be advantageous for reducing or preventing disease morbidity and mortality.
  • the RNA-based detection techniques described Fortem Ref. No. MMS.004WO herein can also provide improved sensitivity compared to conventional imaging techniques and protein-based assays.
  • the inclusion of one or more introns in the payload gene allows for differentiation between gene products resulting from expression of the payload gene in the target cell versus gene products resulting from background expression in the genetically engineered microorganism, which can further improve diagnostic accuracy.
  • 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)
  • mucosa if the tissue is intraperitoneal
  • muscular layer if the tissue is muscular layer
  • serosa if the tissue is intraperitoneal
  • 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 Fortem Ref. No. MMS.004WO 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.
  • 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.
  • 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. Fortem Ref. No.
  • 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 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 a
  • 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 adenoma 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.
  • FAP familial adenomatous polyposis
  • AFAP attenuated FAP
  • MMS.004WO polyposis which is caused by pathogenic variants in the MUTYH gene
  • 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.
  • 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.
  • Patients with MUTYH-associated polyposis (MAP) develop adenomatous polyposis of the colorectum and have an 80% risk of CRC.
  • 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.
  • BilINs 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 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).
  • IPMNs intraductal papillary mucinous neoplasms of the pancreas
  • MNs 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
  • MMS.004WO includes Crohn’s disease, ulcerative colitis (UC), and indeterminate colitis.
  • CACC Colitis-associated colorectal cancer
  • 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.
  • Crohn’s disease is a progressive disease that starts with mild symptoms and gradually gets worse. Early diagnosis is important to help prevent bowel damage such as fistulae, abscesses, or strictures.
  • the genetically engineered microorganisms disclosed herein are used to detect and/or treat irritable bowel syndrome.
  • IBS Irritable bowel syndrome
  • 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 Fortem Ref. No. MMS.004WO Barrett’s esophagus is unknown, but gastroesophageal reflux disease (GERD) increases the risk of developing Barrett’s esophagus. II.
  • 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), an endosomal lysis gene that encodes an endosomal 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), a bacterial lysis gene that encodes a bacterial lysis element that lyses the microorganism to release the payload (e.g., as described in Section II.C below), an auxotrophic mutation (e.g., as described in Section II.D below), a selection mechanism (e.g., as described in Section II.E below), and/or a payload gene encoding a gene product to be delivered to the target cell (e.g., as described in Section III below).
  • a targeting gene that encodes a targeting
  • 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 Fortem Ref. No. MMS.004WO 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).
  • the genetically 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.
  • 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.
  • 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 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 Fortem Ref. No.
  • MMS.004WO 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
  • invasin listeriolysin O
  • a mutant derivative thereof e.g., listeriolysin O
  • the complete genome sequence of Escherichia coli Nissle 1917 is known.
  • the targeting gene e.g., a gene encoding a surface protein such as invasin
  • an endosomal 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. Additionally or alternatively, in some embodiments, a bacterial lysis gene is integrated at the first genomic site, the second genomic site, or a third genomic site of the Escherichia coli Nissle 1917 derivative. In some embodiments, the target gene, the endosomal lysis gene, and/or the bacterial lysis gene are integrated at a single genomic site, optionally, the single genomic site can be an integration site of a bacteriophage.
  • the bacteriophage is bacteriophage Lambda.
  • the targeting gene endosomal lysis gene, and/or the bacterial 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 mechanism (e.g., an auxotrophic marker such as alr as described).
  • the targeting gene is inserted on a plasmid.
  • the endosomal lysis gene is inserted on the plasmid. In some embodiments, the bacterial 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). Fortem Ref.
  • 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 mimic the affinity of the native surface protein.
  • 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 Fortem Ref. No. MMS.004WO 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, 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 Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO
  • 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 produces a targeting element of SEQ Fortem Ref. No. MMS.004WO 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 produces 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 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., endosomal lysis gene(s), bacterial lysis gene(s), payload gene(s), and/or selection mechanism(s)).
  • the targeting gene(s) are integrated at a first genomic site
  • the endosomal lysis 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 targeting gene(s), endosomal lysis gene(s), bacterial lysis gene(s), payload gene(s), and selection mechanism(s) can all be 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 or endosome) of the target cell.
  • the genetically engineered microorganism can include at least one endosomal lysis gene encoding an endosomal 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 endosomal 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). Representative examples of endosomal lysis elements are provided in Table 2 below. Table 2: Endosomal Lysis Elements Fortem Ref. No.
  • the genetically engineered microorganism produces an endosomal 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 produces an endosomal lysis element of any one of SEQ ID NO: 3 or SEQ ID NO: 4.
  • the genetically engineered microorganism includes a single endosomal lysis gene encoding a single endosomal lysis element.
  • the genetically engineered microorganism can include multiple endosomal lysis genes expressing multiple endosomal lysis elements, such as a first endosomal lysis gene encoding a first lysin and a second endosomal lysis gene encoding a second, different lysin.
  • the multiple endosomal lysis elements can be expressed as multiple separate proteins, or can be expressed as a single fusion protein.
  • the endosomal 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 endosomal 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 endosomal 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.
  • Endosomal lysis gene(s) can occur from a plasmid (e.g., an episome) or from a genomic site (e.g., a chromosome).
  • the endosomal lysis gene(s) can be incorporated at the same site or different site as the other genetic elements described herein Fortem Ref. No. MMS.004WO (e.g., targeting gene(s), bacterial lysis gene(s), payload gene(s), and/or selection mechanism(s)).
  • the endosomal 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 bacterial lysis gene(s), payload gene(s), and selection mechanism(s) are inserted in a plasmid.
  • the endosomal lysis gene(s), targeting gene(s), bacterial lysis gene(s), payload gene(s), and selection mechanism(s) can all be inserted in the same plasmid.
  • the endosomal lysis gene(s) are constitutively expressed.
  • the endosomal lysis gene(s) can be conditionally expressed.
  • expression of the endosomal 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.
  • the lysis gene(s) are operably linked to a pH-sensitive promoter, such as a pASR acid-sensitive promoter (SEQ ID NO: 5).
  • the pASR promoter represses expression at pH 7 and induces gene expression at approximately pH 5.
  • the endosomal lysis gene(s) may be advantageous, for example, in embodiments where the endosomal lysis element(s) is toxic to the genetically engineered microorganism and may cause the microorganism to die prematurely (e.g., before internalization by the target cell).
  • Representative examples of promoters responsive to endosomal conditions are provided in Table 3 below.
  • the genetically engineered microorganism includes a promoter 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: 5.
  • the genetically engineered microorganism includes a promoter of SEQ ID NO: 5. Fortem Ref. No. MMS.004WO C. Bacterial Lysis Elements
  • the genetically engineered microorganism is lysed in order to release its payload. Accordingly, the genetically engineered microorganism can include at least one bacterial lysis gene encoding a bacterial lysis element that, when expressed, lyses the genetically engineered microorganism to allow the payload to escape (e.g., into the cytoplasmic space of the target cell or into the extracellular space near the target cell).
  • the bacterial lysis element is or includes a protein that interferes with bacterial cell wall synthesis, e.g., by blocking one or more steps in peptidoglycan biosynthesis, thereby promoting bacterial lysis.
  • the bacterial lysis element is or includes a protein that breaks down one or more components of the bacterial cell wall (e.g., a lysin), thereby promoting bacterial lysis.
  • the bacterial lysis element is or includes a protein that facilitates the lytic activity of a lysin, e.g., such as a holin that creates holes in the bacterial inner cell membrane to allow the lysin to reach the cell wall, or a spanin that aids in degrading the bacterial outer cell membrane after degradation of the cell wall by the lysin.
  • the bacterial lysis element is a phage-derived protein.
  • bacteriophage ⁇ X174 Protein E (LytE) is involved in bacteriophage escape and functions by blocking MraY, which catalyzes the final step in peptidoglycan precursor synthesis.
  • the LysM protein of Levivirus phage M blocks lipid II transport activity mediated by MurJ, which is essential for peptidoglycan synthesis.
  • phage-derived proteins include phage-derived lysins, such as phage-derived lysozymes (e.g., T4 phage Protein E, lambda phage Protein R); and additional phage-derived proteins which aid in the lysis process and/or may have some lytic activity on their own, such as phage-derived holins (e.g., T4 phage Protein T) and phage-derived spanins (e.g., lambda phage Protein Rz, lambda phage Protein Rz).
  • phage-derived lysins such as phage-derived lysozymes (e.g., T4 phage Protein E, lambda phage Protein R)
  • additional phage-derived proteins which aid in the lysis process and/or may have some lytic activity
  • the bacterial lysis element is a non-phage derived protein, such as a eukaryotic lysin (e.g., a eukaryotic lysozyme such as hen egg white lysozyme), a bacterial lysin, etc.
  • the bacterial lysin is a bacteria-derived peptidoglycan hydrolase, such as an E. coli peptidoglycan hydrolase.
  • coli peptidoglycan hydrolase is a peptidoglycan glycosidase, such as N- acetylglucosaminidase or a lytic transglycosylase (e.g., Slt70, MltA, MltB, MltC, MltD, MltE, MltF).
  • the peptidoglycan hydrolyase is a peptidoglycan peptidase, such as a DD-carboxypeptidase (e.g., PBP4b, PBP5, PBP6, PBP6b), a DD-endopeptidase (e.g., Fortem Ref. No.
  • a LD-carboxypeptidase
  • a bacterial lysis element may include or may be modified to include a signal sequence that directs the bacterial lysis element out of the cytoplasm and into the periplasm, e.g., to break down the bacterial cell wall.
  • the signal sequence is a secretion signal, such as a periplasmic secretion signal.
  • the signal sequence is a cell-penetrating peptide sequence that is capable of traversing the bacterial inner cell membrane.
  • multiple bacterial lysis elements may be co-expressed to improve lysis efficiency.
  • a lysin e.g., lysozyme
  • a holin that makes holes in the bacterial inner cell membrane to allow the lysin to reach the cell wall and/or with a spanin that aids in degrading the bacterial outer cell membrane after degradation of the cell wall.
  • two, three, four, five, or more different bacterial lysis elements are co-expressed, e.g., as a single fusion protein or as separate proteins. Representative examples of bacterial lysis elements are provided in Table 4 below. Table 4: Bacterial Lysis Elements Fortem Ref. No. MMS.004WO Fortem Ref. No.
  • the genetically engineered microorganism produces a bacterial 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: 6 – SEQ ID NO: 18.
  • the genetically engineered microorganism produces a bacterial lysis element of any one of SEQ ID NO: 6 – SEQ ID NO: 18.
  • the genetically engineered microorganism includes a single bacterial lysis gene encoding a single bacterial lysis element.
  • the genetically engineered microorganism can include multiple bacterial lysis genes expressing multiple bacterial lysis elements, such as a first bacterial lysis gene encoding a first bacterial lysis element and a second bacterial lysis gene encoding a second, different bacterial lysis element.
  • the multiple bacterial lysis elements can be expressed as multiple separate proteins, or can be expressed as a single fusion protein.
  • the bacterial 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 bacterial 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 bacterial 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.
  • bacterial lysis gene(s) can occur from a plasmid (e.g., an episome) or from a genomic site (e.g., a chromosome).
  • the bacterial 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), endosomal lysis gene(s), payload gene(s), and/or selection Fortem Ref. No. MMS.004WO mechanism(s)).
  • the targeting gene(s) are integrated at a first genomic site, the endosomal lysis gene(s) are integrated at the first genomic site or a second, different genomic site, and the bacterial lysis gene(s), payload gene(s), and selection mechanism(s) are inserted in a plasmid.
  • the targeting gene(s), endosomal lysis gene(s), bacterial lysis gene(s), payload gene(s), and selection mechanism(s) can all be inserted in the same plasmid.
  • the bacterial lysis gene(s) are conditionally expressed, e.g., to avoid premature lysis of the genetically engineered microorganism before internalization by the target cell.
  • expression of the bacterial lysis gene(s) can be induced after endocytosis, e.g., using an inducible promoter that responds to changes in the environment indicative of endosomal localization, such as pH, temperature, and/or addition of a small molecule.
  • the bacterial lysis gene(s) are operably linked to a pH-sensitive promoter, such as a pASR acid-sensitive promoter (SEQ ID NO: 5).
  • the bacterial lysis gene(s) are operably linked to a promoter (e.g., T7 promoter), and the genetically engineered microorganism further includes a sequence element (e.g., T7 RNA polymerase gene) that is operably linked to a pH-sensitive promoter (e.g., a pASR promoter).
