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WO2016054298A1 - Magnétogénétique - Google Patents

Magnétogénétique Download PDF

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Publication number
WO2016054298A1
WO2016054298A1 PCT/US2015/053374 US2015053374W WO2016054298A1 WO 2016054298 A1 WO2016054298 A1 WO 2016054298A1 US 2015053374 W US2015053374 W US 2015053374W WO 2016054298 A1 WO2016054298 A1 WO 2016054298A1
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WIPO (PCT)
Prior art keywords
endosymbiont
host cell
cell
protein
magneto
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English (en)
Inventor
Caleb BELL
Christopher Contag
Brian Rutt
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Bell Biosystems Inc
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Bell Biosystems Inc
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host

Definitions

  • the present invention relates generally to modifying eukaryotic cells with artificial endosymbionts such that function of the eukaryotic cell can be modulated.
  • the artificial endosymbionts are designed to communicate with the host cell such that this interaction directs function of the host ceil.
  • Mitochondria, chloroplasts and other membrane bound organelles add heritable functionalities, such as photosynthesis, to eukaryotic cells.
  • Such organelles are believed to be endosymbioticaily derived.
  • MTB magnetotactic bacteria
  • Fe 3 O 4 magnetite
  • Fe 3 S 4 greigite
  • a targe number of MTB species have been identified since their initial discovery.
  • bacteria are readily modified by genetic and recombinant techniques, allowing for introduction of new functionalities.
  • the present invention is directed to host cells that have been altered or reprogrammed by transfer of chemical information from artificial endosymbionts to the host cell.
  • the present invention relates to changing the phenotype and/or genotype of host cells, n a regulated manner, by introducing into the host cell artificial endosymbionts that can secrete within the host cell polypeptides, nucleic acids, lipids, carbohydrates, amino acids, therapeutic agents, or other factors in response to specific signals, /. «., regulated secretion, to affect host cell function.
  • the artificial endosymbionts comprise magnetic-endosymbionts, which are used to deliver factors into the host cell in a controlled (Le., regulated) manner.
  • the magneto-endosymbiont secretes a polypeptide into the host cell.
  • the secreted protein is a heterologous polypeptide to the magneto-endosymbiont.
  • the secreted polypeptide from the magneto-endosymbiont causes a phenotypic change in the host cell.
  • the magneto-endosymbiont secretes a nucleic acid into the host cell.
  • the nucleic acid is a recombinant nucleic acid.
  • the nucleic acid secreted from the magneto-endosymbiont causes a phenotypic change in the host cell.
  • the artificial endosymbiont can be subjected to any appropriate treatment, e.g., physical or biochemical, that that can control the levels of the secreted polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s).
  • the treatment comprises magnetic hyperthermia, eg., by application of an alternating magnetic field.
  • the treatment comprises a small molecule that controls the level of the secreted polypeptide ⁇ ), nucleic acid(s), Hpid(s), carbohydrate(s), amino acid(s), or other factor(s).
  • the treatment comprises x-ray, ultrasound, light, radiofrequencies, or other electromagnetic signals that act on the endosymbiont
  • the artificial endosymbionts secrete into the host cell potypeptide(s), nucleic acid(s), lipid(s), carbohydrate ⁇ ), amino acid(s), or other factoids) mat contain regions that direct delivery to specific cellular compartments or organelles of the host cell. In some embodiments, the artificial endosymbionts secrete into the host cell
  • the artificial endosymbionts secrete into the host cell polypeptide ⁇ ), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s) that contain regions to direct their delivery to the cytoplasm.
  • the artificial endosymbionts secrete into the host cell polypeptide ⁇ ), nucleic acid(s), lipid(s),
  • the artificial endosymbionts secrete into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factors) mat contain regions that direct delivery to the mitochondria.
  • the magneto- endosymbionts of the invention secrete into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factors) that contain regions to direct delivery to a specific intracellular organelle such as mitochondria, lysosomes, and
  • the artificial endosymbionts secrete into the amino acid(s), or other factors) that contain regions to direct their delivery to the plasma membrane specifically for expression on the surface of the host cell, and these can confer function on the host cell or serve as a marker on the host cell.
  • the secreted polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s) acts at the level of the nucleic acid or chromosomes of the host cell.
  • the secreted polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factoids) changes the phenotype of the host cell.
  • the host cell can comprise any appropriate eukaryotic cell for modification with the artificial endosymbiont.
  • the cell can comprise a plant or animal cell, particularly a mammalian cell.
  • the host cell is a pluripotent, oligopotent, or unipotent cell (e.g., stem cell) and the method induces the cell to differentiate into a desired ceil.
  • the method uses a treatment, e.g., magnetic hyperthermia, to induce a magneto-endosymbiont to secrete polypeptides), nucleic acid(s), or other factor(s) that contribute to the differentiation of the host stem cell into a desired cell.
  • the magneto-endosymbiont secretes a Yamanaka factor (e.g., Oct4, Oct3, Sox2, Klf4, c-Myc, NANOG, and/or Lin28) into the host cell so the host cell becomes an induced pluripotent stem cell ("iPS").
  • a Yamanaka factor e.g., Oct4, Oct3, Sox2, Klf4, c-Myc, NANOG, and/or Lin28
  • the iPS cell is induced to differentiate into a desired cell by other polypeptides or nucleic acids or factors secreted by the artificial endosymbiont.
  • the host cells and methods of the invention are used to make medically and industrially important recombinant peptides/proteins that will be useful for therapeutic, biopharmaceutical, agricultural, and industrial applications.
  • the host cells and methods of the invention are used to make medically and industrially important recombinant peptides/proteins that will be useful for therapeutic, biopharmaceutical, agricultural, and industrial applications.
  • the magneto-endosymbionts and described methods are used to introduce into host cells phenotypes that require the introduction of multiple factors and/or multiple genes.
  • the magneto-endosymbiont introduces multiple phenotypes into the host cell.
  • the magneto-endosymbiont is capable of imparting these multiple phenotypes to the host cell at different desired times.
  • the multiple phenotypes may each be caused by single or multiple polypeptides or factors or nucleic acids secreted from the magneto-endosymbiont into the host cell.
  • the host cells and methods of the invention are used to produce natural or recombinant biologies, e.g., therapeutic peptides/polypeptides, which will be useful for the in situ treatment of diseases.
  • the therapeutic peptides/protein comprises a neurotransmitter expressed in neurons to treat neurological diseases; or a wild- type gene in target tissues to correct a genetic defect in heritable diseases, e.g., enzymes such as a glucosidase, a-galactosidase A and 6-glucocerebrosidase.
  • the artificial endosymbionts and methods disclosed herein are used to introduce into host cells phenotypes mat require the introduction of multiple factors and/or multiple genes.
  • the artificial endosymbiont introduces multiple phenotypes into the host cell.
  • the artificial endosymbiont is capable of imparting these multiple phenotypes to the host cell at different desired times.
  • the multiple phenotypes may each be caused by single or multiple polypeptides or factors or nucleic acids secreted from the magneto-endosymbiont into the host cell.
  • the eukaryotic host cell is a mammalian cell.
  • the host cell is a human, mouse, rat, canine, primate, or rodent cell.
  • the host cell is a fibroblast cell, epithelial cell, keratinocyte, hepatocyte, neuron, immune cell, adipocyte, endothelial cell or other differentiated cell.
  • the host cell is a stem cell, p!uripotent ES cell, pluripotent iPS cell, a muhipotent mesenchymal stem cell, multipotent hematopoietic stem cell, or other pluripotent stem cell.
  • the host cell is a progenitor cell, such as for example, a neural progenitor cell, an angioblast, an osteoblast, a chondroblast, a pancreatic progenitor cell, or an epidermal progenitor cell.
  • the host cell is a solid tumor cell or a hematopoietic cancer cell.
  • the host cell is from a carcinoma, sarcoma, leukemia, lymphoma, or glioma.
  • the host cell is obtained from a prostate cancer, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, melanoma, glioblastoma, liver cancer, or the NCI 60 panel of cancer cell lines.
  • FIG. 1 A, IB, 1C and ID depict MRI images of 231BR cells containing AMB-1 in mice.
  • FIG. 1A shows an image of 10 4 231 BR cells containing AMB-1.
  • FIG. IB shows an image of 10 3 231 BR cells containing AMB-1.
  • FIG. 1C shows an image of 10 2 231 BR cells containing AMB-1.
  • FIG. 1 D shows an image of single 231 BR cells containing AMB-1 in a mouse. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention is directed to host cells that contain an artificial endosymbiont, particularly a magneto-endosymbiont, where the artificial endosymbiont can secrete into the host cell a polypeptide, nucleic acid, or other factor in a regulated manner.
  • polynucleotide'* or "nucleic acid' refers to two or more nucleosides that are covalently linked together.
  • the polynucleotide may be wholly comprised ribonucleosides (i.e., an RNA), wholly comprised of V deoxyribonucleotides (i.e., a DNA) or mixtures of ribo- and 2' deoxyribonucleosides.
  • the polynucleotides may include one or more non-standard linkages.
  • the polynucleotide may be single-stranded or double-stranded, or may include bom single-stranded regions and double-stranded regions.
  • a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (/.e., adenine, guanine, uracil, thymine and cytosine), it may include one or more modified and/or synthetic nucleobases, such as, for example , inosine, xanthine, hypoxanthine, etc.
  • such modified or synthetic nucleobases will be encoding nucleobases.
  • a polymer of at least two amino acids covalently linked by an amide bond regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristilation, ubiquitination, etc.). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids.
  • the term "cellular life cycle” refers to series of events involving the growth, replication, and division of a eukaryotic cell. It is typically divided into stages mat include G ⁇ 3 ⁇ 4 in which the cell is quiescent, Gi and G 2 , in which the cell increases in size, S, in which the cell duplicates its DNA, and M, in which the cell undergoes mitosis and divides.
  • aughter cell refers to cells that are formed by the division of a cell.
  • essential molecule refers to a molecule required by a cell for growth or survival.
  • the term "genetically modified” refers to altering the DNA of a cell so that a desired property or characteristic of the cell is changed.
  • control region refers to a segment of nucleic acid that directs and regulates expression of a nucleic acid, such as a gene that encodes a protein.
  • a control region may include a promoter, operator, enhancer(s), activation binding site(s), attenuators), and other sequences involved in regulation of expression.
  • regulatable control region refers to a control region in which expression of the nucleic acid can be controlled by signals or agents, particularly by external signals or agents, which can be chemical, biological, or physical.
  • regulatable control regions include control regions that can be controlled by biological molecules, chemical agents,
  • regulatable control region is the heat shock control region in which expression can be controlled by hyperthermia. It is to be understood that regulated expression from a control region generally involves one or more accessory molecules (e.g., repressor protein), which in some embodiments, such as a cell, can be endogenous or heterologous. In the embodiments herein, regulatable includes the capability of inhibiting or inducing expression by the control region.
  • accessory molecules e.g., repressor protein
  • the term "localization signal” refers to a molecule or portion of a molecule that directs or mediates localization of another molecule, e.g., protein or nucleic acid, to a particular region of a cell, e.g., of a host cell.
  • the localization signal is a "targeting sequence” which refers to a sequence, either a polypeptide or polynucleotide sequence, that directs or mediates localization of biomolecules to a particular region in a cell when the biomolecule is attached to (e.g., fused to) the sequence.
  • treatment or “treating” in the context of a “control region” refers to contacting or exposing the target (e.g., cell, tissue or organism) to a signal or agent that controls (e.g., induces) expression from the control region.
  • target e.g., cell, tissue or organism
  • nucleic acid or polypeptide refers to a nucleic acid or polypeptide not normally present in nature.
  • a heterologous nucleic acid or polypeptide in reference to a host cell refers to a nucleic acid or polypeptide not naturally present in the given host cell.
  • a nucleic acid molecule containing a non-host nucleic acid encoding a polypeptide operably linked to a host nucleic acid comprising a promoter is considered to be a heterologous nucleic acid molecule.
  • a heterologous nucleic acid molecule can comprise an endogenous structural gene operably linked with a non-host (exogenous) promoter.
  • a peptide or polypeptide encoded by a non-host nucleic acid molecule, or an endogenous polypeptide fused to a non-host polypeptide is a heterologous peptide or polypeptide.
  • magnetic bacteria refers to prokaryotic cells that mineralize iron or other metals into magnetosomes, which are intracellular structures comprising magnetic iron enveloped by a lipid membrane.
  • artificial endosymbiont refers to a single celled organism, particularly bacteria, naturally occurring or modified, that is or has been introduced into a host cell through human intervention.
  • artificial endosymbiont is capable of secreting into the host cell polypeptide(s), nucleic acid(s), lipid(s),
  • the secreted factor may act in the cytoplasm, nucleus, organelle or other sites in the host cell. As described herein, this secretion comprises communication between the artificial endosymbiont, which can result in a phenotypic or epigenetic change of the host cell.
  • the term “magneto-endosymbiont” refers to magnetic bacteria, naturally occurring or modified, that is or has been introduced into a host cell through human intervention.
  • the magneto-endosymbiont is capable of secreting into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), and/or other factor(s).
  • the secreted factor may act in the cytoplasm, nucleus, organelle or other sites in the host cell. As described herein, this secretion comprises communication between the magneto-endosymbiont and the host cell, which can result in a phenotypic or epigenetic change of the host cell.
  • magnetic-genetics refers to modulation of complex cellular functions in targeted single cells, or groups of cells that contain magneto-endosymbionts.
  • epigenetic changes or “epigenetic modification” refers to modulation of simple or complex cellular functions, genetic functions in targeted single cells, or groups of cells without changes to the DNA sequence of the cell, e.g., host cell of the artificial endosymbiont.
  • the term “magnetosome” refers to particles of magnetite (i.e., Fe30 4 ) or greigite (Fe3S 4 ) enclosed by a sheath or membrane. In some embodiments, the particles can be individual particles or chains of particles.
  • the term “mammal” refers to warm-blooded vertebrate animals all of which are characterized by hair on the skin and, in the female, milk producing mammary glands.
  • phenotype refers to an observable characteristic or characteristics at any level - physical, morphological, biochemical, or molecular - of a cell, tissue, or organism.
  • secrete refers to the passing of molecules or signals from one side of a membrane to the other side. Accordingly, in some embodiments, the term “secrete” or “secretion** refers to transport of a molecule from the interior of a bacterium to its exterior, such as for example, periplasrnic space or extracellular environment ⁇ e.g. , internal environment of a host cell).
  • magnetic hyperthermia refers to use of or treatment with a magnetic field to induce hyperthermia in a target containing magnetic particles, such as a magnetosome.
  • the magnetic field applied for inducing hyperthermia is an alternating magnetic field.
  • Artificial endosymbionts of die present invention comprise a single celled organism, particularly bacteria, that are capable of surviving in a eukaryotic cell and maintain copy number such that the functionality ⁇ e.g., phenotype) introduced by the single-celled organism is observed in daughter cells of the eukaryotic cell.
  • the artificial endosymbiont secretes into the host cell a polypeptide(s), nucleic acid(s), other molecule(s), and/or other factor(s) in a regulated manner.
  • the polypeptide and/or nucleic acid are recombinant and heterologous to the artificial endosymbiont.
  • the artificial endosymbiont (e.g., magneto-bacteria) is heritable to the daughter cells of the eukaryotic host cell.
