JP2017522022A - Eukaryotic cells containing artificial endosymbiosis for multimodal detection - Google Patents

Abstract

The present invention generally relates to a eukaryotic cell comprising a unicellular organism that is introduced into a eukaryotic cell by human intervention and transmitted to the daughter cell of the eukaryotic cell, and a method for introducing such a unicellular organism into a eukaryotic cell. About. The present invention provides unicellular organisms that introduce phenotypes maintained in daughter cells into eukaryotic cells. The present invention further provides eukaryotic cells containing magnetic bacteria. The invention further provides eukaryotic cells engineered with unicellular organisms to allow multimodal observation of eukaryotic cells. Each imaging method (or modality) allows visualization of various aspects of anatomy and physiology, and by combining them, allows the image creator to learn more about the object being imaged. [Selection] Figure 1

Description

Reference of related applications
[1] This application is a continuation-in-part of US application Serial No. 13 / 838,717, filed on March 15, 2013. This is a continuation-in-part application, and this application is a continuation-in-part of PCT / US2013 / 021414, 2013, which is part of US application Serial No. 13 / 374,799, January 13, 2012. Are continuation applications and the entire contents of those applications are incorporated herein in their entirety for all purposes.

Field of Invention
[2] The present invention relates generally to the field of endosymbiosis, ie, eukaryotic cells engineered with artificial endosymbiont and magnetotactic bacteria. In particular, the present invention relates to unicellular organisms such as artificial endosymbiosis including magnetotactic bacteria, eukaryotic cells harboring those unicellular organisms, methods of using eukaryotic cells containing unicellular organisms, and unicellular organisms to eukaryotic cells. A method of introducing into a cell is provided. The present invention also provides eukaryotic cells that have been engineered with intracellular single cell organisms to enable multimodal detection of eukaryotic cells.

  [3] Mitochondria, chloroplasts, and other membrane-bound organelles add genetic functions, such as photosynthesis, to eukaryotic cells. Such organelles (identified by their trace amount of circular DNA) are thought to be derived from endosymbiosis.

[4] There are bacteria with a wide range of functionality that do not exist in various eukaryotic cells. For example, in 1975 Blakemore identified a magnetotactic bacterium (MTB) that is oriented and swimming along the geomagnetic field (Blakemore, R., “Magnetotactic bacteria,” Science 24: 377-379 (1975) It is incorporated herein in its entirety for all purposes). These magnetotactic bacteria are magnetized (magnetosome) composed of magnetite (magnetite) (Fe ₃ O ₄ ) or greigite (Greg ore) (Fe ₃ S ₄ ) encapsulated by a lipid membrane. Produces a structure (Id.). A number of MTB species have been identified since their first discovery (ibid.).

  [5] Magnetotactic bacteria have been used to selectively bind and separate substances (U.S. Patent No. 4,677,067, incorporated herein in its entirety for all purposes). Furthermore, attempts have been made to add magnetic functions to cells with external tags (Swiston, AJ et al., “Surface Functionalization of Living Cells with Multilayer Patches,” Nano Lett. 8 (12): 4446-53 (2008). Vandsburger, MH et al., “MRI reporter genes: applications for imaging of cell survival, proliferation, migration and differentiation,” NMR Biomed. 26 (7): 872-84 (2013); Ahrens, ET et al, “Tracking Immune cells in vivo using magnetic resonance imaging, ”Nature Rev Immunol. 13: 755-763 (2013); those publications are incorporated herein in their entirety for all purposes). Bacterial magnetite is also introduced into red blood cells by cell fusion (Matsunaga, T. and Kamiya, S., (1988), In: Atsumi, K., Kotani, M., Ueno, S., Katila T., Williamsen, SJ (Eds) 6th International Conference on Biomagnetisms, Tokyo Denki University Press, Tokyo, pp. 50-51 (1988), which is incorporated herein by reference in its entirety for all purposes), MTB eats granulocytes and monocytes (Matsunaga, T. et al., “Phagocytosis of bacterial magnetite by leucocytes,” Applied Microbiology and Biotechnology 31 (4): 401-405 (1989), which is incorporated herein in its entirety for all purposes. ))). However, none of these changes can be inherited to daughter cells.