  • a promoter e.g., T7 promoter
  • the genetically engineered microorganism further includes a sequence element (e.g., T7 RNA polymerase gene) that is operably linked to a pH-sensitive promoter (e.g., a pASR promoter).
  • the pH-sensitive promoter may also be operably linked to one or more other genes, such as to the endosomal lysis gene(s).
  • the genetically engineered microorganism can include a genomic site including a pH-sensitive promoter operably linked to the endosomal lysis gene(s) and to the T7 RNA polymerase gene, and a plasmid including a T7 promoter operably linked to the bacterial lysis gene(s).
  • a genomic site including a pH-sensitive promoter operably linked to the endosomal lysis gene(s) and to the T7 RNA polymerase gene
  • a plasmid including a T7 promoter operably linked to the bacterial lysis gene(s).
  • Representative examples of polynucleotide sequences encoding a bacterial lysis gene under the control of a T7 promoter are provided in Table 5 below. Table 5: Bacterial Lysis Genes with T7 Promoter Fortem Ref. 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 any one of SEQ ID NO: 19 or SEQ ID NO: 20. In some embodiments, the genetically engineered microorganism includes a sequence of SEQ ID NO: 19 or SEQ ID NO: 20.
  • the genetically engineered microorganism may not express a bacterial lysis element, e.g., if the payload is capable of escaping the genetically engineered microorganism or can otherwise exert its function without lysis of the genetically engineered microorganism, or if the genetically engineered microorganism includes another mechanism for bacterial lysis (e.g., an auxotrophic mutation as described in Section I.D below).
  • 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 Fortem Ref. No.
  • 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 microorganism can include multiple auxotrophic mutations (e.g., a partial or complete deletion of multiple genes).
  • 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 genetically engineered microorganism can be used alternatively or in addition to a bacterial lysis element, as discussed in Section II.C above.
  • 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 nutritional 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), endosomal lysis gene(s), bacterial 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 6 below. Table 6: Selection Markers Fortem Ref. 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: 21 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: 21 or the coding region thereof.
  • 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), endosomal lysis gene(s), and/or bacterial 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, the genetically engineered microorganism can include multiple selection mechanisms.
  • selection mechanisms 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.D 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 R1, parDE 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 Fortem Ref. No.
  • 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: 21 or the coding region thereof.
  • the genetically engineered microorganism includes a selection marker that is encoded by the sequence of SEQ ID NO: 21 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), endosomal lysis gene(s), bacterial lysis gene(s), payload gene(s), and/or selection mechanism(s)).
  • the targeting gene(s) and/or endosomal lysis gene(s) are inserted in a first plasmid
  • the bacterial lysis gene(s) and/or payload gene(s) are inserted in a second plasmid
  • the selection mechanism is inserted in both the first and second plasmids.
  • the endosomal lysis gene(s), bacterial lysis gene(s), targeting gene(s), payload gene(s), and selection mechanism(s) can all be inserted in the same plasmid.
  • the targeting gene(s) and/or endosomal lysis gene(s) can be integrated at a genomic site, and the bacterial lysis gene(s), payload gene(s), and the selection mechanism is inserted in a plasmid.
  • 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 diagnose and/or treat a disease, such as any of the diseases described herein.
  • the payload can facilitate detection and/or treatment 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.
  • precancerous lesions e.g., adenomas
  • GI tract cancers e.g., CRC
  • IBD e.g., UC, Crohn’s disease
  • IBS or Barrett’s esophagus.
  • the payload delivered to the target cell by the genetically engineered microorganism is a nucleic acid molecule that includes one or more payload genes, such as a DNA molecule (e.g., plasmid DNA) or an RNA molecule.
  • the payload gene(s) can encode at least one gene product (e.g., a DNA molecule, RNA molecule, protein, and/or peptide) that, when expressed by the target cell, serves as a detection marker for diagnosing the subject with a disease and/or a therapeutic agent for treating the disease.
  • the payload gene(s) can also include one or more introns, such that the gene product(s) have a first form when expressed by the target cell (e.g., diseased and/or abnormal cells), and a second, different form when expressed by a non-target cell (e.g., the genetically engineered microorganism and/or healthy cells).
  • the first form can be a spliced RNA molecule, or a protein or peptide produced from the spliced RNA molecule
  • the second form can be an unspliced RNA molecule, or a protein or peptide produced from the unspliced RNA molecule.
  • the first form can be a first splice variant of an RNA molecule, or a protein or peptide produced from the first splice variant
  • the second form can be a second, different splice variant of the RNA molecule (e.g., a variant produced by alternative splicing of the same RNA molecule), or a protein or peptide produced from the second splice variant.
  • the two forms can allow for differentiation between gene products that are expressed specifically due to the presence of diseased and/or abnormal cells in the subject versus background expression of the gene products that is unrelated to the presence of diseased and/or abnormal cells (e.g., due to leaky expression, background activity of mammalian promoters in the microorganism, non-specific binding of the microorganism to non-target cells).
  • the methods herein can include diagnosing the subject with a disease based on whether the first form of the gene product is present or absent in a biological sample from the subject; and/or treating the disease with the first form of the gene product.
  • the first form can be a functional form of the functional molecule and the second form can be a nonfunctional form of the functional molecule.
  • the methods herein can include diagnosing the subject with a disease, based on whether the functional molecule is present in a functional or nonfunctional form; and/or treating the disease using the functional form of the functional molecule.
  • FIG. 1 is a schematic illustration of a DNA construct encoding gene products having multiple forms, in accordance with embodiments of the present technology. The DNA Fortem Ref. No.
  • MMS.004WO construct can be part of a DNA molecule (e.g., a plasmid) carried by a genetically engineered microorganism of the present disclosure.
  • the DNA construct can include at least one payload gene under the control of a promoter (e.g., a mammalian promoter).
  • the payload gene can encode a detection marker, a therapeutic agent, or a combination thereof.
  • the payload gene can also include one or more introns (e.g., spliceosomal introns). The intron(s) can be located between the 5’ end of the payload gene and the 3’ end of the payload gene.
  • the RNA and/or protein produced when the payload gene is expressed by the genetically engineered microorganism can differ from the RNA and/or protein produced when the payload gene is expressed by a target cell (e.g., a diseased and/or abnormal mammalian cell).
  • a target cell e.g., a diseased and/or abnormal mammalian cell.
  • the genetically engineered microorganism can be a prokaryotic cell lacking spliceosomal machinery, such that the RNA produced by the microorganism is unspliced and still contains the intron(s).
  • the intron can create a frameshift or premature stop codon in the unspliced mRNA, such that the protein produced by translation of the unspliced mRNA is an inactive (e.g., truncated) form of the protein.
  • the RNA is a catalytic RNA (e.g., a ribozyme)
  • the unspliced RNA can be an inactive form of the catalytic RNA.
  • the target cell can be a eukaryotic cell having spliceosomal machinery, such that the RNA produced by the target cell is spliced to remove some or all the introns.
  • the protein produced by translation of the spliced mRNA can be the functional (e.g., full-length) form of the protein.
  • the spliced RNA can be a functional form of the catalytic RNA. Because the spliced RNA and/or the functional protein are produced exclusively by the target cell, the spliced RNA and/or functional protein can thus serve as a highly specific detection marker for diagnosing the subject with the disease.
  • FIGS.2A–2D are schematic illustrations of a method for detecting diseased GI tissue using genetically engineered microorganisms, in accordance with embodiments of the present technology.
  • certain types of GI diseases e.g., GI cancer and/or precancerous syndromes
  • certain types of cell membrane receptors are Fortem Ref. No. MMS.004WO present only on the basolateral surfaces of healthy GI epithelial cells, and are not present on the apical surfaces facing the GI tract lumen.
  • integrins such as ⁇ 1 integrins
  • diseased GI epithelial cells at 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. Referring next to FIG.
  • the genetically engineered microorganisms of the present disclosure 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.
  • 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). As shown in FIG. 2C, 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 expression of one or more bacterial lysis genes encoding one or more bacterial lysis element that disrupts cell wall synthesis and/or degrade cell wall components, and/or due to a nutritional auxotrophic mutation (e.g., the dapA attenuation mutation) that causes a defect in cell wall synthesis.
  • the genetically engineered microorganism can also include one or more endosomal lysis genes encoding one or more endosomal lysis elements (e.g., Listeriolysin O), as described herein.
  • MMS.004WO lysis element(s) can be released upon lysis of the microorganism and/or can be naturally exported by the microorganism.
  • the endosomal 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 construct) 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.
  • the DNA can be transcribed to RNA, and the RNA can optionally be translated to produce protein expression.
  • the protein can remain within the intracellular space of the diseased cell, can be transported and incorporated into the cellular membrane, and/or can be secreted.
  • the DNA includes one or more introns that are spliced out by spliceosomal machinery of the diseased cell during the transcription process. Accordingly, the RNA and/or protein expressed by the diseased cell can have a different form than RNA and/or protein expressed by the genetically engineered microorganism, as described elsewhere herein. Referring next to FIG.
  • a fecal sample can be collected from the subject.
  • the fecal sample can include food waste, genetically engineered microorganisms that were previously administered to the subject, and/or healthy and/or diseased cells that were shed into the lumen of the GI tract.
  • At least some of the diseased cells in the fecal sample can contain gene products (e.g., RNA and/or proteins) expressed from the DNA payload transferred to the diseased cells by the genetically engineered microorganisms. Accordingly, the presence of gene products in the fecal sample can serve as detection markers for the diseased cells and thus can be used to diagnose the subject with the disease.
  • the testing process involves extracting gene products from the fecal sample, then differentiating between gene products that are in a first form indicative of expression by eukaryotic cells (e.g., spliced RNA, full-length and/or functional protein) and gene products that are in a second form indicative of expression by prokaryotic cells (e.g., unspliced RNA, truncated and/or inactive protein).
  • eukaryotic cells e.g., spliced RNA, full-length and/or functional protein
  • prokaryotic cells e.g., unspliced RNA, truncated and/or inactive protein
  • genetically engineered microorganisms that include an auxotrophic mutation do not survive long in the gut lumen after administration to the subject and will lyse, thus releasing their nucleic acid payload (e.g., DNA molecules such as plasmid Fortem Ref. No. MMS.004WO DNA) into the gut lumen.
  • the released nucleic acid payload can be rapidly degraded and/or excreted (e.g., within 48 hours, 24 hours, or 12 hours after administration of the genetically engineered microorganism to the subject).
  • a nucleic acid payload that was delivered to a target cell (e.g., a diseased and/or abnormal cell) by the genetically engineered microorganism may persist in the subject’s body for longer time frames (e.g., for at least 12 hours, 24 hours, or 48 hours).
  • a biological sample e.g., fecal sample collected after a sufficient delay can include little or no nucleic acid material contained in the genetically engineered microorganism, and most or all of the nucleic acid material within the biological sample is persisting in cells of the subject.
  • FIGS.3A–3C are schematic illustrations of a method for detecting diseased GI tissue using genetically engineered microorganisms, in accordance with embodiments of the present technology. Referring first to FIG.
  • a fecal sample collected from the subject is expected to include large numbers of live uninternalized genetically engineered microorganisms, as well as healthy and/or diseased cells that were shed into the lumen of the GI tract.
  • analysis of fecal samples at the first time point may include a high background signal from the live uninternalized microorganisms, particularly if the analysis is based on the detection of plasmid DNA.
  • genetically engineered microorganisms that include an auxotrophic mutation and that have not been internalized by cells fecal sample collected from the subject may lyse within the GI tract lumen, thus releasing their plasmid payload into the GI tract. Free plasmid may be degraded or excreted rapidly.
  • a fecal sample collected at the second time point is expected to include smaller numbers of live uninternalized genetically engineered microorganism, a small amount of free plasmid, as well as healthy and/or diseased cells that were shed into the lumen of the GI tract.
  • analysis of fecal samples at the second time point may include a lower background signal from the live uninternalized microorganisms and free plasmid.
  • a fecal sample collected from the subject is expected to include mostly healthy and/or diseased Fortem Ref. No. MMS.004WO cells that were shed into the lumen of the GI tract.
  • any plasmid DNA and/or gene products expressed from the plasmid DNA are likely to originate from genetically engineered microorganisms that were internalized by diseased cells, rather than from uninternalized microorganisms.
  • analysis of the fecal sample at the third time point can indicate the presence of the diseased cells in the subject with high specificity and with little or no background signal.