  • the artificial endosymbiont is maintained in host daughter cells through at least 3 cell divisions, or at least 4 division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell divisions or indefinitely for the lifetime of the host cell.
  • the artificial endosymbiont can be maintained in the host daughter cells through 3-5 divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 divisions.
  • the introduction of functionality involves expression of a gene in the single-celled organism.
  • the functionality involves expression of one or more genes or set of genes in the single-celled organism.
  • the functionality involves expression of a protein in the single-celled organism.
  • the functionality involves expression of a set of proteins in the single-celled organism.
  • the functionality involves expression of a gene or gene product that is transferred to the host to express the phenotype.
  • the host cell maintains the phenotype for at least 1 day, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.
  • the single celled organism can stably maintain the phenotype in host daughter cells through at least 3 cell divisions, or at least 4 division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell divisions or indefinitely for the lifetime of the host cell.
  • the single celled organism can stably maintain phenotype in the host daughter cells through 3-5 divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 divisions.
  • the single-celled artificial embodiments are a-Proteobacteria, such as magnetic-bacteria further described below.
  • a large number of a-proteobacterial genomes that cover all of the main groups within a-proteobacteria have been sequenced, providing information that identifies unique sets of genes or proteins that are distinctive characteristics of various higher taxonomic groups (e.g., families, orders, etc.) within ⁇ x- proteobacteria. (Gupta, supra).
  • single-celled organisms useful as artificial endosymbionts include, by way of example and not limitation, Anabaena, Nostoc, Diazotroph,
  • Halanaerobiales (low G+C brand of the Firmicutes), the red aerobic Salinibacter
  • Glaciecola Pseudoalteromonas, Shewanella, Polaribacter, Pseudomonas, Psychrobacter, Athrobacter, Frigoribacterium, Subtercola, Microbacterium, Rhodoccu, Bacillus,
  • the single celled organism for use as artificial endosymbionts clude, by way of example and not limitation, M. frigidum, M. burtonii, C. symbiosum, C.sychrerythraea, P. haloplanktis, Halorubrum lacusprofundi, Vibrio salmonicida,hotobacterium profundum, S. violacea, S. frigidimarina, Psychrobacter sp. 273-4, S.
  • xiguobacterium 255-15 Flavobacterium psychrophilum, Psychroflexus torquis,olaribacter filamentous, P. irgensii, Renibacterium salmoninarum, Leifsonia-relatedHSC20-cl, Acidithiobctcillus ferrooxidans, Thermoplasma acidophilum, Picrophilus rridus, Sulfolobus tokodaii, and Ferroplasma acidarmanus.
  • single-celled organisms useful as artificial endosymbionts are ose known to be intracellular pathogens or intracellular endosymbionts.
  • single celled organism is an intracellular pathogen characterized by genomic lands containing virulence genes encoding, for example, adherence factors that allow the tracellular pathogen to attach to target eukaryotic cells, and trigger phagocytosis of the tracellular pathogen (Juhas, M. et al., FEMS Microbiol Rev. 33:376-393, 2009). Many rulence factors utilize type III or type IV secretion systems.
  • Some virulence factors are creted into the eukaryotic host cell and alter membrane traffic within the target eukaryoticell, some virulence factors interact with host proteins involved in apoptosis. (Dubreuil, R. et ., Cell Logis. 1 :120-124, 2011).
  • the single celled organism useful as artificial endosymbionts clude by way of example and not limitation, endosymbionts found in insects such as uchnera, Wigglesworthia, and Wolhachia; the methanogenic endosymbionts of anaerobic liates; the nitrogen-fixing symbionts in the diatom Rhopalodia; the chemosyntheticndosymbiont consortia of gutless tubeworms (Olavius or Jnanidrillus); the cyanobacterialndosymbionts of sponges; the endosymbionts of all five extant classes of Echinodermata; e Rhizobia endosymbionts of plants; various endosymbiotic algae; the Legionella-like Xacteria endosymbionts of Ameoba proteus; numerous Salmonella sp., Mycobacteriumuberculosis, Legionella pneumophila
  • the single-celled organism useful as artificial endosymbiont is haracterized by a secretory system acquired by phagocytosis to evade the endocytic pathway nd allow the single celled organism to persist in the host cell.
  • the ngle-celled organism is characterized by the Dot-Icm Type IV secretory system, which is mployed by many intracellular bacteria to evade the endocytic pathway and persist in the ost cell.
  • This system has been well-studied in L pneumophila and consists of the proteins: DotA through DotP, DotU, DotV, IcmF, IcmQ through IcmT, IcmV, IcmW and IcmX.
  • the luminescent endosymbiont of nematodes In hotorhabdus lumirtescens, the luminescent endosymbiont of nematodes, the genes encoding TX-like toxins, proteases, type III secretion system and iron uptake systems were shown to upport intracellular stability and replication.
  • the gene bacA and the regulatory system vrRS are essential for maintenance of symbiosis between Rhtzobia and plants as well as the urvival of Brucella abortus in mammalian cells.
  • the PrfA regulon enables some Listeria pecies, e.g., Listeria monocytogenes, to escape the phagesome and inhabit the cytosol.
  • the esired cellular location (e.g., symbiosome or cytosol) of the intracellular MTB will dictate which genes are required to be expressed in the MTB (either directly from the genome or hrough a stable vector) for survival and proliferation in the host environment
  • the ndogenous plasm id pMGT is highly stable in MTB and a number of other broad range ectors (including those of IncQ, IncP, pBBRl, etc.) are capable of stable replication in MTB. hus, any of the foregoing single-celled organisms can be used as an artificial endosymbiont.
  • the single-ceiled organisms are genetically modified.
  • the bacteria are genetically modified to improve weir survivability in ukaryotic host cells, and/or to reduce the toxicity of the single-ceiled organism to the ukaryotic cell, and/or to provide the eukaryotic cell with a useful phenotype.
  • modifications can be directed modifications, random mutagenesis, introduction of eterologous genes, or a combination thereof.
  • molecular biology tools have een developed for genetic manipulations of MTB in AMB and M gryphiswaldense strain MSR-1 (reviewed in Jogler, C. and Schtiler, D., in "Magnetoreception and Magnetosomes in acteria," p 134-138, New York, Springer (2007), incorporated herein by reference).
  • the single-celled organism is genetically modified to express nd secrete polypeptide(s), nucleic acid(s), Hpid(s), carbohydrates), amino acid(s), or other actor(s) in a regulated manner into the host cell, as further described herein.
  • recombinant transport pathways are engineered in whole or in part into the rtificial endosymbiont for the delivery of target protein(s), nucleic actd(s), carbohydrate(s), pid(s), other molecule(s), and/or other factor(s) to the host cells.
  • the rget protein(s), nucleic acid(s), carbohydrate(s), lipid(s), other molecule(s), and/or otheractor(s) are directed to exert an effect on the host cells by acting on cellular componentsresent in the cytoplasm, nucleus, mitochondria, organelle, or other sites in or around the hostell.
  • the single-celled organisms are also genetically modified for able association of the single-celled organism with the host cell and/or selection of the hostell containing the single celled organism. Natural colonization of a host by the symbiontsan follow the following stages: 1) transmission, 2) entry, 3) countering of host defense, 4)ositioning, 5) providing advantage to the host, 6) surviving in host environment, and 7) gulation. Accordingly, in some embodiments, the single-celled organism is genetically odified to affect one or more of the foregoing stages, particularly providing advantage to e host, survivability in the host environment, and regulation. In some embodiments, the ngle celled organism is genetically modified to create mutual nutritional dependence iotrophy) between the single-celled organism and the eukaryotic cell. In some
  • the single-celled organism comprises at least one deletion or inactivation of aene encoding an enzyme for synthesizing an essential molecule, thereby resulting in absencef enzyme or expression of inactive enzyme, wherein said essential molecule is produced by e eukaryotic host cell.
  • An essential molecule can include, but is not limited to, an aminocid, a vitamin, a cofactor, and a nucleotide.
  • biotrophy can be accomplished bynocking-out the ability of the single-celled organism to make an amino acid, which will thene derived from the host.
  • An exemplary target is the metabolic pathway for synthesis of ycine, which is highly abundant in mammalian cells and a terminal product in bacterialmino acid biogenesis.
  • the gene encoding the enzymeerine hydroxymethyltransferase which converts serine into glycine at the terminus of the 3-hosphoglycerate biosynthetic pathway, is mutated (e.g., deletion, insertion, or substitution) eliminate presence of the enzyme or produce inactive enzyme.
  • the ngle-celled organism is an AMB in which the gene amb2339 (which encodes the enzymeerine hydroxymethyltransferase) is genetically modified.
  • genes encoding antibiotic resistance are inserted into theenome of the single-celled organism, and the eukaryotic host cell cultured in mediaontaining the antibiotic will require the single-celled organism for survival.
  • various antibiotic resistance genes can be introduced into the single- celled organism, such as neomycin resistance gene, hygromycin B resistance gene, and puromycin resistance gene.
  • Neomycin resistance is conferred by either one of two aminoglycoside phosphotransferase genes, which also provide resistance against geneticin (G418), a commonly used antibiotic for eukaryotes.
  • Hygromycin B resistance is conferred by a kinase that inactivates hygromycin B by phosphorylation.
  • Puromycin is a commonly used antibiotic for mammalian cell culture, and resistance is conferred by the pac gene encoding puromycin N-acetyl-transferase. External control of the antibiotic concentration allows intracellular regulation of the copy number of the single-ceiled organism. Any other system where resistance or tolerance to an external factor is achieved by chemical modification of this factor can also be employed.
  • the single-celled organism is genetically modified with other selection genes ⁇ e.g., negative or positive selection genes), such as bacteriostatic gene(s), siderophore gene(s), metabolic requirement gene(s), suicide gene(s), life cycle regulation gene(s), transporter gene(s), and escape from the phagosome gene(s).
  • negative or positive selection genes such as bacteriostatic gene(s), siderophore gene(s), metabolic requirement gene(s), suicide gene(s), life cycle regulation gene(s), transporter gene(s), and escape from the phagosome gene(s).
  • the single-celled organisms are randomly mutated and subsequently screened for enhanced integration within the host cell. Random mutation can be accomplished by treatment with mutagenic compounds, exposure to UV -light or other methods known to those skilled in the art.
  • the single-celled organism is genetically modified so that its cell cycle is coordinated with the cell cycle of the eukaryotic host cell so that copy number of the single-celled organism can be maintained at a sufficient level to impart the phenotype to daughter cells.
  • the genes localize artificial endosymbionts to specific subcellular locations.
  • the genes provide enhanced or blocked entry of the artificial endosymbionts to specific host cells.
  • the gene suppresses or alters the host immune system response to the artificial endosymbiont or genes and proteins expressed from it.
  • transgenetic modification(s) are made to counter eukaryotic cell defenses using genes from various parasites or endosymbionts.
  • the population of the single-celled organisms in the eukaryotic host cell is regulated though a balance of intrinsic use of host mechanisms (nutrient availability, control of reproduction, etc.) and antibiotic concentration.
  • the artificial endosymbiont or single cell rganism is an MTB characterized by the presence of magnetic particles, such as those ontaining magnetite (Fe 3 O 4 ) or greigite (Fe 3 S 4 ), enclosed by a sheath or membrane.
  • the single-celled organism is an MTB that produces magnetic particles upon ulturing of the eukaryotic host cell.
  • magneto-endosymbionts are agnetic bacteria capable of surviving in a eukaryotic cell, where the magneto-endosymbiont ecretes into the host cell a polypeptide(s), nucleic acid(s), other molecule(s), and/or otheractor(s) in a constitutive or regulated manner, particularly in a regulated manner.
  • the polypeptide and/or nucleic acid are recombinant and heterologous to the agneto-endosymbiont.
  • the magneto-endosymbiont introduces a phenotype into the ost cell through secretion from the magneto-endosymbiont into the host cell. In some mbodiments, this phenotype introduced by the magneto-endosymbiont is maintained in aughter cells. In some embodiments, the host cell maintains the phenotype for at least 1 day, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days.
  • e magneto-endosymbiont can stably maintain the phenotype in host daughter cells through least 3 cell divisions, or at least 4 division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell divisions or indefinitely for the lifetime of the ost cell.
  • the magneto-endosymbiont can stably maintain phenotype in e host daughter cells through 3-5 divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 visions.
  • the magneto-endosymbiont comprises magnetotactic bacteriaMTB.
  • MTB are a diverse group of bacteria that belong to different subgroups of the roteobacteria and Nitrospirae phylum, and are mostly represented within the a- roteobacteria.
  • Many MTB have a Gram-negative cell wall structure (inner membrane, eriplasm, and outer membrane).
  • Many MTB inhabit water bodies or sediments with vertical hemical concentration gradient, predominantly at the oxic-anoxic interface thus are ategorized as microaerophiles, anaerobic, facultative aerobic or some combination of thehree.
  • Many MTB are chemoorganoheterotrophic and some strains can also grow
  • MTB contain magnetosomes, which are intracellular structures comprising magnetic iron crystals enveloped by a hospholipid bilayer membrane (Gorby et al., J Bacteriol. 170(2): 834-41, 1988).
  • the transport pathways of MTB can deliver endogenous and/or heterologous proteins and/or nucleic acids to the host cell.
  • recombinant transport pathways are engineered in whole or in part into the magneto-endosymbiont for the delivery of target protein(s), nucleic acid(s), carbohydrate(s), lipid(s), other molecule(s), and/or other factor(s) to the host cells.
  • the target protein(s), nucleic acid(s), carbohydrate(s), lipid(s), other molecule(s), and/or other factor(s) are directed to exert an effect on the host cells by acting on cellular components present in the cytoplasm, nucleus, mitochondria, organelle, or other site in or around the host cell.
  • MTB species A large number of MTB species are known to those of ordinary skill in the art (see, e.g., Blakemore, "Magnetotactic bacteria," Science 24: 377-379, 1975; Faivre et al.,
  • MTB Magnetic tactic bacteria and magnetosomes
  • MTB have been identified in different subgroups of the Proteobacteria and the Nitrospira phylum with most of the phylotypes grouping in a-Proteobacteria.
  • Culturable MTB strains assigned as a-Proteobacteria by 16S rRNA sequence similarity include the strains Magnetospirillum magnetotactium (formerly Aquasprillium
  • M. gryphiswaldense M. magneticum strain AMB-1, M. polymorphum, Magnetosprillum sp. MSM-4 and MSM-6, Magnetococcus marinns, marine vibrio strains MV-1 and MV-2, a marine spirillum strain MMS-1 and Magnetococcus sp. strain MC-1, as well as others.
  • the magneto-endosymbiont can introduce multiple factors into the host cell without the need for integration of each individual gene into a host cell chromosome. Instead, the desired genes are introduced into the magneto-endosymbiont, and when the magneto-endosymbiont is introduced into the host cell the gene products of interest are introduced into the host cell. Expression of the gene products in the bacterium, and thus their introduction into the host cell, can be done in a regulated (controlled) or unregulated (uncontrolled, e.g., constitutive) manner. Magneto-endosymbionts may contain numerous constitutive or regulated genes either in operon cassettes, or as individual genes, allowing complete genetic programs to be transiently, or permanently, transferred to host cells.