U.S. Patent No. 4,677,067

Blakemore, R., “Magnetotactic bacteria,” Science 24: 377-379 (1975) Swiston, A.J. et al., “Surface Functionalization of Living Cells with Multilayer Patches,” Nano Lett. 8 (12): 4446-53 (2008) Vandsburger, M.H. et al., “MRI reporter gene: applications for imaging of cell survival, proliferation, migration and differentiation,” NMR Biomed. 26 (7): 872-84 (2013) Ahrens, E.T. et al, “Tracking immune cells in vivo using magnetic resonance imaging,” Nature Rev Immunol. 13: 755-763 (2013) Matsunaga, T. and Kamiya, S., (1988), In: Atsumi, K., Kotani, M., Ueno, S., Katila T., Williamsen, SJ (Eds) 6th International Conference on Biomagnetisms, Tokyo Denki UniversityPress , Tokyo, pp. 50-51 (1988) Matsunaga, T. et al., “Phagocytosis of bacterial magnetite by leucocytes,” Applied Microbiology and Biotechnology 31 (4): 401-405 (1989)

  [6] Currently, there is a need in the imaging field for multimodal probes that can label eukaryotic cells with minimal host cell manipulation and track them with MRI or other types of imaging methods. In certain embodiments, an object of the present invention is to contain a unicellular organism introduced into a eukaryotic cell by human intervention, which is transmitted to the eukaryotic daughter cell, in particular through at least 5 cell divisions, and Is to provide a eukaryotic cell that maintains a sufficient copy number in the daughter cell and thus maintains the target function introduced by the unicellular organism in the daughter cell. It is a further object of the present invention to provide eukaryotic host cells containing artificial endosymbiosis that can be inherited to daughter cells and methods of using these eukaryotic cells. It is also an object of the present invention to provide a method for introducing an artificial endosymbiosis into the cytosol of eukaryotic host cells. Another object of the present invention is to provide eukaryotic cells with an inheritable magnetic phenotype. It is also an object of the present invention to provide a method for tracking, localizing, steering, controlling or damaging eukaryotic cells. Another object of the present invention is to provide a eukaryotic cell containing a unicellular organism capable of multimodal detection of eukaryotic cells. It is a further object of the present invention to provide a eukaryotic host cell containing a unicellular organism with multiple phenotypes inheritable to daughter cells.

  [7] The present invention relates to a eukaryotic cell containing a unicellular organism such as an artificial endosymbiosis, a method of using such a eukaryotic cell, and a method of introducing such a unicellular organism into a eukaryotic cell. In one embodiment, the unicellular organism provides the desired functionality to the eukaryotic cell. In one embodiment, the unicellular organism is an artificial endosymbiosis that can be inherited by daughter cells. In other embodiments, the artificial endosymbiosis is a magnetotactic bacterium that forms a magnetosome and expresses other detectable gene products. In one embodiment, the magnetotactic bacterium provides a magnetic function for eukaryotic cells. In one embodiment, the method of use is a method of detecting eukaryotic cells. In other embodiments, the method of use is a method of manipulating or targeting eukaryotic cells magnetically. In other embodiments, the method of use is a method of damaging eukaryotic cells. In one embodiment, a eukaryotic cell has a multiple phenotype that allows multimodal detection, which can be introduced into a eukaryotic cell by a unicellular organism. In one embodiment, multiple phenotypes can be inherited to eukaryotic daughter cells. In one embodiment, the magnetotactic bacteria provides eukaryotic cells with magnetic function and luminescence or absorbance and / or acoustic properties. In certain embodiments, a unicellular organism produces a gene product that interacts with a gene product from a eukaryotic cell to generate a detectable signal. In one embodiment, a eukaryotic cell containing a unicellular organism is used in a method for detecting a eukaryotic cell.

  [8] In certain embodiments, the artificial endosymbiosis of the present invention can be modified by deletion, addition, and / or mutagenesis of at least one gene, whereby the artificial endosymbiosis is endosymbiotic or biotrophic Acquire useful traits for (biotrophy). Genes that are mutated, added, and / or deleted in an artificial endosymbiosis include genes that encode components of flagellar assembly, as well as enzymes to synthesize essential macromolecules such as amino acids, nucleotides, vitamins, and cofactors. It may be a gene that encodes. In certain embodiments, the MTB may be further modified to express an antibiotic resistance gene or other selectable marker. In certain embodiments, the genes localize the artificial endosymbiosis to specific intracellular locations. In certain embodiments, the gene enhances or blocks the entry of an artificial endosymbiosis into a particular host cell. In other embodiments, the gene suppresses or alters the host immune system response to the artificial endosymbiosis or genes and proteins expressed therefrom.