  • the analysis can involve detection of plasmid DNA, RNA, and/or protein. Additional techniques that can be used to further reduce the background signal include differential centrifugation to remove native bacteria and/or any remaining genetically engineered microorganisms, and/or DNAse treatment before lysing the cells in the biological sample to remove cell free DNA.
  • Additional parameters that can be adjusted to further improve sensitivity include varying the time course of detection, varying the amount of the genetically engineered microorganism administered to the subject, removing the endosomal lysis gene from the genetically engineered microorganism, and/or changing the time from administration of the genetically engineered microorganism to fecal measurements.
  • A. Introns In some embodiments, the genetically engineered microorganisms of the present disclosure include one or more payload genes, each of which can have one or more introns.
  • An intron can be a noncoding polynucleotide sequence within a gene that is spliced or otherwise removed from the RNA molecule produced by transcription, and thus does not form part of the protein or peptide produced by translation of the RNA molecule.
  • Splicing can be carried out by a large ribonucleoprotein complex known as the spliceosome, which includes five small nuclear RNAs (U1, U2, U4, U5, and U6) and associated spliceosomal proteins.
  • the spliceosomal pathway is present in eukaryotes but is absent in prokaryotes.
  • the intron(s) include at least one spliceosomal intron, such that the gene products produced by the expression of the payload genes (e.g., RNA, proteins, and/or peptides) have two or more different forms, depending on the splicing of the intron(s).
  • the different forms of the gene products can be used to differentiate gene products produced by the target cell from gene products produced by non- target cells.
  • RNA e.g., RNA including one or more introns
  • spliced RNA e.g., RNA lacking introns
  • the intron(s) can prevent expression of the functional protein from the unspliced RNA (e.g., by including or creating a frameshift or premature stop codon in an unspliced RNA), such that functional protein is produced only from expression of the payload gene by the target cell, and is not produced from expression of the payload gene by the genetically engineered microorganism.
  • the splicesomal intron(s) can allow for alternative splicing, such as alternative splicing due to exon skipping, intron retention, and/or alternative 3’ and 5’ splicing.
  • the alternative splicing can produce different RNA splice variants, with some RNA splice variants being indicative of expression of the payload gene by the target cell, and other RNA splice variants being indicative of expression of the payload by non-target cells.
  • a first splicing pathway can produce a first RNA splice variant that results in expression of a functional protein
  • a second splicing pathway can produce a second RNA splice variant with a frameshift or premature splice codon that prevents expression of the functional protein or results in expression of a different protein.
  • a spliceosomal intron may be classified as being a major (U2-type) intron or minor (U12-type) intron, depending on whether the intron is spliced by the major spliceosome (U2-type spliceosome) or the minor spliceosome (U12-type spliceosome).
  • a spliceosomal intron generally includes a 5’ donor splice site (the splice site in the upstream region of the intron), a 3’ acceptor splice site (the splice site in the downstream region of the intron), and a branch point region.
  • U2-type introns typically include a GT dinucleotide at the donor splice site and an AG dinucleotide at the acceptor splice site.
  • U12-type introns typically include an AT dinucleotide at the donor splice site and an AC dinucleotide at the acceptor splice site, or an GT dinucleotide at the donor splice site and an AG dinucleotide at the acceptor splice site.
  • the introns described herein can also include other dinucleotides at the donor and/or acceptor splice sites.
  • the 5’ and 3’ dinucleotides can be located within longer consensus sequences that may influence the strength of the splicing sites (e.g., a high degree of similarity to the consensus sequence may produce stronger and/or constitutive splicing, while a lower degree of similarity may produce weaker and/or alternative splicing).
  • the branch point can be located upstream of the 3’ splice site and can usually be in proximity to the 3’ splice site (e.g., within 20 to 200 nucleotides, or within 20 to 25 nucleotides).
  • a single Fortem Ref. No. MMS.004WO intron can include several alternatively used branch points.
  • a pyrimidine enriched sequence polypyrimidine tract
  • the U1 small nuclear ribonucleoprotein binds the 5’ splice site, while the branch point and polypyrimidine tract bind the U2 small nuclear ribonucleoprotein and U2AF, respectively.
  • the spliceosome can catalyze the removal of the intron and ligation of the flanking exons.
  • Splicing events can also be influenced by the presence of cis-acting splicing regulatory elements (SREs), such as exonic splicing enhancers, exonic splicing silencers, intronic splicing enhancers, and intronic splice silencers.
  • SREs cis-acting splicing regulatory elements
  • RNA-binding non-spliceosomal regulatory proteins can promote or hinder the spliceosome activity on adjacent splicing sites.
  • Other factors that may influence splicing include the length of the introns flanking an exon (e.g., alternatively spliced exons may be flanked by longer introns compared to constitutively spliced exons), epigenetic factors, and RNA secondary structures.
  • the intron(s) can include at least one intron that enhances, regulates, or otherwise modifies gene expression of one or more payload genes.
  • the payload gene can include at least one intron that increases the amount of the gene product produced by the target cell, also known as “intron- mediated enhancement.”
  • the presence of the intron can increase RNA and/or protein expression by at least 2X, 5X, 10X, 20X, 50X, or 100X compared to target cells that include the payload gene without the intron.
  • the intron is located at or near the 5’ end of the payload gene. The number of introns in a payload gene can be varied as desired.
  • any of the payload genes described herein can include one, two, three, four, five, or more introns.
  • some or all of the introns can be the same intron, or some or all of the introns can be different introns.
  • a payload gene can include a first intron to increase gene expression via intro- mediated enhancement, and a second intron that is a spliceosomal intron.
  • the first intron can be located at or near the 5’ end of the payload gene, and the second intron can be spaced apart from the 5’ end of the payload gene (e.g., by at least 10 bp, 50 bp, 100 bp, 500 bp, or 1 kb).
  • some of the payload genes described herein may not include any introns. Fortem Ref. No.
  • An intron incorporated into a payload gene can have any suitable length, such as a length within a range from 10 bp to 20 kb, 10 bp to 10 kb, 10 bp to 5 kb, 10 bp to 1 kb, 10 bp to 500 bp, 10 bp to 200 bp, 10 bp to 100 bp, 10 bp to 50 bp, 100 bp to 20 kb, 100 bp to 10 kb, 100 bp to 5 kb, 100 bp to 1 kb, 100 bp to 500 bp, 100 bp to 200 bp, 200 bp to 20 kb, 200 bp to 10 kb, 200 bp to 5 kb, 200 bp to 1 kb, 200 bp to 500 bp, 500 bp to 20 kb, 500 bp to 20 kb, 500 bp to 10 kb, 500 bp to 20
  • the intron can have a length of at least 10 bp, 20 bp, 50 bp, 100 bp, 500 bp, 1 kb, 2 kb, 5 kb, or 10 kb; and/or a length of no more than 20 kb, 15 kb, 10 kb, 5 kb, 2 kb, 1 kb, 500 bp, 100 bp, 50 bp, or 10 bp.
  • the location of an intron within a payload gene can also be varied as desired.
  • a spliceosomal intron can be inserted into the coding region of the payload gene at a location that splits the coding region into two separate regions, such that neither the region downstream of the intron (toward the 3’ end of the gene) or upstream of the intron (toward the 5’ end of the gene) can produce a functional protein.
  • the intron can be inserted into the payload gene such that the payload gene includes at least 10 b, 50 b, 100 b, 500 b, or 1 kb downstream of the intron; and/or at least 10 b, 50 b, 100 b, 500 b, or 1 kb upstream of the intron.
  • an intron that enhances gene expression can be located at or the 5’ end of the gene.
  • an intron is inserted within a consensus splice site that is present in the payload gene, or within a sequence derived from a consensus splice site that is present in the payload gene.
  • the intron can be positioned after one or more bases that are part of an exon portion of a 5’ consensus splice site, and before one or more bases that are part of an exon portion of a 3’ consensus splice site.
  • An example of a 5’ consensus splice site is CAG/GURAGU, where CAG corresponds to the 3’ end of the exon and GURAGU corresponds to the 5’ end of the intron.
  • an example of a 3’ consensus splice site is AG/G, where AG corresponds to the 3’ end of the intron and G corresponds to the 5’end of the next exon.
  • the intron is inserted within a CAG/G sequence present in the payload gene, where “/” denotes the location of the intron (CAG is located before the 5’ end of the intron and G is located after the 3’ end of the intron).
  • the intron is inserted within an AAG/G, GAG/G, CAG/A, AAG/A, or GAG/A sequence present in the payload gene, where “/” denotes the location of the intron.
  • the genetically engineered microorganism includes a payload gene with at least one intron that is a naturally occurring intron or a derivative thereof.
  • the naturally occurring intron can be a mammalian intron, such as a human intron, or a viral intron.
  • the intron 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 intron.
  • one or more introns of a payload gene can be an artificial intron (also known as a synthetic intron).
  • Artificial introns can be generated in various ways, such as by modifying the sequence of a naturally occurring intron (e.g., to insert a stop codon, produce a frameshift in a downstream coding region, increase or decrease splicing activity, increase or decrease gene expression, increase intron length to provide better resolution when comparing spliced versus unspliced RNA) or via de novo design.
  • a library of artificial introns is generated and screened for the desired activity.
  • candidate sequences for artificial introns can be generated by analyzing sequences of known introns in a model organism (e.g., the human genome) to identify consensus sequences for the donor site, acceptor site, branch point, and/or polypyrimidine region.
  • the lengths and/or locations of these intron elements can also be determined based on the most frequent lengths and/or locations observed in the sequences of the known introns.
  • the candidate sequences can be tested by insertion into a reporter gene on a test plasmid, then verifying expression of the spliced reporter gene.
  • the intron is a viral intron.
  • the viral intron is a simian virus 40 (SV40) intron.
  • the viral intron is a human cytomegalovirus (HCMV) intron, such as HCMV intron A.
  • the viral intron is an adenovirus intron.
  • the intron is a eukaryotic intron, such as mammalian intron (e.g., a human intron).
  • the intron is an intron from the APC (adenomatous polyposis coli) gene, which encodes a tumor suppressor protein that acts as an antagonist of the Wnt signaling pathway.
  • the APC intron can be the intron between exons 13 and 14 of the APC gene, the intron between exons 14 and 15 of the APC gene, or any other intron of the APC gene.
  • the intron is from the TRPM2 (transient receptor potential cation channel, subfamily M, member 2) gene, which encodes a protein that forms a tetrameric cation channel that is permeable to calcium, sodium, and potassium, and is regulated by free intracellular ADP-ribose.
  • TRPM2 intron can be the intron between exons 1 and 2 Fortem Ref. No. MMS.004WO of the TRPM2 gene, the intron between exons 2 and 3 of the TRPM2 gene, or any other intron of the TRPM2 gene.
  • the intron is from the CHEF1 (Chinese hamster elongation factor-1 ⁇ ) gene, which catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes.
  • the CHEF1 intron can be intron 1 of the CHEF1 gene or any other intron of the CHEF1 gene.
  • the intron is from the CANX (calnexin) gene, which encodes a calcium-binding, endoplasmic reticulum-associated protein that interacts transiently with newly synthesized N-linked glycoproteins, facilitating protein folding and assembly.
  • the CANX intron can be the intron between exons 8 and 9 of the CANX gene, the intron between exons 10 and 11 of the CANX gene, or any other intron of the CANX gene.
  • the intron is an intron from the PSEN-1 (presenilin-1 gene), which encodes a presenilin protein that forms part of the gamma secretase complex.
  • the PSEN-1 intron can be intron 5 of the PSEN-1 gene or any other intron of the PSEN-1 gene.
  • the intron is an intron from the COL1A1 gene (type I collagen alpha 1 chain gene), which encodes the pro-alpha1 chain of type I collagen.
  • the COL1A1 intron can be intron 17 of the COL1A1 gene or any other intron of the COL1A1 gene.
  • the intron is an intron from the COL1A2 gene (type I collagen alpha 2 chain gene), which encodes the pro-alpha2 chain of type I collagen.
  • the COL1A2 intron can be intron 11 of the COL1A2 gene or any other intron of the COL1A2 gene.
  • the intron is an intron from the COL4A5 gene (type IV collagen alpha 5 chain gene), which encodes the alpha 5 chain of type IV collagen.
  • the COL4A5 intron can be intron 10 of the COL4A5 gene or any other intron of the COL4A5 gene.
  • the intron is an intron from the COL6A1 gene (type VI collagen alpha 1 chain gene), which encodes the alpha1 chain of type VI collagen.
  • the COL6A1 intron can be intron 9 of the COL6A1 gene or any other intron of the COL6A1 gene.