  • Magneto-endosymbionts can utilize operon structures to express multiple genes from a single control region. By incorporating the operon structure, sets of genes can be engineered into the magneto-endosymbiont for coordinated expression, and groups of operons can be used to express sets of genes at different desired times. [0067] In some embodiments, the magneto-endosymbiont circumvents the need for integration of engineered genes into the host genome for long-term expression and provides necessary spatiotemporal control or removal of the genes at desired times.
  • magneto-endosymbiont also enables selective ablation of (a) the engineered magneto- endosymbiont, (b) magneto-endosymbiont and its host cell, or (c) magneto-endosymbiont, its host cell, and surrounding tissues.
  • the magneto-endosymbiont can be used to reprogram and differentiate host cells, thus directing cell fates and function in the body.
  • gene expression and viability of the artificial endosymbionts, particularly magneto-endosymbionts, and the cells that contain them can be controlled by a treatment with an agent that regulates expression of the gene of interest.
  • the treatment can be with a chemical, biological, or physical agent.
  • the chemical or biological agent induces expression of the gene.
  • the physical agent can comprise, among others, thermal, acoustic (e.g., ultrasound), electromagnetic (e.g., infrared-thermal, visible light, radio-frequency, X-ray, etc.), and magnetic radiation.
  • the treatment can be applied locally (e.g., focused), or applied to the organism as a whole.
  • low frequency alternating magnetic fields applied to the entire body can be absorbed by the magnetic structures in the magneto- endosymbiont and subsequently dissipated as heat.
  • This conversion of the alternating magnetic field into heat raises the temperature in the magneto-endosymbiont, and optionally the host cell and further optionally in surrounding tissues.
  • heat shock control regions are linked to the target genes in the magneto-endosymbiont, this system allows direct, noninvasive control of expression in the body via magnetic hyperthermia.
  • expression in the endosymbiont can be controlled with hyperthermia induced by focused ultrasound.
  • the role of host cells with magneto- endosymbionts in tissue regeneration can be followed using MRI, optical imaging, ultra sound, or nuclear medicine imaging methods such as positron emission tomography (PET) or single photon emission tomography (SPECT).
  • PET positron emission tomography
  • SPECT single photon emission tomography
  • magneto- endosymbionts are used to control stem cells while they reside in target tissues of the body.
  • magneto-endosymbiont can be used to deliver, control, and finally silence large sets of developmental genes in host cells resulting in differentiation of host cells into desired cell types at desired locations in the body.
  • magneto- endosymbionts are used to control cells to rebuild damaged tissues while they reside in target organs and tissues of the body.
  • magneto-endosymbionts are used to control stem cells to restore function to damaged tissues while they reside in target organs and tissues of the body. In some embodiments, magneto-endosymbionts are used to control stem cells to become cardiomyocytes, hepatocytes, beta cells, or other tissue specific cell types while they reside in target tissues and organs of the body.
  • magneto-endosymbionts Unlike passive magnetic particles, magneto-endosymbionts have the ability to self- replicate inside the host cells, and are thus retained over numerous cell divisions and maintain their magnetic properties through continued biogenesis of magnetosomes.
  • the artificial endosymbionts is designed to target specific cell types and enter these cells to become endosymbionts.
  • internalins such as InlA or InlB can be coded into the endosymbiont that facilitate entry into human epithelial cells, hepatocytes, fibroblasts, and epithelioid cells by interacting with surface proteins on these cells and inducing
  • the magneto-endosymbiont can be delivered systemically to an animal and localize to, and infect, a specific cell of a target organ or tissue.
  • the magneto-endosymbiont is directed to infect cells of the heart.
  • the magneto-endosymbiont is directed to infect damaged cells for directed repair.
  • the magneto-endosymbiont is directed to cells of the liver. In these embodiments the magneto-endosymbiont is designed for systemic delivery and targeted infection of host cells at a distance from the site of introduction into a host organism.
  • a natural endosymbiont or an intracellular parasite is genetically modified to produce magnetosomes.
  • Endosymbionts of insects such as Buchnera, Wigglesworthia, and Wolbachia; the methanogenic endosymbionts of anaerobic ciliates; the nitrogen-fixing symbionts in the diatom Rhopalodia; the chemosynthetic endosymbiont consortia of gutless tubeworms (Olavius or Inanidrillus); the cyanobacterial endosymbionts of sponges; the endosymbionts of all five extant classes of Echinodermata; the Rhizobia endosymbionts of plants; various endosymbiotic algae; the Legionella-like X bacteria endosymbionts of Ameoba proteus, numerous Salmonella sp., Mycobacterium tuberculosis, Legionella pneumophila belonging to a-
  • a pre-existing organelle can be genetically modified to express one or more magnetosome genes to produce an artificial endosymbiont.
  • mitochondria, plastids, hydrogenosomes, apicoplasts or other organelles, which harbor their own genetic material can be genetically altered.
  • Bacteria modified to produce magnetosomes can include Francisella tularensis, Listeria monocytogenes, Salmonella typhi, Brucella, Legionella, Mycobacterium, Nocardia, Rhodococcus equi, Yersinia, Neisseria meningitidis, Chlamydia, Rickettsia, Coxiella and the like. Methods for engineering cells to express magnetosomes are described in various publications, for example patent publication no. US2009/0311194, incorporated herein by reference).
  • secretion of protein(s), nucleic acid(s), other molecule(s) and other factor(s) from the artificial endosymbiont can make use of the endogenous secretion systems of the artificial endosymbiont.
  • the artificial endosymbiont can be engineered with heterologous secretion systems, or portions thereof, for directed secretion of these target molecules.
  • these transport systems of the invention are used to transport medically and industrially important genes, recombinant peptides/proteins, and/or other factors that will be useful for therapeutic, biopharmaceutical, agricultural, and industrial applications.
  • secretion systems are known in the art and described in the present disclosure, it is to be understood that practice of the present invention does not require knowledge or understanding of a specific secretion system.
  • the signals or sequences that cause secretion of polypeptides or other factors are known for many single celled organisms; they can also be found on known secreted proteins or nucleic acids, or identified based on similarities to sequences found on secreted proteins or nucleic acids.
  • These secretion signals can then be used, particularly as fusions, to direct a target molecule for secretion in the artificial endosymbiont into the host cell without prior knowledge of the specific transport system that directs secretion using the particular secretion signal.
  • a-proteobacteria have transport pathways that include ABC transporter-based pathways including the type I secretion system (T1SS), type II secretion systems (T2SS), type III secretion systems (T3SS), type IV secretion systems (T4SS), type V secretion systems (T5SS), type VI secretion systems
  • T1SS type I secretion system
  • T2SS type II secretion systems
  • T3SS type III secretion systems
  • T4SS type IV secretion systems
  • T5SS type V secretion systems
  • type VI secretion systems type VI secretion systems
  • T6SS type VII secretion systems
  • T7SS type VII secretion systems
  • these transport systems of a-proteobacteria are used to transport proteins, nucleic acids, and other factors from the magneto-endosymbiont into the host cell.
  • secreted proteins are exported across the inner and outer membrane in a single step via the T1SS, T3SS, T4SS, and T6SS pathways. Proteins are exported into the periplasmic space across the inner membrane via Sec or two-arginine (Tat) pathways. Proteins are transported across the outer membrane from the periplasmic space by T2SS, T5SS or less commonly by Tl SS or T4SS.
  • T1SS consists of three proteins; an inner membrane protein with a cytoplasmic ATPase domain operating as an ATP-binding cassette (ABC) transporter (Escherichia coli HlyB), a periplasmic adaptor (also known as membrane fusion protein, MFP; E. coli HlyD), and an outer membrane channel protein of the TolC family (E. coli TolC) (Delepelaire, P., "Type I secretion in gram-negative bacteria," Biochim Biophys Acta 1694: 149-161, 2004). These proteins form a pore in the periplasm through which an unfolded protein may be translocated.
  • ABSC ATP-binding cassette
  • T1SS protein substrates typically contain carboxy-terminal, glycine-and aspartate-rich repeats known as repeat-in-toxin (RTX) (Linhartova, I. et al., "RTX proteins: a highly diverse family secreted by a common mechanism," FEMS Microbiol. Rev.” 34:1076- 1 1 12, 2010; incorporated herein by reference) and are often located close to ABC and MFP genes. Due to its simplicity, T1SS has been used to transport heterologous proteins.
  • RTX repeat-in-toxin
  • HlyBD high level (1.5 g per liter) of protein from an E. coli.
  • the E. coli hemolysin transporter is also known to secrete other heterologous T1SS substrates expressed in E. coli, including exotoxins Cya of Bordatella. pertussis (Sebo et al., "Repeat sequences in the B.
  • pertussis adenylate cyclase toxin can be recognized as alternative carboxy-proximal secretion signals by the E. coli alpha-haemolysin translocator," Mol Microbiol. 9: 999-1009, 1993;
  • LtkA of Aggregatibacter actinomycetemcomitans (Lally et al., "Analysis of the Actinobacillus actinomycetemcomitans leukotoxin gene, Delineation of unique features and comparison to homologous toxins," J Biol Chem. 264: 15451-15456, 1989; incorporated herein by reference); PaxA of Pasteurella aerogenes (Kuhnert et al., "Characterization of PaxA and its operon: a cohemolytic RTXtoxin determined from pathogenic Pasteurella aerogenes," Infect lmmun.
  • Hly hemolysin secretion system
  • HlyA the T1SS substrate
  • HlyB ATP hydrolysis catalyzed by HlyB
  • HlyA is a member of the RTX (repeats in toxin) protein family and contains glycine-rich peptide repeats in the C-terminal domain, which have the consensus sequence GGXGXD (X represents any amino acid) and are important for the binding of Ca 2+ ions. This triggers folding of HlyA in the extracellular medium, which in turn generates the biologically active form of the toxin.
  • the Tl SS substrates contain a translocation signal at the C-terminus (last 27 to 218-amino acids fragment of HlyA), and a minimal secretion signal is located within the last -60 C-terminal amino acids and is both necessary and sufficient to direct secretion.
  • T1SS from Pseudomonas fluorescens is also known and has been used to secrete recombinant proteins.
  • Park et al identified a 105 amino acid polypeptide as the minimal region for recognition and transport by the lipase ABC transporter (Park et al., "Identification of the minimal region in lipase ABC transporter recognition domain of Pseudomonas fluoresceTM for secretion and fluorescence of green fluorescent protein," Microb Cell Fact. 11 :60, 2012; incorporated herein by reference).
  • a fusion of a target protein to this minimal region allowed secretion of a recombinant protein.
  • the versatility of T1SS for protein secretion is seen in its wide array of transport substrates, which vary from small proteins like the hemophore HasA (19 kDa) to huge surface layer proteins up to 900 kDa in size
  • RTX proteins a highly diverse family secreted by a common mechanism, FEMS Microbiol. Rev.” 34:1076-1112, 2010; Satchell, "Structure and function of MARTX toxins and other large repetitive RTX proteins," Ann Rev Microbiol. 65:71 -90, 2011 ; all publications incorporated herein by reference).
  • Other proteins secreted by T1SS include, for example, adenylate cyclases, lipases, and proteases.
  • Tl SS system that can be used in the invention is
  • RaxABC from Xanthomonas oryzae pv. oryzae.
  • Phylogenetic analysis identifies RaxB as an ABC transporter (da Silva et al., "Bacterial genes involved in type I secretion and sulfation are required to elicit the rice Xa21 -mediated innate immune response," Mol Plant Microbe Interact. 17:593-601, 2004; incorporated herein by reference), equivalent to HlyB from E. coli.
  • the RaxABC transport system is used to secrete AvrXa21 molecules (small sulfated polypeptides), metalloproteases, adhesion factors and glycanases (Delepelaire, supra; Reddy et al., "ToIC is required for pathogenicity of Xylella fastidioa in Vitis vinifera grape-vines," Mol Plant Microbe Interact. 20:403-410, 2007; all of which are incorporated herein by reference).
  • T1SS sequences and the others described above are used to promote secretion of target proteins from the magneto-endosymbionts of the invention.
  • an autotransporter comprises an N-terminal Sec-dependent signal sequence, a passenger domain, and a C-terminal beta-motif.
  • translocation is a two-step process.
  • the target protein is transported into the periplasm using .Sec-dependent transport whereupon the beta- motif forms a transmembrane pore through which the passenger domain is secreted out of the periplasm (Dautin et al., "Protein secretion in gram-negative bacteria via the autotransporter pathway," Ann Rev Microbiol.
  • the target protein is fused with the N-terminal signal sequence, the C-terminal signal, and the beta-domain that mediate translocation of a recombinant protein through the inner and outer membranes, respectively.
  • This chimeric gene has the N-terminal signal sequence fused in frame to the N-terminal end of the target gene, and a second, in frame fusion to DNA encoding the beta-domain sequence at the C-terminal end of the target gene.
  • the passenger domain may be replaced in the fusion protein.
  • Jong et al. defined passenger domains of E. coli autotransporter hemoglobin-binding protease (Hbp) that could be replaced in a fusion protein to facilitate secretion, along with an intact beta-domain (Jong et al., "A structurally informed
  • passenger domains that could be replaced by heterologous proteins are: (1) 53-308;(2) 533-608; (3) 657-697; (4) 735-766; (5) 898-922 amino acids.
  • E. coli autotransporters can also be used.
  • the YfaL autotransporter (NCBI accession no. P45508) can be used to secrete proteins ranging from 25.3 to 143 kDa from E. coli (Ko et al., "Functional cell surface display and controlled secretion of diverse agarolytic enzymes by Escherichia coli with a novel ligation- independent cloning vector based on the autotransporter YfaL," Appl Environ Microbiol.
  • a protease e.g., a tobacco etch virus protease
  • a tobacco etch virus protease is used to cleave the C-terminus of the fusion proteins to remove the beta-domain and autotransporter, resulting in secretion from the cell.
  • the E. coli serine protease Pet can be used to cleave fusion proteins and provide for complete secretion of a range of proteins varying in sizes and structures, and including multi-component proteins (Sevastsyanovich et al., "A generalized module for the selective extracellular accumulation of recombinant proteins," Microb. Cell. Fact. 11 :69, 2012; incorporated herein by reference).
  • Pet is one of the serine protease autotransporters of the Enterobacteriaceae (SPATEs) that releases passenger domain from the beta-domain.
  • An application of an autotransporter for consolidated bioprocessing uses an E. coli autotransporter Antigen 43 (Ag43) engineered to secrete a target protein.
  • This autotransporter system is unique in that the passenger domain Ag43alpha is self-cleaved yet the secreted domain is non-covalently attached to the beta-domain, forming an integral outer-membrane protein.
  • the segment of Ag43alpha containing the cleavage mechanism was fused to a target sequence.
  • T5SS A very large number of proteins are secreted via the T5SS, even more than the T2SS (Jacob-Dubuisson et al., "Protein secretion through autotransporter and two-partner pathway,” Biochim Biophy Acta 1694:235-257, 2004; Dautin et al., "Protein secretion in the gram-negative bacteria via autotransporter pathway,” Ann Rev Microbiol. 61 :89-l 12, 2007; all publications incorporated herein by reference). Most of the T5SS secreted proteins characterized to date are virulence factors. Proteins secreted via the T5SS include adhesions, such as AIDA-I and Ag43 of E.