  [9] In some embodiments, eukaryotic cells of the invention are from mammals, such as mice, rats, rabbits, hamsters, humans, pigs, cows, or dogs. In other embodiments, the artificial endosymbiosis is transmitted from the host cell to a progeny daughter host cell. In other embodiments, the method further comprises deleting, inserting, and / or mutating at least one gene from the eukaryotic cell.

  [10] The unicellular organism of the present invention can be introduced into eukaryotic cells by a number of methods known to those skilled in the art, including microinjection, natural-phagocytosis, induction-phagocytosis, macropinocytosis. , Other cellular internalization processes, liposome fusion, erythrocyte ghost fusion, or electroporation.

  [11] The present invention also relates to a method for characterizing eukaryotic cells containing unicellular organisms using multimodal detection. In the methods of the invention, eukaryotic cells, including unicellular organisms, have multiple phenotypes associated with that unicellular organism. In certain embodiments, multiple phenotypes allow for detection of eukaryotic cells using multimodal observation methods. In certain embodiments, multimodal detection is used for non-invasive in vivo imaging of eukaryotic cells. Imaging methods and modalities, respectively, allow visualization of various aspects of anatomy and physiology, and combining them gives more information about eukaryotic cells in an in vivo environment.

  [12] In some embodiments, multimodal detection of eukaryotic cells is used to simultaneously acquire multiple forms of functional data about the cells and / or to enable detection on a wide variety of imaging devices. It is done.

[13] FIG. 1 shows that using a 1.5T instrument over a long scale concentration of about 10 ⁸ MTB / mL for gfp ⁺ AMB-1 suspended in an agar plug to optimize and elucidate imaging properties. _The positive contrast created by _one pulse sequence is shown. [14] FIG. 2 shows a blastocyst mouse embryo microinjected with gfp ⁺ AMB-1 in one of its two cells at the two-cell embryo stage. Embryos are imaged with a Leica SP2 AOBS spectral confocal inverted microscope with a 20 ×, 0.7 NA objective and 3 × optical zoom surrounded by an environmental control chamber for live cell imaging. Panel A shows a differential interference contrast (DIC) image and Panel B shows grayscale fluorescence capture of the same image. [15] FIG. 3 shows changes in total embryo GFP fluorescence of four mouse embryos measured over time by confocal microscopy. Gfp ⁺ AMB-1 was microinjected into one of the two cells from the 2-cell stage of each embryo, and after microinjection, starting from the 8-cell stage, 24 hours, total GFP fluorescence of each embryo Was measured. [16] FIGS. 4A-C show images acquired for MDA-MB-231 human breast cancer cells containing gfp ⁺ AMB-1. FIG. 4A shows a phase contrast image of MDA-MB-231 human breast cancer cells containing gfp ⁺ AMB-1. FIG. 4B shows a fluorescence image of the same cell, demonstrating the fluorescence signal from gfp ⁺ AMB-1. FIG. 4C shows a T2w MRI image of a tube filled with agar (lower half) and phosphate buffered saline (upper half) between which layers of MDA-MB-231 cells containing gfp ⁺ AMB-1 (Black band). FIGS. 4D-F show phase contrast, fluorescence, and MRI images of MDA-MB-231 cells without gfp ⁺ AMB-1. FIG. 5 shows MDA-MB-231 cells containing gfp ⁺ AMB-1 suspended in an agar gel in a cylindrical 100 ml tube. FIG. 5A is an image obtained by visual inspection of MDA-MB-231 cells containing gfp ⁺ AMB-1. FIG. 5B shows an MRI image of MDA-MB-231 cells containing gfp ⁺ AMB-1. FIG. 5C shows a fluorescence image of MDA-MB-231 cells containing gfp ⁺ AMB-1. [18] Figure ^6, a variety of cells without gfp + ^AMB-1, in comparison to cells containing the gfp + AMB-1. Cells are imaged by epifluorescence microscope and fluorescence imaging. iPS (artificial pluripotent stem cells, FIGS. 6A and 6B), MDA-MB-231 (breast cancer cells, FIGS. 6C and 6D), J774.2 cells (mouse macrophages, FIGS. 6E and 6F), BJ (human fibroblasts, 6G and 6H), HEP1 cells (human liver adenocarcinoma, FIGS. 6I and 6J), and MCF7 (human epithelial breast adenocarcinoma, FIGS. 6K and 6L) were used. FIGS. 6A, 6C, 6E, 6G, 6I and 6K are confocal microscopic images of eukaryotic cells containing gfp ⁺ AMB-1; FIGS. 6B, 6D, 6F, 6H, 6J and 6L are gfp ⁺ AMB−. 1 is a fluorescence image of a eukaryotic cell containing 1. Same as above Same as above [19] FIG. 7 shows MDA-MB-231 cells containing lux ⁺ AMB-1 imaged for luminescence or imaged by MRI. FIG. 7A is a chart of luminescence signals obtained from MDA-MB-231 cells with or without lux ⁺ AMB-1. The unit of the chart is light emission / minute. FIG. 7B shows an MRI image (dark band) of MDA-MB-231 cells containing lux ⁺ AMB-1, where MDA-MB-231 cells containing lux ⁺ AMB-1 were stacked on the agar base. Yes.