  • the intron is an intron from the ALDH7A1 gene (aldehyde dehydrogenase 7 family member A1 gene), which encodes a member of subfamily 7 in the aldehyde dehydrogenase gene family.
  • the ALDH7A1 intron can be intron 9 of the ALDH7A1 gene or any other intron of the ALDH7A1 gene.
  • the intron is an intron from the KCNQ1 gene (potassium voltage-gated channel subfamily Q member 1 gene), which encodes a voltage-gated potassium channel involved in the repolarization phase of the cardiac action potential.
  • the KCNQ1 intron can be intron 14 of the KCNQ1 gene or any other intron of the KCNQ1 gene.
  • the intron is an intron from the PDHA1 gene (pyruvate dehydrogenase E1 subunit alpha 1 gene), which encodes an enzymatic component of the pyruvate dehydrogenase complex.
  • the PDHA1 intron can be intron 8 of the PDHA1 gene or any other intron of the PDHA1 gene.
  • the intron is a modified intron that is produced by concatenating two or more introns with each other. Any of the introns described herein can be linked to each other to form a modified intron.
  • a modified PSEN-1 intron can include an adenovirus intron linked to a PSEN-1 intron.
  • a modified COL1A2 intron can include an adenovirus intron linked to a COL1A2 intron.
  • Representative examples of introns are provided in Table 7 below. Table 7: Introns Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No. MMS.004WO Fortem Ref. No.
  • the genetically engineered microorganism includes a payload gene with an intron 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: 22 – SEQ ID NO: 43.
  • the genetically engineered microorganism includes a payload gene with an intron of any one of SEQ ID NO: 22 – SEQ ID NO: 43.
  • Representative examples of payload genes with introns are provided in Table 8 below. Table 8: Payload Genes with Introns Fortem Ref. No. MMS.004WO Fortem Ref. No.
  • the genetically engineered microorganism includes a payload gene 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: 44 – SEQ ID NO: 48.
  • the genetically engineered microorganism includes a payload gene of any one of SEQ ID NO: 44 – SEQ ID NO: 48.
  • 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 Fortem Ref. No. MMS.004WO 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, and/or saliva. Accordingly, the subject can be diagnosed by collecting the biological sample 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 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 Fortem Ref. No. MMS.004WO 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 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 an 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, a 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 Fortem Ref.
  • PET Positron Emission Tomography
  • SPECT Single Photon Emission Computed Tomography
  • 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
  • the mRNA encodes for a near-infrared fluorescent protein selected from iRFP670, miRFP670, iRFP682, iRFP702, miRFP703, miRFP709, iRFP713 (iRFP), 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 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, Fortem Ref. No. MMS.004WO 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 that are spliced out in the RNA molecule to produce spliced RNA and/or different RNA splice variants, as described herein.
  • the intron(s) can include any of the introns described above in Section III.A.1, for example.
  • 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 variant
  • the non-target cell e.g., an unspliced RNA or second splice 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).
  • 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
  • RNA 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 be 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).
  • target cells e.g., a spliced RNA molecule, a first 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 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 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 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 Fortem Ref. No.
  • 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, a 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, a 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 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 (iRFP), 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.
  • 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 change in, e.g., color, fluorescence, or luminescence. Such reactions may use chromogenic, fluorigenic, 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 fluorigenic.
  • 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 Fortem Ref. No. MMS.004WO reporter is chloramphenicol acetyltransferase, and 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 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.
  • a secreted protein or a subunit of peptide therefrom
  • the genetically engineered microorganism delivers one or more payload genes encoding at least 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 Fortem Ref. No. MMS.004WO 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.
  • 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+ ).
  • 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+ ).
  • 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 G
  • 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 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 Fortem Ref. No. MMS.004WO 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 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.
  • a PET probe e.g., a positron emitting radioisotope
  • 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, [18F]fluoropropyl-trimethoprim, Na124I, or a 225Ac-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.
  • Illustrative 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] ⁇ -C
  • 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.
  • a detectable form e.g., a form that causes the accumulation of a SPECT probe
  • 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 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, inhibiting or otherwise interfering with cellular processes in the target cell.
  • diseased cells may be targeted with proteins that sensitize the diseased cells to therapy. Such proteins may function by converting a prodrug to an active metabolite.
  • the therapeutic agent is or includes a prodrug converting enzyme.
  • a prodrug converting enzyme can be a protein having a function of converting an inactive prodrug into an active drug 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, anti-bacterial drug, an anti-parasite drug, a hormone, an immunosuppressive drug, or a combination thereof.
  • the prodrug converting enzyme increases cell permeability to a therapeutic agent, restores hormonal responsiveness, and/or renders the cell more sensitive to radiotherapy or chemotherapeutics. 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. 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.
  • the prodrug converting enzyme remains in the cytoplasm of the target cell.
  • the prodrug converting enzyme is attached to the surface of the target cell (e.g., by a GPI anchor).
  • the prodrug converting enzyme is secreted by the target cell.
  • the nucleic acid encoding the prodrug converting enzyme includes a leader sequence for secretion of the prodrug converting enzyme.
  • the prodrug converting enzyme is an enzyme capable of catalyzing conversion of 5-fluorocytosine (5-FC) into 5-fluorouracil (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), 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 derived from a yeast or bacterial UPRT (yUPRT 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. Fortem Ref. No.
  • 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).
  • PNP purine nucleoside phosphorylase
  • 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 payload gene(s) encoding the therapeutic agent includes one or more introns such that the therapeutic agent is expressed in an active form (e.g., Fortem Ref. No. MMS.004WO a catalytically active form) only in the target cells, as described herein.
  • an active form e.g., Fortem Ref. No. MMS.004WO a catalytically active form
  • the prodrug converting enzyme can be capable of converting the inactive prodrug into the active drug only when the prodrug converting enzyme is expressed from a spliced RNA molecule or a splice variant produced specifically by the target cell.
  • the therapeutic agent is an RNA molecule that has therapeutic activity in the target cell (e.g., a catalytic RNA, a non-coding RNA, etc., that mediates RNA interference or inhibits key cellular processes)
  • the RNA molecule may exhibit therapeutic activity only when expressed in a spliced form or as a splice variant produced specifically by the target cell.
  • the genetically engineered microorganisms of the present disclosure deliver a DNA molecule (e.g., a plasmid DNA, also referred to herein as a payload plasmid) to the target cells (e.g., diseased and/or abnormal cells).
  • 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., proteins, peptides, and/or RNA that may serve as detection markers and/or therapeutic agents).
  • 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.
  • the 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 Fortem Ref. No. MMS.004WO 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). 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) Fortem Ref. No. MMS.004WO 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, 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 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’ (SEQ ID NO: 49).
  • 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) (SEQ ID NO: 50), F2A ((GSG)VKQTLNFDLLKLAGDVESNPGP) (SEQ ID NO: 51), P2A ((GSG)ATNFSLLKQAGDVEENPGP) (SEQ ID NO: 52), or T2A ((GSG)EGRGSLLTCGDVEENPGP) (SEQ ID NO: 53).
  • the GSG sequence (which is included in the parentheses) may be optionally present. Fortem Ref. No.
  • the polyadenylation sequences 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.
  • the one or more payload genes optionally include upstream sequences for display of the corresponding protein or peptide on a mammalian cell surface.
  • 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) (SEQ ID NO: 54).
  • 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 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.
  • IV. Compositions in some embodiments, 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 or Fortem Ref. No. MMS.004WO treated. In some embodiments, 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 prior to detection of the expressed marker.
  • the genetically engineered microorganisms may be administered from 1 to about 21, or from 1 to about 14, or from 1 to about 7 times 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 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) as already described.
  • the genetically engineered microorganisms of the present technology are administered by 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.
  • 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 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 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.
  • 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).
  • 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, glycerinated gelatin).
  • 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.
  • 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 Fortem Ref. No. MMS.004WO 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, 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 detecting and/or treating a disease, such as a disease of the GI tract and/or associated tissue.
  • a pharmaceutical composition for detecting and/or treating 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 detection marker, a therapeutic agent, or a combination thereof.
  • the payload gene can be operably linked to a mammalian promoter for expression by the target cell.
  • the mammalian promoter can be active and/or specific for epithelial expression (e.g., GI tract epithelial cell- specific expression).
  • epithelial expression e.g., GI tract epithelial cell- specific expression.
  • the gene product has a first form when expressed by the target cell and a second, different form when expressed by a non-target cell (e.g., the genetically engineered microorganism).
  • the first form of the gene product can be a spliced RNA or first splice variant lacking one or more introns that were initially present in the payload gene, or a protein or peptide produced from the spliced RNA or first splice variant.
  • the second form of the gene product can be an unspliced RNA or second splice variant including some or all of the introns that were initially present in the payload gene, or a protein or peptide produced from the unspliced RNA or second splice variant.
  • the genetically engineered microorganism includes a targeting gene encoding a targeting element that facilitates binding and 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 genetically engineered microorganism includes an endosomal lysis gene encoding an endosomal 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 endosomal 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 endosomal lysis element may be operably linker to a constitutive promoter or to an inducible promoter (e.g., a pH-sensitive promoter or other promoter that induces expression in response to endosomal conditions).
  • the genetically engineered microorganism includes a bacterial lysis gene encoding a bacterial lysis element that lyses the genetically engineered microorganism in order to release the payload plasmid into the cytoplasm of the target cell.
  • the bacterial lysis element can interfere with bacterial cell wall synthesis and/or degrade the bacterial cell wall, thereby causing lysis of the genetically engineered microorganism.
  • the bacterial lysis element is expressed only when the genetically engineered microorganism has been internalized by the target cell.
  • the bacterial lysis element can be operably linked to an inducible promoter that is activated in response to endosomal conditions (e.g., a pH-sensitive promoter) or can be operably linked to a promoter that is activated by a sequence element that is operably linked to an inducible promoter (e.g., a T7 promoter that is activated by a T7 RNA polymerase under the control of a pH-sensitive promoter).
  • the genetically engineered microorganism can optionally include a nutritional 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 mutated copy of the essential 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 present technology provides methods for detecting and/or treating 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 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 Fortem Ref. No. MMS.004WO (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. 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.
  • 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, 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).
  • 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 including a laxative.
  • the colon cleansing agent including the laxative is administered prior to the administration of the microorganism.
  • the method can include collecting a biological sample from the subject after administration of the 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 Fortem Ref. No. MMS.004WO 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 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.
  • 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 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 (e.g., DNA and/or RNA).
  • the detection marker is a gene product encoded by a payload gene that is transferred to the target cell by the genetically engineered microorganism.
  • the detection marker can be a first form of the gene product that results from expression of the payload gene in the target cell, such a spliced RNA, a splice variant produced by a splicing mechanism specific to the target cell, or a protein or peptide produced from the spliced RNA or splice variant.
  • 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,
  • the fecal sample can contain diseased and healthy epithelial cells sloughed off from the GI tract, Fortem Ref. No. MMS.004WO as well as the 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.
  • 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. In some embodiments, the subject is diagnosed as having the disease or being at risk of developing the disease only if the detection marker is present in a form produced exclusively by eukaryotic cells (e.g., a spliced form). Alternatively or in combination, 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 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
  • the target cells e.g., diseased cells
  • non-target cells e.g., normal cells
  • the method can optionally include administering the substrate the subject, such as a substrate of a bioluminescent protein, a substrate of a 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 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.
  • the treatment is surgery, e.g., to remove diseased tissue.
  • the treatment is 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 genetically engineered microorganism can deliver a payload gene encoding a therapeutic agent for the disease, as described herein. Fortem Ref. No.
  • Example 1 In Vitro Delivery of DNA to Mammalian Cells by Engineered Bacteria
  • E. coli Nissle 1917 were engineered to have the invasin and listeriolysin O (“LLO”) genes integrated on the bacterial chromosome under a ProD bacterial promoter, and an episome containing the fluorescent protein iRFP670 under a CMV mammalian promoter (FIG. 4).
  • LLO listeriolysin O
  • iRFP670 requires biliverdin as a cofactor, which is ubiquitous in mammalian cells but not present in E.
  • the strain is also an auxotroph for diaminopimelic acid (DAP) and D-alanine.
  • DAP auxotrophy prevents the bacteria from proliferating, and the D-alanine auxotrophy maintains the episome containing the payload protein.
  • Bacteria were grown to mid-log phase for 2 hours at 30 °C in LB containing 0.1 mg/mL 2,6-diaminopimelic acid (DAP) (Sigma Aldrich, St. Louis, MO).