  • T5bSS (TPS) secreted proteins include adhesions, such as HecA/HecB of the plant pathogen Dickeya dadantii (Erwinia
  • chrysanthemii chrysanthemii
  • cytolysins such as ShIA/ShIB of Serratia marcescens, HpmA/HpmB of Proteus mirabilis and EthA/EthB of Edwardsiellla tarda.
  • the T5SS sequences described above are used to promote secretion of target proteins from the magneto-endosymbionts of the invention.
  • T4SS type IV secretion system
  • T4SS The type IV secretion system
  • T4SS is a versatile, multi-component secretion system used by both gram-negative and gram-positive bacteria to secrete proteins, DNA, and protein- DNA complexes from a wide range of targeted eukaryotic and bacterial cells
  • Group 1 T4SSs mediate the conjugative transfer of plasmid DNA or transposons into a wide range of bacterial species.
  • E. coli and Agrobacterium tumifaciens can deliver DNA substrates into fungal, plant, or human cells (Grohmann et al., "Conjugative Plasmid Transfer in Gram-Positive Bacteria," Microbiol Mol Biol Rev.
  • T4SSs in group 2 such as those found in Helicobacter pylori and Neisseria gonorrhea, mediate the uptake and release of DNA into the extracellular environment (Smeets et al., "Natural Transformation in Helicobacter Pylori: DNA Transport in an Unexpected Way," Trends Microbiol 10(4): 159-62, 2002; Hamilton et al., "Natural Transformation of Neisseria Gonorrhoeae: From DNA Donation to Homologous
  • T4SSs deliver effector molecules into eukaryotic cells during infection.
  • H. pylori, Brucella suis and Legionella pneumophila are examples of bacteria that use their T4SSs to inject virulence proteins into mammalian host cells (Backert et al., "Type IV Secretion Systems and Their Effectors in Bacterial Pathogenesis,” Curr Opin Microbiol. 9(2):207-17, 2006; Corbel, "Brucellosis: An Overview,” Emerg Infect Dis.
  • H. pylori is an example of a bacterium that encodes multiple T4SSs.
  • H pylori has an effector protein delivery system encoded by the cag pathogenicity island and a DNA release and uptake system encoded by the comB gene cluster (Backert et al., "Type IV Secretion Systems and Their Effectors in Bacterial Pathogenesis," Curr Opin Microbiol. 9(2):207-17, 2006; Smeets et al., "Natural Transformation in Helicobacter Pylori: DNA Transport in an Unexpected Way," Trends Microbiol. 10(4): 159-62, 2002; all of which are incorporated herein by reference).
  • T4SS Depending on the structural components that compose a T4SS, the systems can be broadly classified as either type IVA or type IVB systems (Voth et al., "Bacterial Type IV Secretion Systems: Versatile Virulence Machines," Future Microbiol. 7(2):241-257, 201 1 ; incorporated herein by reference).
  • tumifaciens is the most well characterized T4SS.
  • the A. tumifaciens T4SS transporter complex and others similar to it typically consist of 1 1 VirB proteins (encoded by the virBl- virBll genes) and the coupling protein VirD4, an NTPase (Tegtmeyer et al., "Role of the Cag-Pathogenicity Island Encoded Type IV Secretion System in Helicobacter Pylori Pathogenesis," FEBSJ. 278(8): 1 190-202, 201 1; incorporated herein by reference).
  • Agrobacterial VirB proteins are grouped into three categories: core components, pilus- associated components and energetic components. T4SSs that fall under the type IV B classification were demonstrated in Legionella pneumophilia to consist of twenty-two structural proteins and 5 chaperone proteins (Vogel et al., "Conjugative Transfer by the Virulence System of Legionella Pneumophila," Science 279(5352):873-6, 1998; Segal et al., "Host Cell Killing and Bacterial Conjugation Require Overlapping Sets of Genes within a 22- Kb Region of the Legionella Pneumophila Genome,” Proc Natl Acad Sci USA 95(4): 1669- 74, 1998; all of which are hereby incorporated by reference).
  • Type IVA secretion systems and Type IVB secretion systems recognize different but overlapping translocation signals.
  • a recent study showed that two Brucella effectors can be translocated by L. pneumophila demonstrating that a type IVB secretion system can recognize translocation signals from type IVA secretion system effectors (de Jong et al., "Identification of Vcea and Vcec, Two Members of the Vjbr Regulon That Are Translocated into Macrophages by the Brucella Type IV Secretion System," Mol Microbiol. 70(6): 1378- 96, 2008; incorporated herein by reference).
  • T4SS translocation signals The A. tumifaciens translocation signal resides in a hydrophilic C-terminal region with a consensus R-X(7)-R-X- R-X-R-X-X(n) motif (Vergunst et al., "Positive Charge Is an Important Feature of the C- Terminal Transport Signal of the Virb/D4-Translocated Proteins of Agrobacterium," Proc Natl Acad Sci USA 102(3): 832-7, 2005; incorporated herein by reference).
  • Bartonella has a BID domain and a short positively charged tail sequence that together form a bipartite C- terminal translocation signal (Schulein et al., "A Bipartite Signal Mediates the Transfer of Type IV Secretion Substrates of Bartonella Henselae into Human Cells," Proc Natl Acad Sci USA 102(3):856-61, 2005; incorporated herein by reference).
  • Helicobacter pylori there is evidence that both the N- and C-terminal ends of the CagA protein have translocation signals.
  • Hohlefeld, et al. observed that residues 6-26 of CagA are important for translocation (Hohlfeld et al., "A C-Terminal Translocation Signal Is Necessary, but Not Sufficient for Type IV Secretion of the Helicobacter Pylori Caga Protein," Mol Microbiol. 59(5): 1624-37, 2006; incorporated herein by reference). Hohlfeld et al., also show that CagA translocation depends on the presence of its 20 C-terminal amino acids. These T4SS sequences and others described above are used to promote secretion of target proteins from magneto- endosymbionts of the invention.
  • Type 1, 4, and 5 secretion system genes have also been identified in the MTB
  • Magnetospirillum sp. strain AMB-1 genome by sequence alignments.
  • AMB-1 contains 83 genes that are involved in cell motility and secretion (Matsunga et al., "Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. AMB- 1," DNA Res. 12:157-166, 2005; incorporated herein by reference).
  • Several putative Tl SS substrates NCBI
  • YP_420631.1, YP_420638.1, YP_420640.1, YP_421364.1, YP_422662.1, YP_422785.1, and YP_423419.1) have been identified in MTB (M magneticum AMB-1) (Linhartova et al., "RTX proteins: a highly diverse family secreted by a common MTB (M magneticum AMB-1) (Linhartova et al., "RTX proteins: a highly diverse family secreted by a common
  • T1SS complex genes responsible for constituting the T1SS complex (such as those described by NCBI Accession Nos. YP_422838, YP_421739, homolog of E. coli HlyB; E.
  • MTB proteins that are either secreted or are part of the cells T1SS secretion machinery have been identified in the sequenced genomes of certain MTB, and these are used to secrete target proteins from the magneto-endosymbionts of the invention.
  • bacteria use diverse machinery to secrete proteins as a means for interacting with their environment, which in the case of endosymbionts includes the environment of the host cell.
  • the invention modifies the morphology or the physiology of the host cell (as well as organism) via protein(s), nucleic acid(s), and/or other factor(s) secreted from the artificial endosymbiont through a secretion system.
  • MTB are modified to achieve a higher level of recombinant protein secretion.
  • Methods for genetically modifying magneto-endosymbionts are well known in the art.
  • the magneto-endosymbiont is genetically modified to improve secretion of target molecules from the magneto-endosymbiont. Modifications may also involve increasing production of proteins or RNA by changing promoter or ribosome binding sequences, deletion or silencing of certain genes in the magneto-endosymbiont, or by other means well-known in the art.
  • the flagellar proteins of a magneto-endosymbiont are modified so that the flagellar proteins are no longer expressed.
  • Flagellar proteins have high homology to bacterial secretion systems suggesting a common evolutionary ancestor.
  • the flagellar proteins of a magneto-endosymbiont are modified to create a secretion system for target proteins.
  • the magneto- endosymbiont is modified so that it can no longer synthesize an essential molecule that is preferably provided by the eukaryotic host cell.
  • the magneto- endosymbiont is genetically modified so that its cell cycle is coordinated with the cell cycle of the eukaryotic host cell to maintain copy number of the magneto-endosymbiont at a sufficient level to impart the phenotype to daughter host cells.
  • genes or portions thereof of magneto-endosymbiont such as MTN or other MTB strains, are modified.
  • the genes encoding the magnetosome are modified, for example, such as to control the size of magnetosome for magnetic hyperthermia applications.
  • magnetosome formation involves at least 100 genes, which are organized in the magnetosome island (MAI).
  • Magnetosomes in wild-type AMB-1 range from 30 to 40 nm in diameter, and occur in single chains. Expression of magnetosome genes can be varied to change magnetosome size, shape and content.
  • a magnetosome size range of 35-120 nm can be obtained by genetic engineering of MAI loci (Baumgartner et al., "Magnetite biomineralization in bacteria,” Prog Mol Subcell Biol. 52:3-27, 2011 ; Jogler et al., “Genomics, genetics, and cell biology of magnetosome formation," Ann. Rev. Microbiol. 63:501-521, 2009; Lower et al., "The bacterial magnetosome: a unique prokaryotic organelle," J. Mol. Microbiol. Biotechnol.
  • AMB-1 The amount of iron present in AMB-1 can also be varied through manipulation of AMB-1 genes.
  • various engineering approaches are used to modify magneto- endosymbionts, including: 1) engineering into the magneto-endosymbiont dedicated secretion systems that naturally exist in other bacteria; 2) engineering cell envelope mutations into the magneto-endosymbiont so that it alters the outer membrane or peptidoglycan layer permeability ⁇ e.g., Shin et al., "Extracellular recombinant protein production from and Escherichia coli lpp deletion mutant," Biotechnol Bioeng.
  • the E. coli a-haemolysin transporter genes HlyB and HlyD are recombinantly expressed in the magneto-endosymbiont.
  • Target proteins are then engineered by fusing them with a T1SS substrate secretion signal, which is located in C-terminal of HlyA to target them to the a-haemolysin transporter system.
  • the HlyA secretion signal comprises the sequence:
  • the T1 SS from Pseudomonas fluorescens is used to transport recombinant proteins.
  • the T1SS genes such as TliA for the lipase ABC transporter are engineered into the magneto-endosymbiont.
  • the target protein is fused N-terminally to a 104 residue, minimal region comprising the sequence:
  • the RaxA, RaxB and RaxC genes of Xanthomonas oryzae pv. oryzae are recombinantly expressed in the magneto-endosymbiont.
  • the target protein is fused to RaxST, which is the recognition sequence for the RaxABC secretory system.
  • the RaxST recognition sequence comprises the sequence:
  • the target protein is fused at the N-terminal end to a Sec- dependent signal sequence, and the C-terminal end of the target protein is fused to a ⁇ -motif.
  • Translocation of such target protein fusions is a two-step process.
  • the target protein is transported into the periplasm using Sec-dependent transport whereupon the ⁇ -motif forms a transmembrane pore in the outer-membrane through which the target protein is secreted from the periplasmic space.
  • the ⁇ -motif is cleaved, allowing translocation of the target protein.
  • the target protein is fused to an autotransporter (e.g., target protein replaces the passenger domain region, such as amino acids 29 to 685 of the YfaL autotransporter (Ko et al., "Functional cell surface display and controlled secretion of diverse agarolytic enzymes by Escherichia colt with a novel ligation-independent cloning vector based on the autotransporter YfaL," Appl Environ Microbiol. 78:3051-3058, 2012;
  • an autotransporter e.g., target protein replaces the passenger domain region, such as amino acids 29 to 685 of the YfaL autotransporter
  • the YfaL autotransporter sequence comprises the sequence:
  • a protease is included to cleave the ⁇ -motif from the target protein, such as for example, a tobacco etch virus protease, E. coli serine protease Pet, or a serine protease autotransporter of the Enterobacteriaceae (SPATEs) that releases passenger domains from the ⁇ -domain, without requiring exogenous protease.
  • a tobacco etch virus protease E. coli serine protease Pet
  • SPATEs serine protease autotransporter of the Enterobacteriaceae
  • an E. coli autotransporter Antigen 43 is used with the target protein.
  • Ag43 E. coli autotransporter Antigen 43
  • the Antigen 43 autotransporter sequence comprises the sequence:
  • the translocase of the outer mitochondrial membrane is engineered into the magneto-endosymbiont.
  • the TOM complex includes the receptors Tom20, Tom22, Tom70 and the channel-forming protein Tom40, and several other small subunits (reviewed in Hoogenraad et al., Biochim BiophyActa 1592:97-105, 2002; Neupert et al., Ann. Rev. Biochem. 76:723-749, 2007; and Chacinska et al., Cell 138:628-644, 2009; all publications incorporated herein by reference).
  • Tom20 recognizes the substrate and transfers to centrally located Tom22, thereby the substrate is inserted into the Tom40 channel.
  • TOM Upon substrate import, TOM forms a complex with the translocase of the inner membrane (TIM complex) (Chacinska et al., EMBO J 22:5370-5381, 2003; incorporated herein by reference).
  • the TIM complex consists of four integral membrane proteins, Tim23, Tim 17, Tim50, and Tim21.
  • Tim23 forms the protein-conducting channel of the translocase and is tightly associated with Tom 17, whereas, Tim50 acts as regulator for the Tim23 channel and Tim21 transiently interacts with the TOM complex via Tom22 (Milisav et al., "Modular structure of the Tim23 preprotein translocase of mitochondria," J Biol Chem. 276:25856- 25861, 2001 ; incorporated herein by reference).
  • the artificial endosymbionts secrete into the host cell polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s), where the polypeptide(s), nucleic acid(s), lipid(s), carbohydrate(s), amino acid(s), or other factor(s) that are targeted or delivered to a specific cellular compartment, organelle or other locale of the host cell.
  • the secreted factor particularly polypeptide(s) and nucleic acid(s) contain a region, also referred to as a targeting sequence, which targets or directs the polypeptide or nucleic acid to a specific cellular compartment or organelle of the host cell.
  • a targeting sequence which targets or directs the polypeptide or nucleic acid to a specific cellular compartment or organelle of the host cell.
  • Various intracellular targets include by way of example and not limitation, Golgi, endoplasmic reticulum, nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and cellular membrane (see, e.g., U.S. Patent No. 6,455,247, incorporated herein by reference).
  • the secrete factor is directed to the cellular cytoplasm.
  • the secreted factors contain a nuclear localization sequence for delivery to the nucleus of the host cell(s).
  • a nuclear localization signal is a targeting peptide that directs proteins to the nucleus and is often a unit consisting of short basic, positively-charged amino acids.
  • the NLS normally is located anywhere on the peptide chain. Numerous NLS amino acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (PKKKLRKV; Kalderon et al., Cell 39:499-509, 1984); the human retinoic acid receptor ⁇ -nuclear localization signal
  • the NLS comprises double basic NLS's, as exemplified by that of the Xenopus (African clawed toad) protein, nucleoplasms (AVKRPAATKKAGQAKKKKLD; Dingwall et al., Cell 30:449-458, 1982; and Dingwall et al., J Cell Biol. 107:641-849, 1988).