  [20] The present invention is illustrated by way of example but not limitation. In this disclosure, the expression “an” or “one” or “some” does not necessarily describe the same aspect, and all such expressions mean at least one. It should be noted that.

  [21] It should also be understood that the singular forms “a”, “an”, and “the” include plural references unless the content clearly dictates otherwise. Similarly, the word “or” is intended to include “and” unless the content clearly dictates otherwise (and vice versa). The numerical limits listed for the concentration or level of the substance shall be approximate. Thus, if a concentration is indicated to be at least (for example) 10 μg, then the concentration should be interpreted as at least approximately or about 10 μg.

  [22] In one aspect, the present invention relates to a eukaryotic cell containing a unicellular organism, for example, a host cell containing an artificial endosymbiosis in the cytosol of the host cell, and a method for introducing the unicellular organism into a eukaryotic cell. . In one embodiment, the unicellular organism is a genetically altered artificial endosymbiosis. In certain embodiments, the unicellular organism is a magnetotactic bacterium (MTB). The invention also relates to eukaryotic cells engineered in unicellular organisms to have multiple phenotypes for detection. Multiple phenotypes for such detection allow multimodal observation of eukaryotic cells. In some embodiments, multimodal detection of eukaryotic cells is used for non-invasive in vivo imaging. Each imaging method (or modality) allows visualization of various aspects of anatomy and physiology, and when combined, allows the image creator to know more about the target or object being imaged.

Definition
[23] For purposes of this disclosure, technical and scientific terms used herein will have the meanings that are commonly understood by those of ordinary skill in the art, unless specifically defined otherwise. Thus, the following terms shall have the following meanings:

[24] The term “AMB” or “AMB-1” as used herein refers to the Magnetospirillum magneticum AMB-1 strain.
[25] As used herein, the term "artificial endosymbiosis" is a unicellular organism that has been introduced into the cytosol of a eukaryotic cell by human intervention and has been or could be transmitted to the daughter cell of the eukaryotic cell. Represents. In certain embodiments, the unicellular organism maintains a sufficient copy number in the daughter cell such that the phenotype introduced by the artificial endosymbiosis is maintained in the daughter cell.

[26] As used herein, the term “cell cycle” refers to a series of events involving the growth, replication, and division of eukaryotic cells. In general, it is known as five periods known as G ₀ when the cell is inactive, G ₁ and G ₂ where the size of the cell increases, S where the DNA of the cell doubles, and M where the cell undergoes mitosis and divides. Can be classified.

[27] The term “cytosol” as used herein refers to the cytoplasm of a portion that is not within the membrane-bound substructure of a cell.
[28] The term "daughter cell" as used herein refers to a cell formed by the division of a cell.