  • Bacteria were then washed and resuspended into mammalian culture media containing 0.1 mg/mL DAP, then seeded onto adherent HEK293T cells at various multiplicities of infection (MOI). After 2 hours at 37 °C and 5% CO 2 , plates were washed with phosphate-buffered saline (PBS) to remove the majority of extracellular bacteria. Mammalian culture medium without DAP and containing gentamycin to kill any remaining extracellular bacteria was added to the plates. After 24 hours at 37 °C and 5% CO2, cells were treated with trypsin to detach them from the plates and analyzed for fluorescence using a flow cytometer.
  • PBS phosphate-buffered saline
  • FIG.5A is a graph illustrating the results from the flow cytometry analysis
  • FIG. 5B is a microscopy image of mammalian cells treated with a negative control strain lacking invasin
  • FIG.5C is a microscopy image of mammalian cells treated with the test strain.
  • DNA was successfully transferred from the bacteria to approximately 12% of the mammalian cells.
  • Mammalian cells treated with the test strain exhibited iRFP670 fluorescence indicative of DNA transfer (FIG. 5C), while no fluorescence was observed in mammalian cells treated with the negative control strain (FIG.5B).
  • MMS.004WO engineered bacteria can effectively deliver a DNA construct to mammalian cells, and that the mammalian cells can successfully express the payload protein encoded by the DNA.
  • Example 2 Use of Introns to Prevent Bacterial Expression of Protein This example describes the use of an intron in a payload gene to produce selective protein expression in mammalian cells and prevent expression of the protein in the engineered bacteria.
  • E. coli Nissle 1917 were engineered to have the invasin and LLO genes integrated on the bacterial chromosome under a ProD bacterial promoter, and an episome containing the Renilla luciferase (Rluc) gene split by an intron under a CMV mammalian promoter (SEQ ID NO: 46) (FIG. 6).
  • Rluc is an intracellular luciferase requiring a coelenterazine substrate to produce light.
  • the strain is also an auxotroph for DAP and D- alanine.
  • the DAP auxotrophy prevents the bacteria from proliferating, and the D-alanine auxotrophy maintains the episome containing the payload protein.
  • the following strains were also produced: (1) a strain having an episome containing Rluc split by an intron under a ProD bacterial promoter, (2) a strain having an episome containing wild type Rluc under a CMV mammalian promoter, and (3) a strain having an episome containing wild type Rluc under a ProD bacterial promoter.
  • FIG. 7 is a graph illustrating Rluc expression in bacteria having a payload protein (Rluc protein split by an intron (“Rluc-Int”) or wild type Rluc (“Rluc”)) under either a bacterial promoter (“Bact”) or a mammalian promoter (“Mam”).
  • Rluc protein split by an intron Rluc-Int
  • Rluc wild type Rluc
  • Bact bacterial promoter
  • Mam mammalian promoter
  • a strain lacking either version of the payload protein was used as the negative control, while a strain including wild type Rluc under the ProD bacterial promoter was used as the positive control.
  • Bacteria were grown to log phase at 30 °C in LB containing 0.1 mg/mL DAP, and analyzed for production of Rluc via an in vitro Renilla luciferase assay (Promega). As shown in FIG.
  • strains containing Rluc split by an intron showed only background levels of Rluc production, indicating that the bacteria were unable to splice out the intron to produce the full-length, functional Rluc protein, even when expressed from a ProD bacterial promoter.
  • Wild-type Rluc expressed from the CMV mammalian promoter still showed expression of Rluc above background, suggesting leaky activity of the CMV mammalian promoter in bacteria.
  • Expression of Rluc in mammalian cells was evaluated using bacteria having the strain design in FIG. 6. Strains lacking invasin, LLO, and/or the Rluc-Int gene were used as controls. Bacteria were grown to mid-log phase for 2 hours at 30 °C in LB containing 0.1 Fortem Ref. No.
  • FIG. 8A is a graph illustrating Rluc expression levels in mammalian cells treated with various bacterial strains. As shown in FIG.8A, Rluc transfer to mammalian cells was dependent on the presence of both invasin and LLO genes, which promote transfer of genetic material. Mammalian cells treated with bacteria expressing both invasin and LLO successfully expressed Rluc, indicating that the mammalian cells were able to splice out the intron to produce the full-length function Rluc protein.
  • FIG.8B is a graph illustrating Rluc expression levels in mammalian cells treated with various MOIs of bacteria. As shown in FIG. 8B, increasing MOIs resulted in increased Rluc expression in mammalian cells.
  • FIG.8C is a graph illustrating Rluc expression levels in mammalian cells over time. As shown in FIG.8C, in the presence of both invasin (“Inv”) and LLO, Rluc expression reached its maximum levels in about 24 hours and remained stable for several days.
  • Example 3 Primers for Detection of Spliced RNA in Mammalian Cells This example describes primers designed to detect spliced RNA indicative of mammalian-specific gene expression. As described herein, RNA may be produced from a mammalian promoter even in bacterial cells, due to leaky expression and/or background activity of the mammalian promoter in the bacteria.
  • the bacteria are unable to splice out any introns present in the RNA, such that the bacterially derived RNA is a different RNA species than the RNA produced by mammalian cells (e.g., as previously described in connection with FIG. 1). Accordingly, the presence or absence of the intron can be used to differentiate between RNA produced by a diseased mammalian cell and background RNA produced by a bacterial cell.
  • E. coli Nissle 1917 were engineered to have the invasin and LLO genes integrated on the bacterial chromosome under a ProD bacterial promoter, and an episome Fortem Ref. No.
  • Rluc Renilla luciferase
  • Primers were designed to differentiate between spliced RNA without the intron (mammalian origin) versus unspliced RNA still containing the intron (bacterial origin) (FIG.9A). Specifically, the primers were designed so that one end of the primer would not hybridize to the unspliced intron, resulting in little or no amplification (FIG. 9B). Conversely, the primers were designed to completely hybridize with the spliced RNA sequence (FIG.9C), leading to amplification and subsequent detection by qPCR.
  • FIG. 10A illustrates different primers together with the unspliced Rluc sequence
  • FIG.10B shows the relative DNA expression levels obtained with the different primer sets when tested on plasmids containing the intron versus plasmids lacking the intron.
  • cycle threshold (Ct) values are inversely proportional to the amount of target DNA in the sample.
  • coli Nissle 1917 were engineered to have the invasin and listeriolysin O genes integrated on the bacterial chromosome under a ProD bacterial promoter, and an episome containing the Renilla luciferase (Rluc) protein split by an intron under a CMV mammalian promoter, as described in Example 2 above.
  • the bacteria were seeded on either wild type HCT116 cells (“WT ⁇ 1”) or on HCT116 cells with the ⁇ 1 integrin knocked out (“No ⁇ 1”). The bacteria were only able to invade and transfer DNA to cells which had exposed ⁇ 1 integrin.
  • Bacterial strains expressing or lacking invasin or LLO were also tested with HEK293T cells.
  • FIG.11A is a graph illustrating expression of Rluc protein in mammalian cells with and without ⁇ 1 integrin measured via enzymatic assay
  • FIG.11B is a graph illustrating expression of spliced Rluc RNA in mammalian cells with and without ⁇ 1 integrin measured via qPCR assay.
  • RNA expression was observed only in mammalian cells expressing ⁇ 1 integrin, indicating that DNA transfer from the bacteria was dependent on the presence of exposed ⁇ 1 integrin.
  • the mammalian cells successfully expressed both the Rluc protein and Rluc spliced RNA, RNA expression was detectable at lower MOIs than protein expression, indicating that RNA-based detection is more sensitive than protein- based detection.
  • FIG. 12A is a graph illustrating expression of Rluc protein in HEK293T mammalian cells treated with bacteria expressing or lacking invasin measured via enzymatic assay
  • FIG.12B is a graph illustrating expression of spliced Rluc RNA in mammalian cells treated with bacteria expressing or lacking invasin measured via qPCR assay. As shown in FIGS.12A and 12B, expression was observed only for mammalian cells treated with invasin- expressing bacteria, indicating that DNA transfer from the bacteria was dependent on the presence of invasin.
  • FIG. 13A is a graph illustrating expression of Rluc protein in HEK293T mammalian cells treated with bacteria expressing or lacking LLO measured via enzymatic assay
  • FIG.13B is a graph illustrating expression of spliced Rluc RNA in mammalian cells treated with bacteria expressing or lacking invasin measured via qPCR assay. As shown in FIGS.
  • Example 5 In Vivo Delivery of DNA to Diseased Cells by Engineered Bacteria This example describes the use of engineered bacteria to deliver DNA encoding an RNA detection marker to diseased cells in a mouse model. Apc l/l ;VilCreErt2 mice are split into control and tumor induced groups.
  • mice The tumor induced group mice are injected twice with 50 ⁇ L of 100 ⁇ M 4-(1-[4- (dimethylaminoethoxy)phenyl]-2-phenyl-1-butenyl)phenol (4-hydroxytamoxifen) (MedChem Express, Monmouth Junction, NJ, USA) in the mid to distal colon and given 1 week for tumor growth.
  • E. coli Nissle 1917 strain SRX1139 carrying an episomal plasmid encoding the Renilla luciferase gene disrupted by a mammalian intron (pSRX468), is inoculated into 10 mL total volume of LB with 2,6-diaminopimelic acid (DAP) (Sigma Aldrich, St.
  • DAP 2,6-diaminopimelic acid
  • mice are allowed to wake and rest overnight in the cage. Feces from each mouse is collected prior to the start of the experiment, and then once a day prior to each dosing, which is carried out once per day for 3 days. Each fecal sample is immediately placed into 500 ⁇ L of DNA/RNA Shield (Zymo Research, Irivine, CA) in a 1.5 mL Eppendorf tube, flash frozen in liquid N2, and then stored at -80°C until processing. Samples are thawed on ice, 200 mg of fecal material is Fortem Ref. No.
  • RNAse I DNAse I
  • supplied buffer 1 ⁇ L
  • RNAse free water 2 ⁇ L
  • primers CH103 SEQ ID NO: 63
  • CH90 SEQ ID NO: 59
  • NEB N-Step RT-qPCR reaction
  • the mixture is run for 45 cycles and the data analyzed in comparison to a standard curve and non-RT controls run concurrently with samples.
  • Uninjected mice which should have no dysplastic tissue
  • the expression levels in uninjected mice are near or at background levels established by the no RT control, or at least significantly less than the mice with induced dysplasia.
  • Injected mice (which contain dysplastic tissue) show significantly increased levels of spliced RNA indicating successful invasion of the dysplastic cell, transfer of DNA from the bacterial cell to the nucleus of the dysplastic cell, transcription of the target RNA by the dysplastic cell, and splicing of the RNA by the dysplastic cell. Feces from the injected mice also show levels of enzymatic activity above background when tested by standard methods (e.g., Renilla luciferase assay), although the qPCR may be more sensitive.
  • Example 6 Detection of Diseased Cells Using DNA This example describes DNA-based detection of diseased cells in a mouse model.
  • FIG. 14A is a schematic illustration of the study protocol.
  • FIGS.14B and 14C illustrate detection of the DNA payload in the fecal sample in mice with and without induced tumors.
  • DNA detection was performed using primers for the plasmid origin of replication (FIG. 14B) or primers for the synthetic Rluc gene (FIG. 14C). These results demonstrated that animals with early adenomas could be differentiated by assaying fecal DNA. Sensitivity was improved when using primers for the synthetic gene versus the origin of replication. Fortem Ref. No.
  • Example 7 Inducible Lysis of Engineered Bacteria This example describes studies to develop and evaluate a system for inducible lysis of engineered bacteria after internalization by mammalian cells. Bacterial lysis enables effective transfer of genetic payloads (e.g., plasmids) from within the bacteria into the intracellular space of the target cell for expression. In initial constructs, bacterial lysis was achieved using a dapA auxotrophy to prevent effective cell wall synthesis, resulting in loss of bacterial envelope integrity and lysis.
  • genetic payloads e.g., plasmids
  • FIG.15 schematically illustrates a non-inducible bacterial lysis system (top) and an inducible bacterial lysis system (bottom).
  • the bacteria e.g., E.
  • EcN coli Nissle
  • EcN coli Nissle
  • the bacteria may begin lysing immediately following in vivo administration, resulting in fewer bacteria being endocytosed by the target cells and leading to lower payload transfer and gene expression levels.
  • bacteria undergo lysis in response to an activation signal (e.g., a change in pH indicative of endosomal localization).