  • the targeting sequence is a nucleolar localization signal
  • nucleolar targeting sequences include, among others: SQDSKKKKKKKEKKKHKKHKKHKKHKKH,
  • the targeting sequence is a lysosomal targeting sequence, including, for example, a lysosomal degradation sequence such as Lamp-2 (KFERQ; Dice, Ann. N. Y. Acad Sci. 674:58, 1992); or lysosomal membrane sequences from Lamp-1 (MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI, Uthayakumar et al., Cell Mol Biol Res. 41 :405, 1995) or Lamp-2 (LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF; Konecki et la., Biochem Biophys Res Comm. 205:1-5, 1994).
  • Lamp-2 KFERQ; Dice, Ann. N. Y. Acad Sci. 674:58, 1992
  • Lamp-2 LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF
  • the secreted factors contain a mammalian mitochondrial localization signal for delivery of the secreted factor to the mitochondria of the host cell(s).
  • the mitochondrial targeting sequence is about 10-70 amino acid long peptide that directs a newly synthesized proteins to the mitochondria. It can be found at the N-terminus and can consist of an alternating pattern of hydrophobic and positively charged amino acids to form what is called an amphipathic helix. Mitochondrial targeting signals can contain additional signals that subsequently target the protein to different regions of the
  • mitochondrial targeting signals are cleaved once targeting is complete.
  • Exemplary mitochondrial localization sequence include, among others, mitochondrial matrix sequences ⁇ e.g., yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST; Schatz, Eur J Biochem. 165:1- 6, 1987); mitochondrial inner membrane sequences (yeast cytochrome c oxidase subunit IV; MLSLRQSIRFFKPATRTLCSSRYLL; Schatz, supra); mitochondrial intermembrane space sequences (yeast cytochrome c 1 ;
  • the secreted factors contain a Golgi/endoplasmic reticulum localization signal to target or deliver the secreted factor to the Golgi/endoplasmic reticulum of the host cell(s).
  • the Golgi/endoplasmic reticulum targeting sequence comprises an amino acid ER retention sequence, such as that found in calreticulin (KDEL; Pelham, Royal Society London Transactions B; 1-10, 1992) or adenovirus E3/19K protein (LYLSRRSFIDEKKMP; Jackson et al., EMBOJ. 9:3153, 1990).
  • KDEL calreticulin
  • LYLSRRSFIDEKKMP adenovirus E3/19K protein
  • the secreted factors contain a localization signal to target or deliver the factor to a peroxisome of the host cell(s).
  • a localization signal to target or deliver the factor to a peroxisome of the host cell(s).
  • At least two types of targeting sequences have been identified for targeting to peroxisome, also referred to as peroxisomal targeting signals (PTS).
  • PTS peroxisomal targeting signals
  • the peroxisomal targeting sequence is PTS1, which is typically comprised of three amino acids located on the C-terminus.
  • An exemplary PTS1 comprises the sequence SKL.
  • the peroxisomal targeting sequence is PTS2, having in some embodiments, a general sequence (R/K)-(L/V/I)-XXXX-(H/Q)-(L/A) (see, e.g., Rachubinski et al., Cell 83: 525-528, 1995).
  • An exemplary PTS2 comprises RQQVLLGHL or RLQVVLGHL.
  • Other variations of peroxisomal targeting sequences are described in Lazarow, P.B., Biochim Biophy Acta 1763(12): 1599— 1604, 2006; incorporated herein by reference.
  • the secreted factors contain signals to deliver the secreted factor to the plasma membrane of the host cell(s).
  • secretory signal sequences which are placed 5' to a polypeptide, and are cleaved from the polypeptide region to target the polypeptide to the secretory pathway.
  • Secretory signal sequences and their transferability to unrelated proteins are well known (see, e.g., Silhavy, et al., Microbiol Rev. 49, 398-418, 1985; incorporated herein by reference).
  • a secreted extracellular polypeptide is useful for binding to the surface of, or affecting the physiology of, a target cell that is other than the host cell.
  • the cell surface membrane localization signal comprises a signal peptide, which is generally a short (about 5-30 amino acids long) peptide present at the N-terminus of the majority of synthesized proteins that are destined towards the secretory pathway. Proteins that contain such signals are destined for either extra-cellular secretion, the plasma membrane, the lumen or membrane of either the (ER), Golgi or endosomes.
  • the core of the signal peptide contains a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • signal peptides begin with a short positively charged stretch of amino acids, which may help to enforce proper topology of the polypeptide during translocation.
  • signal peptidase there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • Suitable signal peptides are well known, including sequences from IL-2 (MYRMQLLSCIALSLALVTNS; Villinger et al., J Immunol 155:3946, 1995), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT; Roskam et al., Nucleic Acids Res. 7:30, 1979); preproinsulin
  • the secreted factors contain membrane anchoring domains along with signals sequences to target the secreted factor to the plasma membrane of the host cell(s) for directed expression on the cell surface of the host cell(s).
  • the targeting sequence is a membrane anchoring signal sequence.
  • a membrane anchoring region is provided at the carboxyl terminus of the polypeptide.
  • the transmembrane proteins are inserted into the membrane such that the regions encoded 5' of the transmembrane domain are extracellular and the sequences 3' become intracellular.
  • the transmembrane domains are placed 5' of the polypeptide, thus serving to anchor it as an intracellular domain, which may be desirable in some embodiments.
  • Exemplary membrane-anchoring sequences include, but are not limited to, those derived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.
  • Useful sequences include sequences from: (1) class I integral membrane proteins such as IL-2 receptor beta-chain (residues 1-26 are the signal sequence, 241-265 are the transmembrane residues; see
  • CD8 and ICAM-2 are particularly preferred.
  • the signal sequences from CD8 and ICAM-2 lie at the extreme 5' end of the transcript. These comprise the amino acids 1-32 in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; Nakauchi et al., Proc Natl Acad Sci USA 82:5126, 1985) and 1-21 in the case of ICAM-2 (MSSFGYRTLTVALFTLICCPG; Staunton et al., Nature 339:61, 1989).
  • These leader sequences deliver the construct to the membrane while the hydrophobic transmembrane domains, placed 3' of the random candidate region, serve to anchor the construct in the membrane.
  • Transmembrane domains are encompassed by amino acids 145-195 from CD8 (PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR; Nakauchi, supra) and 224-256 from ICAM-2 (MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR; Staunton, supra).
  • the membrane anchoring sequence comprises a GPI anchor sequence, which results in a covalent bond formation between the molecule and a glycosyl- phosphatidylinositol moiety, thus anchoring the protein to the lipid bilayer.
  • GPI anchor sequence is contained in the sequence
  • PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT see, e.g., Homans et al., Nature 333(6170):269-72, 1988; and Moran et al., J Biol Chem. 266:1250, 1991).
  • the membrane anchoring domain comprises a myristylation sequence.
  • myristylation of c-src recruits the protein to the plasma membrane.
  • the first 14 amino acids of the protein are solely responsible for this function:
  • Palmitoylation can be used to anchor polypeptides in the plasma membrane; for example, palmitoylation sequences from the G protein-coupled receptor kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL; Stoffel et al., J Biol Chem. 269:27791, 1994); from rhodopsin (KQFRNCMLTSLCCGK PLGD; Barnstable et al., J Mol Neurosci. 5(3):207, 1994); and the p21 H-ras 1 protein
  • a method for introducing a phenotype into a host cell can comprise expressing a polypeptide, nucleic acid or other factor in an artificial endosymbiont in a eukaryotic host cell, where the polypeptide, nucleic acid or other factor comprises a intracellular localization signal, e.g., targeting sequence.
  • the polypeptide, nucleic acid or other factor expressed in the artificial endosymbiont is secreted into the host cell, and induces a phenotype in the host cell by localization to specific intracellular target(s), e.g., an organelle or cellular membrane.
  • the expression can be constitutive, without the use of a regulatable control region.
  • expression in the artificial endosymbiont can be constitutive or regulatable.
  • the nucleic acids of the invention include those that encode at least in part the individual peptides, polypeptides and proteins secreted in the method of the disclosure.
  • the peptides, polypeptide and proteins can be natural, synthetic or a combination thereof.
  • the nucleic acids of the invention also include the nucleic acids that are secreted from the magneto-endosymbiont into the host cell.
  • the nucleic acids of the invention may be RNA, mRNA, microRNA, siRNA, shRNA, DNA or cDNA.
  • the nucleic acids of the invention also include expression constructs, such as plasmids, or viral vectors, or linear vectors, or vectors that integrate into chromosomal DNA.
  • Expression constructs can contain a nucleic acid sequence that enables the construct to replicate in one or more selected host cells. Such sequences are well known for a variety of cells. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria. In eukaryotic host cells, e.g., mammalian cells, the expression construct can be integrated into the host cell chromosome and then replicate with the host chromosome.
  • constructs can be integrated into the chromosome of prokaryotic cells.
  • Expression constructs also generally contain a selection gene, also termed a selectable marker.
  • Selectable markers are well-known in the art for prokaryotic and eukaryotic cells, including host cells of the invention. This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the constructs containing the selection gene will not survive in the culture medium.
  • Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • antibiotics or other toxins e.g., ampicillin, neomycin, methotrexate, or tetracycline
  • c supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.
  • One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen.
  • Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well-known in the art.
  • the expression construct for producing a heterologous polypeptide contain an inducible control region that is recognized by the host RNA polymerase and is operably linked to the nucleic acid encoding the protein or polypeptide, or expression of the nucleic acid that is to be secreted into the host cell.
  • Inducible or constitutive promoters (or control regions) with suitable enhancers, introns, and other regulatory sequences are well-known in the art.
  • the control regions comprise regulatable control regions, where expression from the control region can be induced or inhibited.
  • the inducible control regions that may be used include heat shock control regions that are induced to express gene products when a cell is undergoing a heat shock response.
  • heat shock control regions include, for example, the magneto-endosymbiont homologues of the mammalian Hsp60 or Hsp70 proteins, that is, the chaperones GroE or DnaK in E. coli (see also, e.g., U.S. Patent No.
  • the expression constructs of the invention may also utilize temperature-sensing RNA structures from the 5' untranslated regions of thermally regulated mRNAs. These RNA structures respond to temperature changes, and can be used in tandem with heat shock control regions to thermally regulate expression in the magneto- endosymbiont (Klinkert et al., "Microbial thermosensors," Cell. Mol. Life Sci. 66:2661-2676, 2009; incorporated herein by reference).
  • expression constructs with thermally regulated control regions and RNA hairpins are used for spatiotemporal control of gene expression from magneto-endosymbionts inside host cells.
  • Gene expression based on heat shock control regions can be regulated by methods that can raise the temperature sufficiently to induce expression from the heat shock control region. These include, among others, alternating magnetic fields, ultrasound, thermal (infrared) radiation, laser illumination (e.g., 532 nm irradiation; see, e.g., Ramos et al., BMC Dev Biol. 6:55-70, 2006), radio- frequency, and microwave radiation.
  • the heat shock control region can be natural or synthetic, including hybrid heat shock control regions.
  • the regulatable control region can be controlled with a chemical agent.
  • a chemical agent e.g., IPTG
  • trp promoter e.g., indoleacrylic acid
  • P. putida cmt promoter e.g., cumate; see aczmarczyk et al., Appl Environ Microbiol.
  • tet promoter -tTA/TetR e.g., doxycycline
  • rapamycin inducible promoters e.g., rapamycin and analogs; see Wang et al., Gene Ther. 13: 187-190, 2006
  • ecdysone regulatable promoters e.g., ecdysone; see, e.g., U.S. Patent No. 7,091,038.
  • the regulatable control region is a control region that can be controlled using light, also referred to as light-switchable expression systems.
  • light-switchable expression systems include, among others, those based on photoreceptor phytochrome, flavin chromophore, and photolyase like crytochromes (Shimizu-Sato et al., Nature Biotech. 20:1041-1044, 2002; U.S. Patent No. 6,858,429; U.S. patent publication 20130345294; all publications incorporated herein by reference).
  • regulated expression from a control region generally requires presence of one or more accessory molecules, such as a repressor protein, that controls expression from the control region.
  • accessory molecules such as a repressor protein
  • the bacterial host cell of the invention expresses these accessory proteins necessary for controlling expression.
  • Such accessory proteins can be encoded on
  • extrachromosomal nucleic acids e.g., plasm ids
  • Methods, genes and vectors for expressing such accessory molecules in a host cell are well known and well within the skill of those in the art. In some embodiments, it may be necessary to de-repress expression as a way of controlling expression.
  • polypeptides of the invention it may be desirable to modify the polypeptides of the invention.
  • One of skill will recognize many ways of generating alterations in a given nucleic acid construct encoding a polypeptide. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques (see, e.g., Giliman and Smith, Gene 8:81-97, 1979; Roberts et al., Nature 328: 731-734, 1987; each publication incorporated herein by reference).
  • the recombinant nucleic acids encoding the polypeptides of the invention are modified to provide preferred codons which enhance translation of the nucleic acid in a selected organism.
  • the polynucleotides of the invention also include polynucleotides including nucleotide sequences that are substantially equivalent to the polynucleotides of the invention. Polynucleotides according to the invention can have at least about 80%, more typically have at least about 90%, and even more typically have at least about 95%, sequence identity to a polynucleotide of the invention.
  • the invention also provides the complement of the polynucleotides including a nucleotide sequence that has at least about 80%, more typically at least about 90%, and even more typically at least about 95%, sequence identity to a polynucleotide encoding a polypeptide recited above.
  • the polynucleotides of the invention also encompass those nucleic acids which will hybridize under stringent conditions to a polynucleotide of the invention.
  • the polynucleotide can be DNA (genomic, cDNA, amplified, or synthetic) or RNA. Methods and algorithms for obtaining such polynucleotides are well known to those of skill in the art and can include, for example, methods for determining hybridization conditions which can routinely isolate polynucleotides of the desired sequence identities.
  • Nucleic acids which encode protein analogs in accordance with this invention may be produced using site directed mutagenesis or PCR amplification in which the primer(s) have the desired point mutations.
  • site directed mutagenesis or PCR amplification in which the primer(s) have the desired point mutations.
  • suitable mutagenesis techniques see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), and/or Ausubel et al., editors, Current Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, N.Y. (1994), which are hereby incorporated by reference in its entirety for all purposes.
  • Chemical synthesis using methods described by Engels et al., Angew Che Intl Ed. 28:716-734, 1989; which is hereby incorporated by reference in its entirety for all purposes, may also be used to prepare such nucleic acids.
  • Recombinant variant refers to any polypeptide differing from naturally occurring polypeptides by amino acid insertions, deletions, and substitutions, created using recombinant DNA techniques. Guidance in determining which amino acid residues may be replaced, added, or deleted without abolishing activities of interest, such as enzymatic or binding activities, may be found by comparing the sequence of the particular polypeptide with that of homologous peptides and minimizing the number of amino acid sequence changes made in regions of high homology. [0129] Preferably, amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements.
  • Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine
  • polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine
  • positively charged (basic) amino acids include arginine, lysine, and histidine
  • negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • “Insertions” or “deletions” are typically in the range of about 1 to 5 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.
  • insertions, deletions or non- conservative alterations can be engineered to produce altered polypeptides or chimeric polypeptides.
  • Such alterations can, for example, alter one or more of the biological functions or biochemical characteristics of the polypeptides of the invention.
  • such alterations may change polypeptide characteristics such as ligand-binding affinities or degradation/turnover rate.