[29] As used herein, the term "essential molecule" refers to a molecule that a cell needs for growth or survival.
[30] The term "genetically modified" as used herein refers to altering cellular genetic material such that desired cellular properties or characteristics are altered. The term includes introducing heterologous genetic material into the cell.

  [31] The term "fluorescent protein" as used herein refers to a protein that can emit light when excited by appropriate electromagnetic radiation. Fluorescent proteins include proteins with natural or engineered amino acid sequences.

  [32] As used herein, the term “bioluminescent protein” refers to an energy capture chemistry in which a specific biochemical, eg, luciferin (natural fluorophore) is oxidized by an enzyme (eg, luciferase) to produce chemiluminescence. Represents the form of chemiluminescence resulting from the reaction.

[33] The term “host cell” as used herein refers to a eukaryotic cell in which an artificial endosymbiosis can reside.
[34] As used herein, the term “image modality” refers to electromagnetic radiation, sound waves, nuclear particles, or other types of energy (eg, electricity) that allow detection or query of the system or target that contains it. Or it represents the absorption or release of the combination.

[35] As used herein, the term "intracellular-internal symbiosis" refers to a unicellular organism that spends at least part of its natural life cycle in eukaryotic cells.
[36] As used herein, the terms "intracellular pathogen" and "intracellular parasite" refer to a bacterium that infects a host organism and causes disease in the host organism in nature, with some bacteria being host Invade cells.

[37] The term “liposome-mediated” as used herein refers to the use of an artificial vesicle having an aqueous core encapsulated in one or more lipid layers for transport of an artificial endosymbiosis to a host cell.
[38] The term "luciferase" as used herein refers to a protein that produces photons using a chemical substrate. In certain embodiments, luciferase represents an enzyme or photoprotein, such as an oxygenase, that catalyzes a reaction that produces bioluminescence. The luciferase may be recombinant or natural, or a variant or variant thereof.

[39] As used herein, the term "magnetosome" refers to magnetic mineral particles encapsulated by a sheath or membrane as individual or chained particles. In certain embodiments, the magnetic mineral in the magnetosome can comprise magnetite (magnetite) (ie, Fe ₃ O ₄ ) or greigite (Greg ore) (Fe ₃ S ₄ ).

[40] The term "magnetic bacterium" as used herein refers to a bacterium that can respond to an external magnetic field.
[41] As used herein, the term "geotactic bacterium" or "MTB" refers to a bacterium having a gene encoding a magnetosome.

[42] The term "mammal" as used herein refers to a warm-blooded vertebrate, all of which have body hair, and a female has a mammary gland that produces milk.
[43] The term "microinjection" as used herein refers to the injection of an artificial endosymbiosis into a host cell.

[44] As used herein, the term “parent cell” refers to a cell that divides to form two or more daughter cells.
[45] As used herein, the term “phenotype” refers to an observable set of characteristics of an organism or cell.

  [46] The term "receptor mediated" as used herein refers to the molecular structure or site of the host cell surface binding to bacteria or tagged bacteria followed by internalizing the bacteria.

  [47] As used herein, the term "reporter" or "reporter molecule" refers to a moiety that can be detected indirectly or directly. Reporters include, but are not limited to, chromophores, fluorophores, fluorescent proteins, receptors, haptens, enzymes, and radioisotopes.

  [48] The term "reporter gene" as used herein refers to a polynucleotide that encodes a reporter molecule that can be detected directly or indirectly. Exemplary reporter genes encode, inter alia, enzymes, fluorescent proteins, bioluminescent proteins, receptors, antigenic epitopes, and transporters.

  [49] As used herein, the term "reporter probe" refers to a molecule that includes a detectable label and that is used to detect the presence (eg, expression) of a reporter molecule. The detectable label on the reporter probe can be any detectable moiety and includes, but is not limited to, isotopes (such as those detectable by PET, SPECT, etc.), chromophores, and fluorophores. The reporter probe may be any detectable molecule or composition that binds to or is acted upon by the reporter probe to allow detection of the reporter molecule.