  • an activation signal e.g., a change in pH indicative of endosomal localization
  • E. coli Nissle 1917 strains were prepared: (1) bacteria containing the wild type dapA gene without an invasin gene, (2) bacteria containing the wild type (“WT”) dapA gene with the invasin gene integrated on the bacterial chromosome under a ProD bacterial promoter, and (3) bacteria containing a dapA auxotrophy with the invasin gene integrated on the bacterial chromosome under a ProD bacterial promoter.
  • WT wild type
  • All strains also included the LLO gene and the T7 RNA polymerase gene integrated on the bacterial chromosome under the acid inducible pASR promoter, and an episome containing the Renilla luciferase (Rluc) gene split by an intron under a CMV mammalian promoter and an mNeonGreen fluorescent marker gene under a ProB bacterial promoter.
  • the episomes in these strains did not contain a functional bacterial lysis gene.
  • Bacteria were seeded onto HEK293T cells and allowed to invade for 2 hours at 37°C 5% CO2. After this period, the cells were washed twice with PBS. At this point, cells were either Fortem Ref. No.
  • E. coli Nissle 1917 were engineered to include an mNeonGreen gene integrated on the bacterial chromosome under a pASR acid- sensitive promoter. Cells were cultured in LB and incubated at 37 °C overnight with shaking.
  • coli Nissle 1917 strains were prepared (FIG.19A): (1) a negative control strain including only the invasin (“INV”) gene alone under a ProD promoter (“Neg”), (2) a positive control strain including both invasin and LLO genes under a ProD promoter (“Pos”), and (3) an inducible strain including the invasin gene under a ProD promoter and the LLO gene under the pASR promoter (“pASR”).
  • Bacterial cultures were grown overnight shaking with at 37 °C in LB, and then subcultured 1:100 in LB and grown to mid-log phase with shaking at 37 °C.
  • coli Nissle 1917 strain was engineered to include: (1) a bacterial chromosome including the invasin gene under a ProD promoter, and a truncated LLO (“LLO-trunc”) and T7 RNA polymerase (“T7 pol”) genes under a pASR promoter, and (2) an episome including an mNeonGreen gene under a T7 promoter (FIG.20).
  • LLO-trunc truncated LLO
  • T7 pol T7 RNA polymerase
  • FIG.20 The truncated LLO was a fragment of LLO that is not catalytically active, and was used to eliminate endosomal disruption associated with active LLO.
  • Negative control strains without the T7 polymerase gene or without the mNeonGreen gene were also prepared.
  • Bacterial cultures were grown to mid-log phase with shaking at 30 °C, then combined with HEK293T cells at an MOI of approximately 1000, spun down at 500xg for 5 minutes to bring the bacteria in closer contact Fortem Ref. No. MMS.004WO to the HEK293T cell monolayer, and incubated for 30 minutes at 37°C 5% CO2. Cells were then washed twice in PBS and incubated with media containing gentamycin to kill extracellular bacteria. At various time points, cells were washed with PBS, detached, and analyzed with flow cytometry for mNeonGreen fluorescence. To evaluate the in vitro performance of an inducible lysis system based on pASR, an E.
  • coli Nissle 1917 strain was engineered to include: (1) a bacterial chromosome including the invasin gene under a ProD promoter, and the LLO-trunc and T7 RNA polymerase genes under a pASR promoter, and (2) an episome including a gene encoding a bacterial lysis element under a T7 promoter (bacteriophage ⁇ X174 Protein E (“LytE,” SEQ ID NO: 6) or lysozyme fused to a 19 amino acid membrane-penetrating amphipathic peptide (“CPP- Lysozyme” or “CPP-Lys,” SEQ ID NO: 8)), the Renilla luciferase (Rluc) gene split by an intron under a CMV mammalian promoter, and the mNeonGreen gene under a ProB bacterial promoter (FIG.22).
  • a bacterial chromosome including the invasin gene under a ProD promoter and the LLO-t
  • bacterial strains containing a non-inducible lysis system (dapA auxotrophic mutation) and bacteria lacking a single circuit component (invasin, CPP- Lysozyme, listeriolysin O, or T7 RNA polymerase) were also prepared.
  • bacteria grown overnight in LB were subcultured into LB and grown with shaking at 30°C to mid-log phase. Bacteria were then washed and subcultured into M9 media at the indicated pH with aliquots removed for colony forming unit (CFU) counting at the indicated times.
  • CFU colony forming unit
  • bacteria were subcultured 1:50 in LB media with DAP and grown for three hours with shaking at 30°C.
  • FIG.16 is a graph illustrating the efficacy of in vitro transfer of a Rluc payload plasmid to mammalian cells using different bacterial strains. As shown in FIG.
  • FIG. 17 is a graph illustrating the in vitro invasion rate of different bacterial strains into target cells. As shown in FIG. 17, bacteria with the dapA auxotrophy had significantly lower invasion rates compared to bacteria containing the wild type dapA and Fortem Ref. No. MMS.004WO invasin gene, as demonstrated by the significantly lower mNeonGreen expression levels.
  • FIG.18 is a graph showing pH-inducible gene expression over time in bacteria including an mNeonGreen fluorescent marker under the control of a pASR acid-sensitive promoter.
  • the pASR promoter is repressed at pH 7 and inducted at pH ⁇ 5.
  • bacteria grown in media at pH 7 showed little mNeonGreen expression over time, while bacteria grown in media at pH 5 showed increased expression of mNeonGreen.
  • FIG. 19A schematically illustrates bacterial strains for assessing pH-induced expression of LLO in vitro
  • FIG.19B is an image showing a Western blot to evaluate LLO expression under different pH conditions.
  • the inducible pASR strain only expressed LLO at pH 5 and not at pH 7.
  • the negative control strain exhibited no LLO expression at either pH values
  • the positive control strain exhibited LLO expression at both pH values.
  • FIG. 21A is a series of flow cytometry plots illustrating mNeonGreen expression over time after co-culture of engineered bacteria with HEK293T cells
  • FIG.21B is a graph showing mNeonGreen expression over time.
  • cells co-cultured with bacteria including the T7 polymerase gene under the control of the pASR promoter (“+T7”) exhibited increasisng fluorescence over time, showing successful induction of pASR following endocytosis.
  • little or no fluorescence was observed in cells co-cultured with bacteria lacking the T7 polymerase (“-T7”) or lacking the mNeonGreen reporter (“-Ctr”).
  • FIG.22 schematically illustrates a bacterial strain including an inducible lysis system.
  • the bacterial chromosome included the invasin gene under a ProD promoter and LLO and T7 RNA polymerase genes under a pASR promoter.
  • the bacteria also included an episome including a bacterial lysis gene under a T7 promoter (SEQ ID NO: 19 or SEQ ID NO: 20), an Rluc gene under a CMV promoter, and an mNeonGreen gene under a ProB promoter.
  • T7 promoter SEQ ID NO: 19 or SEQ ID NO: 20
  • Rluc gene under a CMV promoter
  • mNeonGreen gene under a ProB promoter.
  • Two different bacterial lysis elements were tested: LytE and CPP-Lysozyme. LytE is involved in Fortem Ref. No.
  • FIG.23 is a graph showing in vitro survival of engineered bacteria including an inducible lysis system based on LytE under various pH conditions. Bacteria with the LytE inducible lysis system exhibited survival rates comparable to wild type bacteria at pH 7, and exhibited increasing bacterial lysis over time at pH 5.
  • FIG.24 is a graph showing in vitro survival of engineered bacteria including an inducible lysis system based on LytE or CPP-Lysozyme under various conditions. Bacteria with the LytE or CPP-Lysozyme inducible lysis system exhibited survival rates comparable to wild type bacteria lacking a bacterial lysis system at pH 7, and exhibited increasing bacterial lysis over time at pH 5. These results demonstrate that both LytE and CPP-Lysozyme systems can functionally induce bacterial lysis under acidic conditions.
  • FIG.25 is a graph illustrating the efficacy of in vitro transfer of a Rluc payload plasmid to mammalian cells using bacteria including inducible or non-inducible lysis systems.
  • bacteria including a LytE or CPP-Lysozyme inducible lysis system were compared to bacteria including a non-inducible lysis system based on the dapA auxotrophy (“Aux”).
  • BactE or CPP-Lysozyme inducible lysis system outperformed bacteria including the LytE inducible lysis system and the non-inducible lysis system, as evidenced by significantly higher luciferase expression.
  • FIG.26 is a graph illustrating the efficacy of in vitro transfer of a Rluc payload plasmid to mammalian cells using bacteria including a CPP-Lysozyme inducible lysis system. All four circuit components—invasin, CPP-Lysozyme, LLO, and T7 RNA polymerase—were required for the most efficient payload transfer. Controls that lacked one of these components produced significantly reduced levels of luciferase expression.
  • FIG.27 is a series of graphs illustrating the efficacy of in vitro transfer of a Rluc payload plasmid to mammalian cells using bacteria including inducible or non-inducible lysis systems.
  • bacteria including a LytE or CPP-Lysozyme inducible lysis system were Fortem Ref. No. MMS.004WO compared to bacteria including a non-inducible lysis system based on the dapA auxotrophy.
  • Bacteria were incubated with mammalian cells at four different MOIs: 12.5 (upper left, 6.25 (upper right), 3.125 (lower left), and 0.78 (lower right).
  • Intron A a short artificial intron
  • PSEN1 an artificial intron composed of an adenovirus intron concatenated to PSEN-1 intron 5
  • COL1A2 an artificial intron composed of an adenovirus intron concatenated to COL1A2 intron 11
  • Introns were inserted at one of two locations in the Rluc gene: after base 438 (“Original position”) or after base 215 (“New position”) (FIG.28).
  • Bases 213–216 in the Rluc gene are a “CAGG” sequence corresponding to a consensus splice site; the intron was inserted after the “CAG” and before the “G” in the sequence.
  • a control strain without an intron in the Rluc gene was also tested.
  • HEK293T and HCT116 cells were seeded in DMEM with 10% FBS without antibiotics at 37 °C with 5% CO2.100 ng of purified plasmid was prepared with Lipofectamine2000 (Thermo Fisher Scientific) and Opti-MEM (Thermo Fisher Scientific) according to the manufacturer’s instructions and added to each well.
  • FIG.29 is a graph illustrating expression of the Rluc payload gene with different introns resulting from Lipofectamine transfection of HEK239T or HCT116 mammalian cells.
  • FIGS. 30 and 31 are graphs illustrating expression levels of the Rluc payload gene with different introns resulting from payload transfer from engineered bacteria to HEK293T (FIG.30) or HCT116 (FIG.31) cells.
  • FIG.32 is a series of graphs illustrating invasion rate (left graph) and payload transfer efficiency (right graph) of engineered bacteria. As shown in the left graph, all bacterial strains expressing invasin were successfully internalized by the cells. Improved payload transfer and luciferase expression was observed for all introns at the new position, compared to Intron A in the original position. Furthermore, Intron A in the new position showed the highest payload transfer and gene expression levels.
  • MMS.004WO administering a genetically engineered microorganism to the subject, wherein the genetically engineered microorganism delivers a payload gene specifically to the target cell, the payload gene encoding at least one gene product that has a first form when expressed by the target cell and a second form when expressed by the genetically engineered microorganism, the second form being different from the first form; collecting a biological sample from the subject; and detecting whether the first form of the at least one gene product is present in the biological sample.
  • Clause 2 The method of Clause 1, wherein the payload gene comprises at least one intron.
  • Clause 3. The method of Clause 2, wherein the at least one intron comprises a spliceosomal intron.
  • Clause 2 or 3 wherein the first form comprises a spliced RNA without the intron, and the second form comprises an unspliced RNA including the intron.
  • Clause 5 The method of any one of Clauses 2 to 4, wherein the first form comprises a first protein produced from a spliced RNA without the intron, and the second form comprises a second protein produced from an unspliced RNA including the intron.
  • Clause 6. The method of Clause 5, wherein the first protein is a full-length protein, and the second protein is a truncated protein.
  • Clause 7 The method of Clause 5 or 6, wherein the first protein is a functional protein, and the second protein is a nonfunctional protein.
  • Clause 2 The method of Clause 2 or 3, wherein the first form comprises a first RNA splice variant, and the second form comprises a second RNA splice variant.
  • Clause 9 The method of any one of Clauses 2, 3, or 8, wherein the first form comprises a first protein produced from a first RNA splice variant, and the second form comprises a second protein produced from a second RNA splice variant.