  • such alterations can be selected so as to generate polypeptides that are better suited for expression, scale up and the like in the host cells chosen for expression.
  • recombinant variants encoding these same or similar polypeptides may be synthesized or selected by making use of the "redundancy" in the genetic code.
  • Various codon substitutions such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a particular prokaryotic or eukaryotic system.
  • Mutations in the polynucleotide sequence may be reflected in the polypeptide or domains of other peptides added to the polypeptide to modify the properties of any part of the polypeptide, to change characteristics such as ligand-binding affinities, or degradation/turnover rate.
  • polynucleotides encoding the novel nucleic acids are changed via site-directed mutagenesis.
  • This method uses oligonucleotide sequences that encode the polynucleotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.
  • site-directed mutagenesis is well known to those of skill in the art and this technique is exemplified by publications such as, Edelman et al., DNA 2:183, 1983.
  • a versatile and efficient method for producing site-specific changes in a polynucleotide sequence was published by Zoller et al., Nucleic Acids Res. 10:6487-6500, 1982.
  • PCR may also be used to create amino acid sequence variants of the novel nucleic acids.
  • primer(s) that differs slightly in sequence from the corresponding region in the template DNA can generate the desired amino acid variant.
  • PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the target at the position specified by the primer. The product DNA fragments replace the corresponding region in the plasm id and this gives the desired amino acid variant.
  • a further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., Gene 34:315, 1985; which is hereby incorporated by reference in its entirety for all purposes; and other mutagenesis techniques well known in the art, such as, for example, the techniques in Sambrook et al., supra, and Current Protocols in Molecular Biology, Ausubel et al., supra.
  • the present invention provides a eukaryotic host cell containing a artificial endosymbiont, particularly a magneto-endosymbiont, wherein the artificial endosymbiont imparts a phenotype to the host cell through secretion of proteins, nucleic acids, and/or other factors from the artificial endosymbiont into the host cell.
  • the artificial endosymbiont is heritable.
  • the host cells of the invention are animal cells.
  • the host cells are mammalian, such as mouse, rat, rabbit, hamster, human, porcine, bovine, or canine. Mice routinely function as a model for other mammals, most particularly for humans (see, e.g., Hanna et al., "Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin,” Science 318:1 20-1923, 2007;
  • Exemplary animal cells include, among others, fibroblasts, epithelial cells (e.g., renal, mammary, prostate, lung), keratinocytes, hepatocytes, adipocytes, endothelial cells, and hematopoietic cells.
  • epithelial cells e.g., renal, mammary, prostate, lung
  • keratinocytes e.g., hepatocytes
  • adipocytes e.g., endothelial cells
  • endothelial cells hematopoietic cells.
  • the host cell is a cancer cell, including human cancer cells.
  • the cancer cells are cancer cell lines, many of which are well known to those of ordinary skill in the art, including common epithelial tumor cell lines such as Coco- 2, MDA-MB231 and MCF7; and non-epithelial tumor cell lines, such as HT-1080 and HL60, and the NCI60-cell line panel (see, e.g., Shoemaker, "The NCI60 human tumor cell line anticancer drug screen," Nature Reviews Cancer 6:813-823, 2006; incorporated herein by reference). Additionally, those of ordinary skill in the art are familiar with obtaining cancer cells from primary tumors.
  • Cancer cells also include, for example, solid tumor cell types, hematopoietic cancer cells, carcinomas, sarcomas, leukemias, lymphomas, gliomas, as well as specific tissue related cancers such as prostate cancer, breast cancer, lung cancer, colorectal cancer, pancreatic cancer, melanoma, glioblastoma, and liver cancer.
  • the host cells are stem cells.
  • stem cells include embryonic stem cells, inducible pluripotent stem cells, hematopoietic stem cells, neural stem cells, epidermal neural crest stem cells, mammary stem cells, intestinal stem cells, mesenchymal stem cells, olfactory adult stem cells, testicular cells, and progenitor cells (e.g., neural, angioblast, osteoblast, chondroblast, pancreatic, epidermal, etc.).
  • stem cells of the invention may be pluripotent, oligopotent, or unipotent.
  • the host cell is a cell of the circulatory system of a mammal, including humans.
  • these cells are defined to be circulating host cells of the invention.
  • the present invention may be used with any of these circulating cells.
  • the host cell is a T-cell.
  • the host cell is a B-cell.
  • the host cell is a neutrophil. In some
  • the host cell is a megakaryocyte.
  • the host cell is a cell that is resident in the tissues and organs of a living animal.
  • the host cell is a fungal cell, including, but not limited to, the genera Saccharomyces, Klyuveromyces, Candida, Pichia, Debaromyces, Hansenula,
  • At least one gene from the host cell is genetically altered, as described herein.
  • mutual nutritional dependence sometimes referred to as biotrophy
  • biotrophy may be established between the artificial endosymbiont and the host cell by genetic modification of the host cell, using appropriate molecular biology techniques specific to the target host cell type known to those of ordinary skill in the art, creating host cell dependence on the artificial endosymbiont for some essential
  • nutritional dependence for a artificial endosymbiont on the host cell may be established by genetically altering the host cell to eliminate the ability of the host cell to synthesize various metabolites, cofactors, vitamins, nucleotides, or other essential molecules.
  • the essential molecule may be provided by the artificial endosymbiont.
  • hydroxymethyltransferase which converts serine into glycine at the terminus of the 3- phosphoglycerate biosynthetic pathway for amino acid production, may be modified.
  • the single-celled organisms of the invention can be introduced into host cells by a number of methods known to those of skill in the art including, but not limited to, microinjection, natural phagocytosis, induced phagocytosis,
  • a single-celled organism is introduced to the host cell by microinjection into the cytoplasm of the host cell.
  • microinjection techniques are known to those skilled in the art. Microinjection is the most efficient transfer technique available (essentially 100%) and has no cell type restrictions (Microinjection and Organelle Transplantation Techniques, 1986; Xi et al., "Characterization of Wolbachia transfection efficiency by using microinjection of embryonic cytoplasm and embryo homogenate," Appl Environ Microbiol.
  • Naturally phagocytotic cells have been show to take up bacteria, including MTB (Burdette et a!., "Vibrio VopQ induces PI3-kinase independent autophagy and antagonizes phagocytosis," Molecular microbiology 73:639, 2009; Wiedemann, et al., "Yersinia enterocolitica invasin triggers phagocytosis via ⁇ integrins, CDC42Hs and WASp in macrophages," Cellular Microbiology 3:693, 2001 ;
  • Hackam et al. "Rho is required for the initiation of calcium signaling and phagocytosis by Fey receptors in macrophages," J Exp Med.
  • non-phagocytotic cell types can be induced to endocytose bacteria when co-cultured with various factors, such as media, chemical factors, and biologic factors, for example, baculovirus, protein factors, genetic knock-ins, etc.
  • factors such as media, chemical factors, and biologic factors, for example, baculovirus, protein factors, genetic knock-ins, etc.
  • differentiated/committed stem cell progenies Potential advantages over transfection with marker genes, fluorescence-activated and magnetic affinity cell-sorting," Med Hypotheses 65(2):334-336, 2005; Potrykus, Ciba Found Symp. 154:198, 1990; each publication incorporated herein by reference). This method is inexpensive, relatively simple and scalable. Additionally, liposome uptake can be enhanced by manipulation of incubation conditions, variation of liposome charge, receptor mediation, and magnetic enhancement (see, e.g., Pan et al., Int. J. Pharm.
  • Erythrocyte-mediated transfer is similar to liposome fusion and has been shown to have high efficiency and efficacy across all cell types tested (Microinjection and Organelle Transplantation Techniques, Celis et al. Eds., Academic Press: New York (1986);
  • erythrocytes are loaded by osmotic shock methods or electroporation methods (Schoen et al., "Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles," Gene Therapy 6:823-832, 1999; Li et al., "Electrofusion between heterogeneous-sized mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation," BiophyJ. 71:479-486, 1996;
  • erythrocytes may be loaded indirectly by loading hematopoietic progenitors with single-celled organisms and inducing them to differentiate and expand into erythrocytes containing single-celled organisms.
  • Electroporation is a commonly used, inexpensive method to deliver factors to cells. (Potrykus, "Gene transfer methods for plants and cell cultures,” Ciba Found Symp 154: 198- 208, 1990; Wolbank et al., "Labeling of human adipose-derived stem cells for non-invasive in vivo cell tracking," Cell Tissue Bank 8: 163-177, 2007; each publication incorporated herein by reference).
  • a host cell that naturally endocytoses bacteria e.g., Chinese hamster ovary (CHO)
  • CHO Chinese hamster ovary
  • the modified single-celled bacteria are added to the CHO culture directly.
  • CHO cells can be cultured by standard procedures, for example, in Ham's F-12 media with 10% fetal calf serum media prior to infection with the MTB. Post infection, the media is augmented with additional iron (40 to 80 ⁇ ) as either ferric malate or FeCl 3 .
  • symbiosomes from one cell can be transplanted to another cell type (e.g., one incapable of endocytosis of magneto-endosymbionts) using microinjection, organelle transplantation, and chimera techniques. These host cells are cultured in typical media and techniques for the specific cell type.
  • a single-celled organism is introduced to the host cell by a liposome mediated process.
  • Mitochondria and chloroplasts which are larger than MTB, have been efficiently introduced into eukaryotic cells when encapsulated into liposomes (see, e.g., Bonnett, H. T. Planta 131 :229, 1976; Giles et al., "Liposome-mediated uptake of chloroplasts by plant protoplasts," In Vitro Cellular & Developmental Biology - Plant 16(7) : 581 -584, 1976; each publication incorporated herein by reference).
  • liposome fusion protocols and agents are available and can be used by the skilled artisan without undue experimentation (see, e.g., Ben-Haim et al., "Cell-specific integration of artificial organelles based on functionalized polymer vesicles," Nano Lett. 8(5): 1368-1373, 2008; Lian et al., "Intracellular delivery can be achieved by bombarding cells or tissues with accelerated molecules or bacteria without the need for carrier particles," Exp Cell Res.
  • a single-celled organism is introduced to the host cell by an infectious process much like naturally occurring intracellular pathogens.
  • a single-celled organism is introduced to the host cell by a mechanism related to Listeria infection.
  • a single-celled organism is introduced to the host cell by a mechanism related to Salmonella infection.
  • a single-celled organism is introduced to the host cell by a mechanism related to Rickettsia infection.
  • a single-celled organism is introduced to the host cell by a mechanism related to Chlymidia infection.
  • the artificial endosymbionts of the invention introduce into host cells nucleic acids, peptides/polypeptides, and/or other factors. These polypeptides, nucleic acids, or other factors can alter gene transcription or translation, post translational modifications, host cell differentiation, remodeling, proliferation, sensitivity, cell surface proteins or response to external and/or internal stimuli, metabolic, anabolic or other biochemical processes.
  • the artificial endosymbiont can control host cells through expression, availability, and delivery of certain transcription factors, growth factors, cell surface markers or other recombinant proteins.
  • the artificial endosymbiont may also introduce a desirable phenotype to the host cell through the polypeptides, nucleic acids or other factors that are secreted into the host cell from the artificial endosymbiont.
  • the artificial endosymbiont comprises a magneto-endosymbiont, e.g., a magnetotactic bacterium.
  • the proteins, nucleic acids, or other factors secreted from an artificial endosymbiont into the host cell can be used for cell viability, proliferation, differentiation, de- differentiation, growth, detoxification, cell labeling, treating a pathology or deficiency, creating energy, inducing cell death, inducing angiogenesis, neurogenesis, osteogenesis, or wound healing, modifying cell signaling, modifying gene expression, neutralizing intracellular proteins or nucleic acids, or modifying cell function by providing: nutrients, growth factors, proteins, minerals, nucleic acids, therapeutic agents, small molecules, ions, chemokines, polysaccharides, lipids, metals, cofactors, or hormones.
  • the artificial endosymbiont and host cells may also be used to manufacture bioremediation agents, enzymes, neurotransmitters, polypeptides, carbohydrates, pesticides, fertilizers, etc.
  • the artificial endosymbiont can secrete a protein or other factor that provide a beacon for the host cell from a reporter such as a fluorescent protein (e.g., GFP, RFP, YFP, CFP), and/or luciferase.
  • a reporter such as a fluorescent protein (e.g., GFP, RFP, YFP, CFP), and/or luciferase.
  • the artificial endosymbiont can secrete a protein or other factor that provide a cell surface marker for the host cell from a protein such as a nerve growth factor, immune cell markers, adhesion molecules or other proteins present on the cell surface.
  • the artificial endosymbiont can secrete an amino acid into the host cell, including, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or combinations of these amino acids.
  • amino acid including, alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, or combinations of these amino acids.
  • the artificial endosymbiont can secrete into a host cell a nucleic acid, RNA, mRNA, hnRNA, shRNA, siRNA, microRNA, antisense RNA or DNA, or DNA.
  • Genetic material exchange between an artificial endosymbiont and a host cell can be used for gene therapy, to silence a gene, to transcribe a gene, to replace a gene, to modify expression of a gene, to modify a gene, to introduce a gene or nucleic acid fragment, to bind nucleic acids, to interact with nucleic acids, or the like.
  • the artificial endosymbiont may introduce into the host cell signal pathway molecules such as, for example, receptors, ligands (hormones,
  • ion channels ion channels
  • kinases kinases
  • phosphatases DNA binding proteins
  • an artificial endosymbiont and its host cell are used to deliver therapeutic proteins to locations in an organism's body.
  • immune cells that migrate to sites of autoimmune disease such as T cells that migrate to the pancreas in diabetics, or to myelin in multiple sclerosis, could be engineered with artificial
  • endosymbionts to express immunomodulatory proteins, IL-12p40 or other immune proteins.
  • host cells with artificial endosymbionts could be implanted into sites in the organism.
  • cells that produce insulin from an artificial endosymbiont could be transplanted under the kidney capsule, or other tissue site, to make an artificial pancreas to treat diabetes.
  • the artificial endosymbiont expression of insulin can be controlled using magnetic hyperthermia, and/or other means, providing genetic control in a location that is difficult to access repeatedly.
  • an artificial endosymbiont can secrete transcription factors used to reprogram a cell like Oct4.
  • an artificial endosymbiont can secrete an enzyme to replace deficient enzymes in a lysosomal storage disease or ALDH2.
  • a magneto-endosymbiont can secrete a moiety to label a cell such as: Calcein, superparamagnetic iron oxide, gadolinium containing reagents, fluorescent proteins, luminescent proteins, magnetic reporters, other reporter proteins (e.g., Gilad et al., "MRI Reporter Genes," JNucl Med. 49: 1905-1908, 2008; incorporated herein by reference in its entirety).
  • a magneto-endosymbiont can secrete proteins related to energy generation or exchange such as hydrogenase, nitrogenase, laccase.
  • gene expression in the artificial endosymbiont in a eukaryotic host cell can be controlled using various methods.
  • the method of controlling gene expression is selected based on the type of control region employed to regulate expression of the nucleic acid or polypeptide of interest (e.g., a gene encoding a polypeptide) in the bacterial cell.
  • control region comprises a heat shock control region, which as described above, can be controlled by hyperthermia and can be induced by treatments with various external signals, such as magnetic fields, ultrasound, laser illumination, radio-frequency, microwave, and thermal (infrared) radiation.