  [50] The term "heterologous" as used herein refers to a nucleic acid or polypeptide that does not normally exist in nature when used with respect to a nucleic acid or polypeptide. Thus, a nucleic acid or polypeptide that is heterologous with respect to a host cell refers to a nucleic acid or polypeptide that is not naturally present in that host cell. For example, a nucleic acid molecule comprising a non-host nucleic acid encoding a polypeptide operably linked to a host nucleic acid comprising a promoter is considered a heterologous nucleic acid molecule. Conversely, a heterologous nucleic acid molecule can contain an endogenous structural gene operably linked to a non-host (exogenous) promoter. Similarly, a peptide or polypeptide encoded by a non-host nucleic acid molecule, or a fusion of a non-host polypeptide to an endogenous polypeptide is a heterologous peptide or polypeptide.

[51] The term "secreting" as used herein refers to the passage of a molecule or signal from one side of the membrane to the other.
[52] The term "selective agent" as used herein refers to unicellular organisms and / or artificial endosymbiosis and / or host cells in the absence of a selectable agent. Represents a molecule, polypeptide, or set of culture conditions that are lethal or inhibitory.

[53] The term “tagged artificial endosymbiosis” as used herein refers to an artificial endosymbiosis having a ligand on the surface of the endosymbiosis.
Artificial endosymbiosis
[54] The unicellular organisms of the present invention include bacteria that can survive in eukaryotic cells and maintain a copy number such that the phenotype introduced by the unicellular organism is observed in daughter cells. In certain embodiments, a unicellular organism does not kill eukaryotic host cells without further human intervention. In certain embodiments, a unicellular organism has a functionality that is acquired by a eukaryotic cell upon introduction of the unicellular organism. In certain embodiments, one or both modalities in the multi-modality provide additional functionality other than just imaging. In certain embodiments, the functionality of a unicellular organism is magnetic, nutrient, metabolite, vitamin, cofactor, DNA molecule, RNA molecule, macromolecule, industrial precursor, prodrug, hormone, fatty acid, carbohydrate, monosaccharide, signal Production of molecules, pharmacologically active compounds, bioactive compounds, desalinization, cryoprotectants, nitrogen fixation, photosynthesis, response to environmental attacks, or resistance to severe environmental attacks. In certain embodiments, functionality involves gene expression in a unicellular organism. In certain embodiments, functionality involves the expression of one or more genes or a set of genes in a unicellular organism. In certain embodiments, functionality involves protein expression in a unicellular organism. In certain embodiments, functionality involves the expression of a set of proteins in a unicellular organism. In certain embodiments, functionality involves the expression of a gene or gene product that is transmitted to a host to express its phenotype.

  [55] Magnetism includes diamagnetism and paramagnetism. In some embodiments, magnetism includes ferromagnetism. In some embodiments, the eukaryotic cell maintains its functionality for at least 48 hours. In some embodiments, the unicellular organism has a phenotype of at least 2 cell divisions, or at least 3 cell divisions, or at least 4 divisions, or at least 5 divisions, or at least 6 in a eukaryotic daughter cell. , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cell divisions. In other embodiments, the unicellular organism has a phenotype between 3-5 divisions, or 5-10 divisions, or 10-15 divisions, or 15-20 divisions in a eukaryotic daughter cell. Can be kept stable.

  [56] In some embodiments, the unicellular organism of the invention is genetically modified. Methods for genetically modifying bacteria are well known in the art. In some embodiments, the bacteria have a phenotype useful for improving their survival in eukaryotic host cells and / or for reducing the toxicity of unicellular organisms to eukaryotic cells, and / or for eukaryotic cells. It will be genetically modified to confer. In one embodiment, the flagellar protein of a unicellular organism is modified such that the unicellular organism can no longer express the flagellar protein in a eukaryotic host cell. In other embodiments, the unicellular organism is modified so that it can no longer synthesize the essential molecule, which is preferably supplied by a eukaryotic host cell. In certain embodiments, a unicellular organism is genetically modified so that its cell cycle is synchronized with the cell cycle of a eukaryotic host cell, thereby causing the copy number of the unicellular organism to a level sufficient to confer that phenotype to the daughter cell. Can be maintained. In certain embodiments, the gene localizes the artificial endosymbiosis at a specific intracellular location. In certain embodiments, the gene provides enhanced or blocked entry of the artificial endosymbiosis into a particular host cell. In certain embodiments, the gene suppresses or alters the host immune system response to an artificial endosymbiosis or gene and protein expressed therefrom.