  • Clause 10 The method of Clause 9, wherein the first protein is a full-length protein, and the second protein is a truncated protein. Fortem Ref. No. MMS.004WO Clause 11.
  • the method of Clause 9 or 10 wherein the first protein is a functional protein, and the second protein is a nonfunctional protein.
  • Clause 15 wherein the sequence corresponding to the consensus splice site is CAG/G, AAG/G, GAG/G, CAG/A, AAG/A, or GAG/A.
  • Clause 17 The method of any one of Clauses 2 to 16, wherein the at least one intron comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 22 – SEQ ID NO: 43.
  • Clause 18 The method of any one of Clauses 2 to 17, wherein the at least one intron comprises a sequence of any one of SEQ ID NO: 22 – SEQ ID NO: 43.
  • Clause 20 The method of any one of Clauses 1 to 18, wherein the at least one intron comprises an intron that increases expression of the gene product by the target cell.
  • Clause 20 The method of any one of Clauses 1 to 19, wherein the payload gene comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 44 – SEQ ID NO: 48.
  • Clause 21 The method of any one of Clauses 1 to 20, wherein the payload gene comprises a sequence of any one of SEQ ID NO: 44 – SEQ ID NO: 48.
  • Clause 22 The method of any one of Clauses 1 to 21, wherein the at least one gene product comprises an RNA molecule.
  • detecting whether the first form of the at least one gene product is present in the biological sample comprises detecting whether a spliced form of the RNA molecule is present in the biological sample.
  • RNA sequencing RNA sequencing, fluorescent in situ hybridization (FISH, including mRNA fluorescent in situ hybridization (RNA-FISH)), or chromogenic in situ hybridization (CISH).
  • FISH fluorescent in situ hybridization
  • RNA-FISH mRNA fluorescent in situ hybridization
  • CISH chromogenic in situ hybridization
  • detecting whether the spliced form of the RNA molecule is present in the biological sample comprises: collecting RNA in the biological sample, converting the RNA in the biological sample into cDNA, and amplifying the cDNA using a primer set that hybridizes to the spliced form of the RNA molecule.
  • Clause 26 The method of any one of Clauses 22 to 25, wherein the RNA molecule comprises an mRNA.
  • Clause 27 The method of any one of Clauses 22 to 25, wherein the RNA molecule comprises a catalytic RNA.
  • Clause 32. The method of any one of Clauses 22 to 31, wherein the RNA molecule comprises at least one feature configured to increase stability of the RNA molecule. Fortem Ref.
  • Clause 33 The method of any one of Clauses 1 to 32, wherein the at least one gene product comprises a protein or peptide.
  • Clause 34 The method of Clause 33, wherein the protein or peptide is an intracellular protein, an intracellular peptide, a surface protein, or a surface peptide.
  • Clause 35 The method of Clause 33 or 34, wherein the protein or peptide is an enzyme, a hormone, an antigen, a fluorescent molecule, or a bioluminescent molecule.
  • any one of Clauses 33 to 35, wherein detecting whether the first form of the at least one gene product is present in the biological sample comprises detecting whether a full-length form of the protein or peptide is present in the biological sample.
  • Clause 37. The method of any one of Clauses 33 to 36, wherein detecting whether the first form of the at least one gene product is present in the biological sample comprises detecting whether a functional form of the protein or peptide is present in the biological sample.
  • Clause 38. The method of any one of Clauses 1 to 37, wherein the target cell comprises a diseased epithelial cell.
  • GI gastrointestinal
  • Clause 39 wherein the genetically engineered microorganism is administered to a lumen of the GI tract of the subject.
  • Clause 41 The method of Clause 39 or 40, wherein the genetically engineered microorganism is administered orally, rectally, or a combination thereof.
  • Clause 42 The method of any one of Clauses 38 to 41, wherein the genetically engineered microorganism expresses a surface protein that binds to a mislocalized surface marker on the diseased epithelial cell.
  • Clause 43 The method of Clause 42, wherein the surface protein comprises an invasin, and the mislocalized surface marker comprises an integrin.
  • the precancerous lesion comprises a polyp selected from one or more of the following: a sessile polyp, a serrated polyp, a sessile serrated polyp, a flat polyp, a sub-pedunculated polyp, a pedunculated polyp, or a diminutive polyp.
  • a polyp selected from one or more of the following: a sessile polyp, a serrated polyp, a sessile serrated polyp, a flat polyp, a sub-pedunculated polyp, a pedunculated polyp, or a diminutive polyp.
  • Clause 48 The method of any one of Clauses 45 to 47, wherein the precancerous lesion comprises a biliary intraepithelial neoplasm (BilIN) selected from one or more of the following: BilIN-1, BilIN-2, BilIN-3, or cholangiocarcinoma.
  • the precancerous lesion comprises a pancreatic intraepithelial neoplasm (PanIN) selected from one or more of the following: PanIN-1, PanIN-2, PanIN-3, or pancreatic ductal adenocarcinoma.
  • PanIN pancreatic intraepithelial neoplasm
  • Clause 50 The method of any one of Clauses 45 to 49, 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.
  • Clause 52 The method of any one of Clauses 44 to 50, wherein the presence of the diseased epithelial cell is indicative of the cancer.
  • Clause 52 The method of Clause 51, wherein the cancer is selected from 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.
  • Clause 53 The method of any one of Clauses 1 to 52, wherein the biological sample comprises one or more of feces, blood, serum, plasma, mucus, urine, or saliva.
  • Clause 55 The method of any one of Clauses 1 to 53, wherein the biological sample comprises a fecal sample, and the target cell is present in the fecal sample.
  • Clause 55 The method of any one of Clauses 1 to 54, wherein the genetically engineered microorganism comprises a targeting gene encoding a targeting element that facilitates binding and entry into the target cell.
  • 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.
  • any one of Clauses 1 to 56 wherein the genetically engineered microorganism comprises an endosomal lysis gene encoding an endosomal lysis element that lyses an endocytic vacuole of the target cell.
  • Clause 58 The method of Clause 57, wherein the endosomal lysis element comprises a lysin.
  • Clause 59 The method of Clause 58, wherein the lysin comprises listeriolysin O.
  • Clause 60 The method of any one of Clauses 1 to 59, wherein the genetically engineered microorganism comprises a bacterial lysis gene encoding a bacterial lysis element that facilitates lysis of the genetically engineered microorganism within the target cell.
  • Clause 61 The method of Clause 60, wherein the bacterial lysis element interferes with bacterial cell wall synthesis, breaks down bacterial cell wall components, or a combination thereof.
  • Clause 62 The method of Clause 60 or 61, wherein the bacterial lysis element comprises one or more of the following: a lysin, a holin, or a spanin.
  • Clause 63 The method of any one of Clauses 60 to 62, wherein the bacterial lysis element comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 6 – SEQ ID NO: 18.
  • Clause 65 The method of any one of Clauses 60 to 64, wherein the bacterial lysis element comprises a sequence of any one of SEQ ID NO: 6 – SEQ ID NO: 18.
  • Clause 65 The method of any one of Clauses 60 to 64, wherein the bacterial lysis element is operably linked to an inducible promoter or is operably linked to a promoter that is activated by a sequence element operably linked to the inducible promoter.
  • Clause 66 The method of Clause 65, wherein the inducible promoter is a pH-sensitive promoter. Fortem Ref. No. MMS.004WO Clause 67.
  • Clause 68 The method of Clause 67, wherein the nutritional auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell.
  • Clause 69 The method of Clause 68, wherein the payload gene is located on a plasmid that is delivered to the target cell after the lysis of the genetically engineered microorganism.
  • Clause 70 The method of any one of Clauses 67 to 69, wherein the nutritional auxotrophic mutation comprises a mutation in one or more of the following: dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF.
  • a genetically engineered microorganism comprising: a targeting gene encoding a targeting element that facilitates binding and entry into a target cell in a subject; and a payload gene encoding at least one gene product that serves as a detection marker for the presence of the target cell in the subject, wherein the at least one gene product has a first form when expressed by the target cell and a second form when expressed by the genetically engineered microorganism, the second form being different from the first form.
  • the genetically engineered microorganism of Clause 74 or 75 wherein the first form comprises a spliced RNA without the intron, and the second form comprises an unspliced RNA including the intron.
  • Fortem Ref. No. MMS.004WO Clause 77 The genetically engineered microorganism of any one of Clauses 74 to 76, wherein the first form comprises a first protein produced from a spliced RNA without the intron, and the second form comprises a second protein produced from an unspliced RNA including the intron.
  • Clause 78 The genetically engineered microorganism of Clause 77, wherein the first protein is a full-length protein, and the second protein is a truncated protein.
  • Clause 79 The genetically engineered microorganism of Clause 77 or 78, wherein the first protein is a functional protein, and the second protein is a nonfunctional protein.
  • Clause 80 The genetically engineered microorganism of Clause 74 or 75, wherein the first form comprises a first RNA splice variant, and the second form comprises a second RNA splice variant.
  • Clause 81 The genetically engineered microorganism of any one of Clauses 74, 75, or 80, wherein the first form comprises a first protein produced from a first RNA splice variant, and the second form comprises a second protein produced from a second RNA splice variant.
  • Clause 82 The genetically engineered microorganism of any one of Clauses 74, 75, or 80, wherein the first form comprises a first protein produced from a first RNA splice variant, and the second form comprises a second protein produced from a second RNA splice variant.
  • the genetically engineered microorganism of Clause 81 wherein the first protein is a full-length protein, and the second protein is a truncated protein.
  • Clause 83 The genetically engineered microorganism of Clause 81 or 82, wherein the first protein is a functional protein, and the second protein is a nonfunctional protein.
  • Clause 84 The genetically engineered microorganism of any one of Clauses 74 to 83, wherein the at least one intron splits a coding region of the payload gene into two separate regions.
  • Clause 85 The genetically engineered microorganism of any one of Clauses 74 to 84, wherein the at least one intron creates a premature stop codon in the payload gene.
  • Clause 86 The genetically engineered microorganism of any one of Clauses 74 to 84, wherein the at least one intron creates a premature stop codon in the payload gene.
  • the genetically engineered microorganism of any one of Clauses 74 to 90, wherein the at least one intron comprises an intron that increases expression of the gene product by the target cell.
  • Clause 93. The genetically engineered microorganism of any one of Clauses 73 to 92, wherein the payload gene comprises a sequence of any one of SEQ ID NO: 44 – SEQ ID NO: 48.
  • Clause 94 The genetically engineered microorganism of any one of Clauses 73 to 93, wherein the at least one gene product comprises an RNA molecule. Clause 95.
  • RNA molecule comprises an mRNA.
  • Clause 96 The genetically engineered microorganism of Clause 94, wherein the RNA molecule comprises a catalytic RNA.
  • Clause 97 The genetically engineered microorganism of Clause 96, wherein the catalytic RNA comprises a ribozyme.
  • Clause 98 The genetically engineered microorganism of Clause 96 or 97, wherein the first form comprises an active form of the catalytic RNA, and the second form comprises an inactive form of the catalytic RNA. Fortem Ref. No. MMS.004WO Clause 99.
  • Clause 101. The genetically engineered microorganism of Clause 100, wherein the protein or peptide is an intracellular protein, an intracellular peptide, a surface protein, or a surface peptide.
  • Clause 103. The genetically engineered microorganism of any one of Clauses 73 to 102, wherein the target cell comprises a diseased epithelial cell.
  • Clause 104. The genetically engineered microorganism of Clause 103, wherein the diseased epithelial cell is located in a gastrointestinal (GI) tract of the subject.
  • GI gastrointestinal
  • the genetically engineered microorganism of Clause 103 or 104 wherein the genetically engineered microorganism expresses a surface protein that binds to a mislocalized surface marker on the diseased epithelial cell.
  • Clause 106 The genetically engineered microorganism of Clause 105, wherein the surface protein comprises an invasin, and the mislocalized surface marker comprises an integrin.
  • Clause 107 The genetically engineered microorganism of any one of Clauses 73 to 106, further comprising a targeting gene encoding a targeting element that facilitates binding and entry into the target cell.
  • the genetically engineered microorganism of Clause 107 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.
  • Clause 109 The genetically engineered microorganism of any one of Clauses 73 to 108, further comprising an endosomal lysis gene encoding an endosomal lysis element that lyses an endocytic vacuole of the target cell.
  • the endosomal lysis element comprises a lysin.
  • Clause 111 The genetically engineered microorganism of Clause 110, wherein the lysin comprises listeriolysin O.