  • various external signals such as magnetic fields, ultrasound, laser illumination, radio-frequency, microwave, and thermal (infrared) radiation.
  • an alternating magnetic field of sufficient strength, duration and frequency is used to activate expression from the heat shock control region.
  • magneto- endosymbionts selectively absorb energy from alternating magnetic fields relative to surrounding tissues, which is subsequently dissipated as heat, a process called magnetic hyperthermia.
  • the alternating magnetic field can be applied at a low frequency.
  • the, alternating magnetic field comprises magnetic fields at frequencies in the range of about 100kHz to about 500kHz. Generally, magnetic fields in these frequency ranges are weakly absorbed by biological tissues and thus highly penetrating. It allows essentially infinite depth of action, but limiting absorption to those cells engineered with magneto-endosymbionts.
  • selective hyperthermia is used to control expression from a bacterial heat shock control region that expresses a gene of interest.
  • the extent of heating from magnetic hyperthermia can be modulated for gene expression, or alternatively, increased to ablate the magneto-endosymbionts, or magneto-endosymbiont and its host cell, or the magneto-endosymbiont, its host cell, and surrounding tissue, at any location in the body.
  • the magneto-endosymbiont are directed to specific target tissues, and the entire body treated with alternating magnetic fields such that only the tissues containing the magneto-endosymbiont are heated.
  • this local hyperthermia is used to control gene expression, protein folding and function, or other naturally occurring or engineered thermally sensitive cellular process (including controlled release of any molecule) such that function can be specifically manipulated noninvasively.
  • MRI is used for real time temperature mapping.
  • proton resonance frequency shift PRFS
  • PRFS proton resonance frequency shift
  • the amount of temperature rise and the area affected are controlled by varying the amount of iron in the magnetosomes, the frequency of the alternating magnetic field, and/or the time of exposure to the alternating magnetic field.
  • magneto-endosymbionts In some embodiments, exposure of magneto-endosymbionts to higher amplitude alternating magnetic fields for short periods of time raises the temperature in smaller areas, e.g., just the magneto-endosymbiont, whereas lower amplitude magnetic fields used for longer periods of time raise the temperature in a larger area, e.g., the host cell and/or surrounding tissues.
  • the magneto-endosymbiont delivers, controls, permits visualization, and elimination of modulators that control cellular fates and function in vivo.
  • different genes or groups of genes are expressed at different times through differential response to temperature or other factors (physical or biochemical).
  • RNA hairpins can be designed to inhibit expression up to different temperature ranges, and when heat shock is induced, the different RNAs will produce gene products at different temperatures.
  • the temperature is controlled by magnetic hyperthermia so that desired genes are expressed at desired times.
  • the frequency and intensity of alternating magnetic fields are modulated to achieve specific temperatures. Lower levels will target control of gene expression and hence the developmental fates and functions of the host cells. At higher levels, heating can be used to ablate the magneto-endosymbionts or host cells without adversely affecting the surrounding tissue, unless ablation of the surrounding tissue is desired.
  • other treatments can be used to regulate expression from heat shock control regions in an artificial endosymbiont.
  • ultrasound is used to induce expression of genes under the control of heat shock control regions (see, e.g., Sontag et al., Ultrasound in Medicine and Biology 35(6):1032-1041, 2009; Eker et al., Radiology 258(2):496-504, 2011 ; all publications incorporated herein by reference).
  • selective hyperthermia can be induced by focused ultrasound. Ultrasound is demonstrated to cause hyperthermia in treated cells and tissues.
  • ultrasound, particularly focused ultrasound, of sufficient frequency, amplitude and duration is used to induce expression from the heat shock control regions.
  • the ultrasound is used at a frequency from 10 to about 20 MHz, particularly at about 10 MHz.
  • the duration of ultrasound treatment can vary from 1 min to about 30 min or more, particularly, about 5 min to about 20 min.
  • the ultrasound treatment can be continuous or pulsed.
  • the focused ultrasound treatment can be based on temperature imaging with MRI, which allows delivery of ultrasound to specified temperatures.
  • an MRI feedback system is used to control the ultrasound treatment to a specific temperature or temperature range.
  • radio-frequency (RF) radiation is used to induce expression from heat shock control regions (see, e.g., U.S. Patent Publication No. 20080140063).
  • the theoretical basis for RF radiation based hyperthermia is that RF radiation absorbed by matter causes molecules to vibrate, which in turn causes heating. More specifically, RF waves interact with matter by causing molecules to oscillate with the electric field. Generally, the interaction is highly effective for molecules that are polar, i.e., having their own internal electric field, such as water. Radio frequency waves have low tissue specific absorption, which can provide for whole body radiation, in some instances.
  • RF radiation of sufficient frequency, amplitude, and duration is used to induce expression of genes under the control of heat shock control regions.
  • the RF spectrum is generally between 3 kHz and 300 GHz, but for hyperthermia it generally refers to frequencies below the microwave range. Microwaves occupy the general EM frequency spectrum between 300 MHz and 300 GHz. In some embodiments, the RF frequency used for inducing hyperthermia is between 10 to 20 MHz. Common RF frequencies used include 13.56 and 27.12 MHz, which have been used in diathermy applications. The treatment with RF frequencies can be continuous or pulsed to produce the desired hyperthermia and subsequent activation of expression from the heat shock control regions.
  • electromagnetic radiation in the microwave range is used to regulate expression from the heat shock control regions.
  • microwave radiation of sufficient frequency, amplitude, and duration is used to induce expression of genes under the control of heat shock control regions.
  • microwave in the range of about 400 to about 3000 MHz is used. Commonly used microwave frequencies in hyperthermia include 433, 915, and 2450 MHz.
  • the microwaves can be coupled into tissues by waveguides, dipoles, microstrips, or other radiating devices.
  • a probe that generates microwave radiation in the desired frequency is used to direct and focus energy into tissues by direct radiation from the probe.
  • an array of microwave generating probes can be used to increase the volume of cells treated. Similar to use in ultrasound applications to measure and control temperature, MRI thermography can be adapted to monitor and control microwave based hyperthermia (Wlodarczyk et al., JMagn Reson Imaging 8:165, 1998; incorporated herein by reference).
  • laser illumination of sufficient frequency, amplitude and duration is used to induce hyperthermia and activate expression from heat shock control regions.
  • Laser mediated induction of heat shock genes are described in, for example, Ramos, supra; Halfon et al., Proc Natl Acad Sci USA 94:6255-6260, 1997; Due et al., Mol Vis.
  • Laser illumination in the range of about 400 nm to about 1000 nm wavelength ⁇ e.g., 440, 532, and 810 nm) have been used successfully to induce hyperthermia in cells and subsequent expression from heat shock promoters.
  • cells can be heat shocked with nanosecond bursts of laser, e.g., 2 200 ns, at a frequency of 1-10 Hz, for a duration of 0.2 to 5 min.
  • the laser can be used for in vivo situations by use of a catheter or endoscope to direct the illumination to a specific target tissue or cells.
  • the heat shock control region is regulated using thermal radiation.
  • the thermal radiation can be from a heated element or probe, infrared light source (e.g., infrared laser), or any other known sources of infrared radiation.
  • the thermal radiation can be directed to a specific target cell or tissue.
  • the thermal probe, heating element, or infrared laser is applied to the target cell or tissue in sufficient frequency, amplitude and duration to induce expression from the heat shock control region.
  • the thermal radiation is delivered by an infrared laser (Deguchi et al., Dev Growth Differ. 51 :769-775, 2009; Kamei et al., Nat Methods 6:79-81, 2009; all publications incorporated herein by reference).
  • control regions inducible by chemical agents are used to regulate gene expression in the artificial endosymbiont
  • the gene expression can be regulated by contacting the cell or tissue, or administering to the organism a specific chemical agent that induces expression.
  • control regions e.g., promoters
  • regulatable with chemical agents include, among others, lac promoter (inducible with IPTG), trp promoter (inducible with indoleacrylic acid), P.
  • the chemical agent can be provided to the cells, tissues or animals at a sufficient dose, as a single bolus or by repeated administration, to induce expression from the specific control region.
  • chemical agents that have minimal or no adverse effects on the eukaryotic cell or animal is used to control gene expression in the bacterial cell (see, e.g., Freundlich et al., J Gene Med. 1 :4 -12, 1999; U.S. Patent No. 7,807,417).
  • control regions regulatable with light are used to control gene expression
  • the gene expression is regulated by illumination with light of sufficient duration, amplitude (intensity) and frequency, depending on the type of light switchable control region used, for example phytochrome, flavin chromophore (e.g., phototropin), or crytochromes (e.g., aureochrome).
  • the light sources include, but are not limited to LED, incandescent, fluorescent, and laser light sources. Exemplary illumination intensities can vary from 0-0.8 W/m 2 , and illumination can vary from 1 s (illumination) every 30 s, 1 s every 60 s, or 1 s every 120 s.
  • the artificial endosymbiont particularly a magneto
  • iPS induced pluripotent stem
  • a fibroblast host cell contains a artificial endosymbiont that expresses Hnf4a and Foxal, 2, and 3 to direct differentiation of the fibroblast into a hepatocyte.
  • a fibroblast host cell contains an artificial endosymbiont that expresses myoD to convert the fibroblast into a myocyte.
  • Other transcription factors for differentiation of cells include those for cardiomyocytes (Gata4, Mefic, Tbx5, and others), and neuronal progenitors (TRA2B, Ascll, SHCBP1). Host cells with artificial endosymbiont expressing these factors can be tested in 1) treatment of cardiac infarcts with cardiomyocytes, 2) use of neuronal progenitors to treat stroke, and 3) implantation of myocytes into skeletal muscle.
  • the artificial endosymbionts can provide carbon, energy (like the endosymbiotically derived mitochondria, hydrogensomes, plastids, mitosomes and mitochondrion-derived organelles) or other metabolites that could be very useful
  • ATP production using an electron donor other than oxygen could be used to enable various host cells to inhabit new niches, potentially even extraterrestrial.
  • Microorganisms have many ways to produce ATP: phototrophy, chemotrophy, photolithotrophy (examples: cyanobacteria, Chromatiaceae, Chlorobiceae), photoorganotrophy (example Rhodospirillaceae), chemolithotrophy
  • the artificial endosymbionts are used to affect epigenetic changes in the host cell. These epigenetic changes can affect various biological processes, such as cellular differentiation, response to environmental agents, genomic imprinting, tumorigenesis, etc. In some embodiments, the epigenetic changes can be effected by expressing in a regulated manner genes and corresponding gene products in the magneto- endosymbiont to induce epigenetic changes in a host cell.
  • the host cell for example a stem cell or contains a magneto-endosymbiont that expresses a DNA methylase (e.g., CpG methylase), RNA methylase, DNA demethylase, RNA demethylase, histone acetylase, histone deacetylase, histone methylase, and the like.
  • the gene is induced to express or expression repressed during a specific stage in development of an organism containing the host cell.
  • T1SS type I secretion systems in Gram-negative bacteria
  • T1SS type I secretion systems
  • the type I secretion systems (T1SS) in Gram-negative bacteria can be used to export a variety of proteins of various sizes and diverse functions (their cognate substrates).
  • MTB genome encodes T1SS genes (Matsunga et al., "Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. AMB-1," DNA Res.
  • Target protein green fluorescent protein (GFP), or red fluorescent protein (RFP) is N-terminally fused to C-terminal 200 amino acids of protein describe by NCBI Accession Nos. YP_420640 (RTX toxins and related Ca 2+ binding protein); YP 423419 (RTX toxins and related Ca 2+ binding protein); YP_422785 (amb3422); or other MTB T1 SS substrates.
  • YP_420640 RTX toxins and related Ca 2+ binding protein
  • YP 423419 RTX toxins and related Ca 2+ binding protein
  • YP_422785 amb3422
  • the proteins in MTB such as those described by NCBI Accession Nos.
  • YP_420502; YP_420631.1 ; YP_420638.1 ; YP_420640.1 ; YP_421364.1 ; YP_422662.1 ; YP_422785.1; and YP 423419.1; may be used for the fusion by taking their C-terminus (the 200 C-terminal amino acids, which contains the secretion signal) to target the recombinant fusion protein to the secretion system.
  • C-terminus the 200 C-terminal amino acids, which contains the secretion signal
  • telomere sequence The DNA encoding the fusion of GFP or RFP with 200 C-terminal amino acids of YP_420640 (RTX toxins and related Ca 2+ binding protein), YP_423419 (RTX toxins and related Ca 2+ binding protein), YP_422785 (amb3422), or other MTB T1SS substrates is cloned into pBBR-MSC (Kovach et al., "pBBRlMCS: a broad-host- range cloning vector," Biotechniques 16:800-2, 1994; incorporated herein by reference). Translocation of the fusion protein out of the magneto-endosymbiont is detected by fluorescence, or immunofluorescence, and/or immunoblotting.
  • the hemolysin (Hly) secretion system of E. coli is one of the best studied type I secretion systems (T1SS).
  • Secretion of the hemolysin A toxin (HlyA) is catalyzed by a membrane protein complex (Bakkes et al., "The rate of folding dictates substrate secretion by the Escherichia coli hemolysin type 1 secretion system," J Biol Chem. 285(52): 40573-40580, 2010; incorporated herein by reference) that consists of HlyB, an inner membrane ATP binding cassette transporter (Davidson et al., "ATP-binding cassette transporters in bacteria," Ann. Rev. Biochem.
  • TolC the outer membrane protein (Koronakis et al., "Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export," Nature 405(6789):914-919, 2000;
  • HlyD the membrane fusion protein that is anchored to the inner membrane
  • HlyB the membrane fusion protein that is anchored to the inner membrane
  • the protein, GFP for example (GenBank: ABG78037.1) is fused to the last 218 C-terminal amino acids of HlyA (HlyAN-term 218, UniProtKB/Swiss- Prot: P09983.1, bolded-underlined sequence):
  • the GFP-HlyAC-term 218 fusion is secreted out of the bacterial cell into the host cell via the HlyB-HlyD-TolC complex.
  • HlyB-HlyD-TolC complex is engineered into the pBBRlMCS-2 plasmid and expressed under the control of the tac promoter. Translocation of the GFP-HlyAC-term 218 fusion into target host cells is monitored by fluorescence microscopy.
  • Translocation/Secretion assays to demonstrate that proteins are secreted from MTB can use the following procedure. Overnight cultures of MTB strains harboring the appropriate recombinant plasmids are diluted (1 : 10) into fresh MG media supplemented with antibiotics (for culture conditions, see Greene, et al., "Analysis of the CtrA pathway in
  • Magnetospirillum reveals an ancestral role in motility in alphaproteobacteria," J itoc/.
  • Cells carrying the defined plasmid combinations are grown to an optical density of 400 nm (OD400) of 0.2 before IPTG or arabinose is added to induce the expression of target protein fusions. Proteins in the supernatants are precipitated with 10-20% trichloroacetic acid for 30 min at 4°C. The precipitated proteins are collected by centrifugation and washed in 80% acetone. Cell pellets are washed once in 20 mM Tris (pH 8.0), 1 mM EDTA.
  • Cell pellets and precipitates are resuspended in IX sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and amounts equivalent to 0.2 OD unit are analyzed by SDS-PAGE and immunoblotting. Proteins are stained with Coomassie brilliant blue and/or probed by immunoblotting using specific antibodies. MTB isolates that secrete GFP are introduced to the mammalian cell line MDA-MB231 , using the magnet assisted entry method. Cells with magneto-endosymbionts are obtained, and translocated protein is detected by fluorescence, or immunofluorescence or immunoblotting.