  • Clause 112. The genetically engineered microorganism of any one of Clauses 73 to 111, wherein the genetically engineered microorganism comprises a bacterial lysis gene encoding a bacterial lysis element that facilitates lysis of the genetically engineered microorganism within the target cell.
  • Clause 113 The genetically engineered microorganism of Clause 112, wherein the bacterial lysis element interferes with bacterial cell wall synthesis, breaks down bacterial cell wall components, or a combination thereof.
  • Clause 115. The genetically engineered microorganism of any one of Clauses 112 to 114, wherein the bacterial lysis element comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 6 – SEQ ID NO: 18.
  • Clause 116 The genetically engineered microorganism of any one of Clauses 112 to 115, wherein the bacterial lysis element comprises a sequence of any one of SEQ ID NO: 6 – SEQ ID NO: 18.
  • Clause 117 The genetically engineered microorganism of any one of Clauses 112 to 116, wherein the bacterial lysis element is operably linked to an inducible promoter or is operably linked to a promoter that is activated by a sequence element operably linked to the inducible promoter.
  • Clause 118. The genetically engineered microorganism of Clause 117, wherein the inducible promoter is a pH-sensitive promoter.
  • Clause 119 The genetically engineered microorganism of any one of Clauses 73 to 118, further comprising a nutritional auxotrophic mutation.
  • Clause 120 The genetically engineered microorganism of any one of Clauses 73 to 118, further comprising a nutritional auxotrophic mutation.
  • the genetically engineered microorganism of Clause 119 wherein the nutritional auxotrophic mutation facilitates lysis of the genetically engineered microorganism within the target cell.
  • Fortem Ref. No. MMS.004WO Clause 121 The genetically engineered microorganism of Clause 119 or 120, wherein the nutritional auxotrophic mutation comprises a mutation in one or more of the following: dapA, dapD, dapE, murA, alr, dadX, murI, thyA, aroC, ompC, or ompF. Clause 122.
  • Clause 124. A pharmaceutical composition comprising the genetically engineered microorganism of any one of Clauses 73 to 123 and a pharmaceutically acceptable carrier.
  • Clause 125. A method of detecting a disease in a subject, the method comprising administering the pharmaceutical composition of Clause 124 to the subject.
  • a method for detecting a target cell in a subject comprising: administering a plurality of genetically engineered microorganisms to the subject, wherein at least some of the genetically engineered microorganisms are internalized by the target cell and deliver a nucleic acid molecule specifically to the target cell, and wherein the genetically engineered microorganisms comprise an auxotrophic mutation that causes lysis of the genetically engineered microorganisms in vivo; collecting a biological sample from the subject after genetically engineered microorganisms that were not internalized by the target cell have lysed; and detecting whether the nucleic acid molecule is present in the biological sample. Clause 127.
  • nucleic acid molecule comprises a DNA molecule.
  • Clause 128 The method of Clause 127, wherein the DNA molecule comprises plasmid DNA.
  • Fortem Ref. No. MMS.004WO Clause 129 The method of any one of Clauses 126 to 128, wherein the biological sample is collected at least 12 hours, 24 hours, or 48 hours after the administration of the genetically engineered microorganisms to the subject. Clause 130.
  • any one of Clauses 126 to 129 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.
  • Clause 131 The method of any one of Clauses 126 to 130, wherein the detecting comprises detecting an amount of the nucleic acid molecule in the biological sample.
  • Clause 132. The method of any one of Clauses 126 to 131, wherein the detecting comprises detecting an amount of a gene product expressed from the nucleic acid molecule in the biological sample.
  • any one of Clauses 126 to 132 wherein the detecting is performed 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-quant
  • Clause 134 The method of any one of Clauses 126 to 133, wherein the biological sample comprises one or more of feces, blood, serum, plasma, mucus, urine, or saliva. Clause 135. The method of any one of Clauses 126 to 134, wherein the biological sample comprises a fecal sample, and the target cell is present in the fecal sample. Clause 136. The method of any one of Clauses 126 to 135, wherein presence of the nucleic acid molecule is indicative of one or more of the following: a precancerous Fortem Ref. No. MMS.004WO lesion, a cancer, an inflammatory bowel disease, irritable bowel syndrome, or Barrett’s esophagus. Clause 137.
  • Clause 138 A method for detecting or treating a target cell in a subject, the method comprising: administering a genetically engineered microorganism to the subject, wherein the genetically engineered microorganism is internalized specifically by the target cell to deliver a payload to the target cell, and wherein the genetically engineered microorganism comprises a bacterial lysis gene encoding a bacterial lysis element that facilitates lysis of the genetically engineered microorganism after internalization by the target cell.
  • GI gastrointestinal
  • Clause 138 wherein the bacterial lysis element interferes with bacterial cell wall synthesis, breaks down bacterial cell wall components, or a combination thereof.
  • Clause 140 The method of Clause 138 or 139, wherein the bacterial lysis element comprises one or more of the following: a lysin, a holin, or a spanin.
  • Clause 141 The method of any one of Clauses 138 to 140, wherein the bacterial lysis element comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to any one of SEQ ID NO: 6 – SEQ ID NO: 18.
  • Clause 143 The method of any one of Clauses 138 to 142, wherein the bacterial lysis gene is operably linked to an inducible promoter.
  • Clause 144 The method of any one of Clauses 138 to 142, wherein the bacterial lysis gene is operably linked to a promoter that is activated by a sequence element operably linked to an inducible promoter.
  • Clause 145 The method of Clause 144, wherein the promoter is a T7 promoter and the sequence element is a T7 RNA polymerase gene.
  • Clause 151 The method of any one of Clauses 138 to 150, wherein the genetically engineered microorganism comprises a targeting gene encoding a targeting element that facilitates binding and entry into the target cell.
  • Clause 152 The method of any one of Clauses 138 to 151, wherein the genetically engineered microorganism comprises an endosomal lysis gene encoding an endosomal lysis element that lyses an endocytic vacuole of the target cell.
  • Clause 153 The method of any one of Clauses 143 to 149, wherein the inducible promoter comprises a sequence of SEQ ID NO: 5.
  • Clause 157 The method of any one of Clauses 138 to 152, wherein the genetically engineered microorganism comprises an auxotrophic mutation.
  • Clause 154 The method of any one of Clauses 138 to 153, wherein the payload comprises a nucleic acid, a protein, or a peptide.
  • Clause 155 The method of any one of Clauses 138 to 154, wherein the payload comprises a detection marker.
  • Clause 156 The method of any one of Clauses 138 to 154, wherein the payload comprises a therapeutic agent.
  • Clause 157 The method of any one of Clauses 138 to 152, wherein the genetically engineered microorganism comprises an auxotrophic mutation.
  • a genetically engineered microorganism comprising: a targeting gene encoding a targeting element that facilitates binding and entry into a target cell in a subject; a payload gene encoding a payload for delivery to the target cell; and a bacterial lysis gene encoding a bacterial lysis element that facilitates lysis of the genetically engineered microorganism after entry into the target cell.
  • a targeting gene encoding a targeting element that facilitates binding and entry into a target cell in a subject
  • a payload gene encoding a payload for delivery to the target cell
  • a bacterial lysis gene encoding a bacterial lysis element that facilitates lysis of the genetically engineered microorganism after entry into the target cell.
  • Clause 163. The genetically engineered microorganism of any one of Clauses 157 to 161, wherein the bacterial lysis gene is operably linked to a promoter that is activated by a sequence element operably linked to an inducible promoter.
  • Clause 164. The genetically engineered microorganism of Clause 163, wherein the promoter is a T7 promoter and the sequence element is a T7 RNA polymerase gene.
  • the genetically engineered microorganism of Clause 166, wherein the pH-sensitive promoter is a pASR promoter.
  • Clause 168. The genetically engineered microorganism of any one of Clauses 162 to 167, wherein the inducible promoter comprises a sequence having at least 80%, 85%, 90%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 5.
  • Clause 169 The genetically engineered microorganism of any one of Clauses 162 to 168, wherein the inducible promoter comprises a sequence of SEQ ID NO: 5.
  • Clause 170 The genetically engineered microorganism of any one of Clauses 157 to 169, further comprising an endosomal lysis gene encoding an endosomal lysis element that lyses an endocytic vacuole of the target cell.
  • Clause 171 The genetically engineered microorganism of any one of Clauses 157 to 170, wherein the genetically engineered microorganism comprises an auxotrophic mutation.
  • Clause 173 The genetically engineered microorganism of any one of Clauses 157 to 172, wherein the payload comprises a detection marker.
  • Clause 174 The genetically engineered microorganism of any one of Clauses 157 to 173, wherein the payload comprises a therapeutic agent.
  • a pharmaceutical composition comprising the genetically engineered microorganism of any one of Clauses 157 to 174 and a pharmaceutically acceptable carrier. Clause 176.
  • a method of detecting or treating a disease in a subject comprising administering the pharmaceutical composition of Clause 175 to the subject.
  • the various embodiments described herein may also be combined to provide further embodiments.
  • 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).
  • 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. Fortem Ref. No.
  • 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 determined by conventional methods, such as 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 associated technology can encompass other embodiments not expressly shown or described herein.

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  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biomedical Technology (AREA)
  • Animal Behavior & Ethology (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne des procédés d'utilisation de micro-organismes génétiquement modifiés pour détecter et/ou traiter des maladies. Dans certains modes de réalisation, un procédé comprend l'administration d'un micro-organisme génétiquement modifié à un sujet. Le micro-organisme génétiquement modifié peut administrer un gène de charge utile spécifiquement à une cellule cible chez le sujet. Le gène de charge utile peut coder au moins un produit génique qui a une première forme lorsqu'il est exprimé par la cellule cible et une seconde forme lorsqu'il est exprimé par le micro-organisme génétiquement modifié, la seconde forme étant différente de la première forme. Le procédé peut en outre consister à collecter un échantillon biologique provenant du sujet, et à détecter si la première forme du ou des produits géniques est présente dans l'échantillon biologique.
PCT/US2024/034854 2023-06-21 2024-06-20 Micro-organismes génétiquement modifiés pour la détection de cellules malades Pending WO2024263805A1 (fr)

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Citations (8)

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WO1997038087A2 (fr) 1996-04-05 1997-10-16 Chiron Corporation Vecteurs a base d'alphavirus de recombinaison, presentant une inhibition reduite de la synthese macromoleculaire cellulaire
US20170173092A1 (en) 2014-03-30 2017-06-22 Benevir Biopharm, Inc. Exogenous tap inhibitor armed oncolytic viruses and therapeutic uses thereof
US20190038727A1 (en) 2015-09-09 2019-02-07 Tvax Biomedical I, Llc Methods for combining adoptive t cell therapy with oncolytic virus adjunct therapy
US20200157518A1 (en) 2017-06-30 2020-05-21 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy for cancer
US20200215111A1 (en) 2017-06-30 2020-07-09 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy for cancer
US20200268794A1 (en) 2017-06-30 2020-08-27 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy
WO2021247480A1 (fr) 2020-06-02 2021-12-09 Sanarx Biotherapeutics, Inc. Microorganismes modifiés pour imagerie diagnostique
WO2022261290A1 (fr) 2021-06-09 2022-12-15 Microbial Machines, Inc. Micro-organismes modifiés pour la détection de cellules malades

Patent Citations (8)

* Cited by examiner, † Cited by third party
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WO1997038087A2 (fr) 1996-04-05 1997-10-16 Chiron Corporation Vecteurs a base d'alphavirus de recombinaison, presentant une inhibition reduite de la synthese macromoleculaire cellulaire
US20170173092A1 (en) 2014-03-30 2017-06-22 Benevir Biopharm, Inc. Exogenous tap inhibitor armed oncolytic viruses and therapeutic uses thereof
US20190038727A1 (en) 2015-09-09 2019-02-07 Tvax Biomedical I, Llc Methods for combining adoptive t cell therapy with oncolytic virus adjunct therapy
US20200157518A1 (en) 2017-06-30 2020-05-21 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy for cancer
US20200215111A1 (en) 2017-06-30 2020-07-09 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy for cancer
US20200268794A1 (en) 2017-06-30 2020-08-27 Memorial Sloan Kettering Cancer Center Compositions and methods for adoptive cell therapy
WO2021247480A1 (fr) 2020-06-02 2021-12-09 Sanarx Biotherapeutics, Inc. Microorganismes modifiés pour imagerie diagnostique
WO2022261290A1 (fr) 2021-06-09 2022-12-15 Microbial Machines, Inc. Micro-organismes modifiés pour la détection de cellules malades

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ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410

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