  • SDS-PAGE IX sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • CagA is secreted through a T4SS system by engineering a CagA protein by replacing the last 20 amino acids with 24 amino acids (residues 684-709) from the C-terminal end of the RSFIOIO MobA protein. Translocation of CagA-MobA into the host cell is monitored by observation of the hummingbird phenotype caused by CagA in the host cell.
  • hummingbird phenotype is "characterized by spreading and elongated growth of the cell, the presence of lamellipodia (thin actin sheets present at the edge of the cell), and filapodia (finger-like protrusions containing a tight bundle of actin filaments; see Segal, et al. "Altered States: Involvement of Phosphorylated Caga in the Induction of Host Cellular Growth Changes by Helicobacter Pylori," Proc Nail Acad Sci USA 96(25): 14559-64, 1999;
  • the hummingbird phenotype resembles the morphological changes induced by hepatocyte growth factor (HGF) or platelet-derived growth factor (PDGF) in epithelial cells (Sugiyama, T., "Development of gastric cancer associated with Helicobacter pylori infection,” Cancer Chemother Pharmacol. 54(Suppl. 1):S12-S20, 2004; incorporated herein by reference).
  • HGF hepatocyte growth factor
  • PDGF platelet-derived growth factor
  • Hepatocyte growth factor also known as scatter factor, evokes a unique morphogenic activity, e.g., induces kidney, or mammary gland-derived epithelial cells, to form branching ducts in three-dimensional collagen gels (Ohmichi, H., et al., "Hepatocyte growth factor (HGF) acts as a mesenchyme-derived morphogenic factor during fetal lung development," Development 125: 131 -1324, 1998; incorporated herein by reference).
  • HGF Hepatocyte growth factor
  • GFP GFP
  • Other proteins including GFP, are fused to the C-terminal residues of MobA (see sequence below) in order to be translocated from Helicobacter pylori with a T4SS into a MDM-MB231 host cell.
  • translocation of this protein into the MDM- MB231 host cells are monitored by looking for the presence of GFP fluorescence outside of the magneto-endosymbiont.
  • GFP amino acid sequence (non-underlined sequence; GenBank: ABG78037.1) fused to MobA residues 684-709 (bolded-underlined sequence; GenBank: AAA26445.1):
  • GFP is fused to the C-terminal 50aa residues of VirD5 in order to be translocated from A. tumifaciens with a T4SS into a MDM-MB231 host cell.
  • translocation of this protein into the MDM-MB231 host cell is monitored by looking for the presence of GFP fluorescence.
  • GFP amino acid sequence (non-underlined sequence; GenBank: ABG78037.1) fused to VirD5 residues 787-836 (bolded-underlined sequence; NCBI Reference Sequence:
  • Example 3 Translocation of a Nucleic Acid into a Host Cell Using a Type IV
  • T4SS type 4 secretion system
  • Plasmid pBBR-MSC is engineered to include (oriT+trwABC) for transfer of the plasmid by a T4SS system.
  • the plasmid is also engineered to contain an expression cassette encoding the target protein under the control of the HCMV IE1 promoter-enhancer-first intron.
  • the target protein is a selectable marker such as, DHFR or glutamate synthetase, or a reporter such as GFP, or a transcription factor such as cMyc.
  • a mammalian selectable marker is used as the target gene. These include puromycin N-acetyl-transferase gene for puromycin resistance; blasticidin S deaminase for balstadin S resistance; and aminoglycoside 3 '-phosphotransferase for G418 resistance. If MDA-MB231 is used as a host cell line, 2 ug/ml puromycin, 5 ug/ml blastacidin S, or 1 mg/ml G418 is used for selection.
  • Plasmid DNA harboring a DNA fragment of interest along with an antibiotic resistance cassette can be introduced into MTB via conjugation with a mating strain of E. coli that is auxotrophic to diaminopimelic acid (DAP).
  • DAP diaminopimelic acid
  • Successful transfer of the plasmid DNA to MTB will result in growth of MTB on MG agar plates in the presence of antibiotic.
  • the E. coli mating strain auxotrophic to (DAP) will ensure that any growth seen on the MG agar plates + antibiotic will be strictly MTB as no E. coli colonies should survive on plates not containing DAP.
  • MTB colonies harboring the plasmid DNA will be cultured in liquid MG growth medium to be used in subsequent experiments where MTB will be introduced into mammalian cells.
  • MTB with the plasmid are introduced into mammalian cells, such as the human breast cancer cell line MDA-MB231.
  • the plasmid will then integrate into the host cell chromosome via non-homologous recombination. Transfer of plasmid nucleic acid from MTB, the magneto- endosymbiont, to the host cell (MDA-MB231) is detected by selection for host cells that grow in the presence of 2 ug/ml puromycin, 5 ug/ml blastacidin S, or 1 mg/ml G418. Selected cells are grown and then suitable assays are performed to detect chromosomal integration of the plasmid into the host cell genome, such as for example, in situ hybridizations, or Southern hybridization.
  • Mammary carcinoma host cells with magneto-endosymbionts are injected into FVB mice subcutaneously in the flank. Mice are imaged with 7T MRI using a cell tracking pulse sequence to confirm cell cluster location. Surface skin temperature is mapped using a thermal imaging camera, for example a FLIR Systems Infrared Camera, temperature is also mapped internally using MR thermography methods. Peak and average values of temperature as a function of AMF application time, for various field amplitudes and frequencies, are recorded. Engraftment and viability are assessed using luciferase/GFP reporters expressed from the magneto-endosymbionts and/or the host cells.
  • the magneto-endosymbionts are also followed using multiplexed BLI through expression of the bacterial Lux operon that encodes luciferase and the biosynthetic enzymes for the substrate, (see Contag et al., "Photonic detection of bacterial pathogens in living hosts," Mol Microbiol. 18:593-603, 1995; incorporated herein by reference) and survival of the labeled cells through expression of Luc (Contag et al., "Bioluminescent indicators in living animals," Nat Med. 4:245-247, 1998; Contag et al., "In vivo patterns of heme oxygenase-1 transcription,” JPerinatol. 21(suppl):Sl 19-S124, 2001; Contag et al., "Advances in in vivo bioluminescence imaging of gene expression,” Ann Rev Biomed Eng. 4:235-260, 2002; all publications incorporated herein by reference).
  • Mammary carcinoma host cells containing magneto-endosymbionts are embedded in matrigel and transplanted subcutaneously into the flanks of mice. AMF is applied at selected frequencies and amplitude, and for a time period to induce gene expression by raising the temperature. The effectiveness of gene expression is assessed using optical reporter genes. Temperatures are mapped on the skin surface by a thermal imaging camera, and throughout the 3D volume of the mice by MR thermography.
  • mice are transplanted with host cells containing magneto-endosymbionts, as described above.
  • AMF is applied at the desired frequency and amplitude, and for a time period to ablate the host cells containing the magneto-endosymbionts.
  • the ablation of host cells is assessed by histological analysis of postmortem tissue. Temperatures are mapped on the skin surface of the mice by thermal imaging camera, and throughout the 3D volume of the mouse by MR thermography.
  • Magneto-endosymbiont are engineered to express a secreted tet activator (tTA) with a host cell nuclear translocation signal into the cytoplasm of host cells.
  • the mammalian host cell is engineered to contain a tet-regulated control region that expresses a GFP-luciferase reporter gene.
  • the tet-reporter construct is derived from a human ubiquitin C control region (huUbiqC) with seven binding sites for the tTA (tet-70) upstream of GFP fused to click beetle luciferase (CBL). In the presence of the small molecule doxycycline, the tTA will bind to the tTA binding sites and activate expression of the GFP-luciferase reporter.
  • the tet activator is placed under the control of a heat shock control region such as the Hsp control region.
  • This engineered magneto-endosymbiont is placed inside a host cell and the host cell is subjected to AMF at selected frequencies and amplitude and time period, for example about 100kHz frequency, lOkA/m amplitude, with times ranging from a 2 min to more than 30 min, to induce gene expression by raising the temperature.
  • Localization of the tet activator to the nucleus of the host cell is identified by GFP-luciferase expression induced by the tet activator.
  • Magneto-endosymbiont are engineered to express the transcription factors, Hnf4a and Foxal, -2 and -3. These magneto-endosymbionts are placed into mammary carcinoma host cells.
  • Gene expression and translocation to the host nucleus by these transcription factors is observed using adult hepatic markers (e.g., ALB, CX32, CYP1A1, CYP1A2, CYP2B6 and CYP3A4), and liver progenitor markers (e.g., DKK1, DPP4, DSG2, CX43 and K19), capacity to form colonies in vitro, and cellular function (Buyl et al., "Characterization of hepatic markers in human Wharton's Jelly-derived mesenchymal stem cells," Toxicol in Vitro 28:113-1 19, 2013; incorporated herein by reference).
  • adult hepatic markers e.g., ALB, CX32, CYP1A1, CYP1A2, CYP2B6 and CYP3A4
  • liver progenitor markers e.g., DKK1, DPP4, DSG2, CX43 and K19
  • hepatocytes derived from fibroblasts are transplanted into livers of normal mice to assess longevity and control of gene expression. These hepatocytes are also transplanted into a murine model of hereditary tyrosinemia type 1 (HTl) (see Vogel et al., "Chronic liver disease in murine hereditary tyrosinemia type I induces resistance to cell death," Hepatology 39:433-443, 2004; incorporated herein by reference). In this model, healthy
  • Fah-/- mice fumarylacetoacetate hydrolase deficient mice (Fah-/-) are protected from liver injury by the drug 2-(2-nitro-4-trifluoromethylbenzoyl)-l ,3-cyclohexanedione (NTBC), and the tyrosine metabolite homogentisic acid (HGA) causes rapid hepatocyte death.
  • NTBC 2-(2-nitro-4-trifluoromethylbenzoyl)-l ,3-cyclohexanedione
  • HGA tyrosine metabolite homogentisic acid
  • the Fah-/- mice are available through Jackson Laboratories (stock number 018129), and fine-tuning of the model is possible through the use of the small molecules, NTBC and HGA.
  • magneto-endosymbiont-tagged hepatocytes expressing luciferase are used in a murine model of hereditary tyrosinemia type 1 (HTl).
  • HGA homogentisic acid
  • engineered hepatocytes with magneto- endosymbionts are introduced into the mice, and BLI is used to localize the transplanted cells, MRI is used to assess tissue volume and to localize the magneto-endosymbionts, and liver enzyme assays are used as an indicator of restored liver function.
  • Example 7 Using Magneto-Endosymbionts to Make Induced Pluripotent Stem
  • Magneto-endosymbiont are engineered to express the Yamanaka factors (sox2, klf-4, c-myc, and oct-3/4).
  • the expression constructs for the Yamanaka factors use heat shock inducible control regions, such as bacterial heat shock proteins, or DNAK.
  • Magneto-endosymbiont are engineered to be infectious to target cells and can be introduced systemically and target specific cells, including stem cells, in the body.
  • AMB-1 expressing luciferase (“ME”) was introduced into 231 BR cells. These 231 BR cells containing ME were administered by intracranial injection into mice. Mice received 10,000, 1,000, or 100 231 BR cells containing MEs. The mice receiving 10,000, 1,000, or 100 231 BR cells containing MEs were imaged my MRI and a bSSFP sequence was used to acquire images with 150um isotropic resolution, TR TE 10, 8 phase cycles, 1 average, FA 15. The mice receiving single 231 BR cells containing MEs fixed, and a GRE MRI sequence was used with resolution of 80x80x100 urn, slice thickness of 100 Mm, TE 17, TR 35, FA 12, BW 3, NEX 4.
  • FIG. 1 A-D MRI images of 231 BR cells containing ME that were injected into mice are shown in FIG. 1 A-D.
  • FIG. 1A shows an image from a mouse that received 104 231 BR cells containing ME. The arrow indicates the location of the 231 BR cells containing ME.
  • FIG. IB shows an image from a mouse that received 103 231 BR cells containing ME. The arrow indicates the location of the 231 BR cells containing ME.
  • FIG. 1C shows an image from a mouse that received 102 231 BR cells containing ME. The arrow indicates the location of the 231 BR cells containing ME.
  • FIG. IC demonstrates that as few as 100 231 BR cells with ME can be visualized in vivo in a mouse with MRI.
  • FIG. ID shows an MRI image with detection of single cells of 231 BR with ME in mouse brain.
  • the arrow indicates the location of single cells of 231 BR with ME.
  • FIG. 2 shows an induction of about a 6 oC increase in temperature by placing a sample containing 100 ⁇ of magnetite nanoparticles in a
  • Magnelles (AMB-1) were placed into 0.5 mL of media to give a concentration of 4 X 1011 Magnelles per mL. These Magnelles were subjected to an RF field of 25 mT at a frequency of 1 11 kHz. The RF field was generated by Magnatherm system from
  • FIG. 3 shows the relative light units (RLU) of lux+AMB-1 in increasing concentration from left to right as follows: 3.91E+05, 7.81E+05, 1.56E+06, 3.13E+06, 6.25E+06, 1.25E+07, 2.50E+07, 5.00E+07, and 1.00E+08 AMB-1 per well. Triplicates at each concentration were assessed across three rows shown. The image was taken using an I VIS Lumina system.
  • FIG. 3 shows that AMB-1 expressed luciferase and the recombinant luciferase produced detectable light.

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Abstract

La présente invention concerne d'une façon générale des cellules hôtes comprenant des endosymbiotes artificiels, en particulier des magnéto-endosymbiotes, l'endosymbiote pouvant être commandé et/ou régulé par un moyen externe. La présente invention porte sur des endosymbiotes artificiels qui sécrètent un polypeptide, un acide nucléique, un lipide, un glucide ou un autre facteur dans la cellule hôte d'une manière régulable. Dans certains modes de réalisation, le polypeptide, l'acide nucléique, le lipide, le glucide ou un autre facteur sécrété dans la cellule hôte par le magnéto-endosymbiote confère un phénotype à la cellule hôte. Le polypeptide sécrété peut être un marqueur de sélection, une protéine rapporteuse, un facteur de transcription, une protéine de voie de signalisation, un récepteur, un facteur de croissance ou une molécule effectrice.
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CN108456694A (zh) * 2017-02-22 2018-08-28 浙江和也健康科技有限公司 一种融合定位系统、磁感应系统和目标蛋白的遗传学操作方法及应用
CN108456695A (zh) * 2017-02-22 2018-08-28 浙江和也健康科技有限公司 一种通过引入磁性介质进行遗传操作的方法
CN108456693A (zh) * 2017-02-22 2018-08-28 浙江和也健康科技有限公司 一种进行遗传操作的方法和装置
WO2019222569A1 (fr) * 2018-05-17 2019-11-21 The Uab Research Foundation Thérapie génique basée sur l'optogénétique mitochondriale pour le traitement du cancer
US11481578B2 (en) 2019-02-22 2022-10-25 Neuropace, Inc. Systems and methods for labeling large datasets of physiological records based on unsupervised machine learning
US11842255B2 (en) 2019-02-22 2023-12-12 Neuropace, Inc. Systems and methods for labeling large datasets of physiological records based on unsupervised machine learning

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