  [57] Embodiments of the present invention include unicellular organisms that are α-Proteobacteria. In the current classification scheme based on 16S rRNA, α-proteobacteria are recognized as a class within the Proteobacteria, with seven major subgroups or eyes (Caulobacterales, Rhizobiales, Rhodobacterales). ), Rhodospirillales, Rickettsiales, Sphingomonadales, and Parvularculales) (Gupta, RS, “Phylogenomics and signature proteins for the alpha Proteobacteria and its main groups, ”BMC Microbiol. 7: 106 (2007), incorporated herein in its entirety for all purposes).

  [58] Numerous α-proteobacterial genomes, including all of the major groups within α-proteobacteria, have been sequenced to identify higher taxonomic groups within α-proteobacteria (eg, families, eyes, etc.) Information has been obtained that can be used to identify a specific set of genes or proteins that are distinct features (Gupta, supra; incorporated herein in its entirety for all purposes).

  [59] Unicellular organisms useful as artificial endosymbiosis include, but are not limited to, the following: Anabaena, Nostoc, Diazotroph, Indigo Cyanobacteria, Trichodesmium, Beijerinckia, Clostridium, Green sulfur bacteria, Azotobacteraceae, Rhizobia, Frankia, flavobacteria, Methanosarcinales, Aerobic halophilic archaea of Halobacteriales, Anaerobium of Halanaerobiales Firmicutes (low G + C brand), red aerobic Salinibacter (Bacteroidetes branch), Marinobacter, Halomonas nas), Dermacoccus, Kocuria, Micromonospora, Streptomyces, Williamsia, Tskamurella, Alteromonas, Colwellia, Glaciecola, Pseudoalteromonas, Shewanella, Polaribacter, Pseudomonas, Psychrobacter, Anthrobacter (Athrobacter), Frigoribacterium, Subtercola, Microbacterium, Rhodoccu, Bacillus, Bacteroides, Propionibacterium Genus (Propionibacterium), Fusobacterium, Klebsiella, Recital Ze-positive Clostridia, Clostridia, Veillonella, Fusobacteria, Chromatiaceae, Chlorobiceae, Rhodospirillaceae, Thiobacilli, Nitromonas, Nitromonas Nitrobacter, methanogen, acetic acid, sulfate reducer, and lactic acid bacteria.

  [60] The genomes of these many unicellular organisms have been sequenced or are being determined, including: M. frigidum, M. burtonii (M. burtonii) ), Clostridium symbiosum, C. psychrerythraea, P. haloplanktis, Halorubrum lacusprofundi, Vibrio salmonicida salmonicida), Photobacterium profundum, S. violacea, S. frigidimarina, Psychrobacter sp. 273-4, Shuwanella benshika (S. violacea) benthica), Psychromonas sp. CNPT3, Moritera (Morit ella species, Desulfotalea Psychrophila, Exiguobacterium 255-15, Flavobacterium psychrophilum, Psychroflexus torquis, Polaribacter filamentous, Polaribacter filamentous P. irgensii, Renibacterium salmoninarum, Leifsonia-related PHSC20-c1, Acidithiobacillus ferrooxidans, Thermoplasma acidophilum acidophilum Picrophilus torridus, Sulfolobus tokodaii, and Ferroplasma acidarmanus.

  [61] In some embodiments, an artificial endosymbiosis excludes unicellular organisms that are known to be intracellular pathogens or intracellular-intrasymbiosis. The genomes of many intracellular pathogens include, for example, genomic islands that contain virulence genes that encode adhesion factors that attach to target eukaryotic cells and induce phagocytosis of intracellular pathogens. (Juhas, M. et al., “Genomic islands: tools of bacterial horizontal gene transfer and evolution,” FEMS Microbiol Rev. 33: 376-393 (2009), incorporated herein in its entirety for all purposes. ). Many virulence factors utilize type III or type IV secretion systems. Some virulence factors are secreted into eukaryotic host cells to alter membrane traffic in target eukaryotic cells, and some virulence factors interact with host proteins involved in apoptosis (Dubreuil, R. et al ., “Bringing host-cell takeover by pathogenic bacteria to center stage,” Cell Logis. 1: 120-124 (2011), incorporated herein in its entirety for all purposes).

  [62] Embodiments of the invention include unicellular organisms that are magnetotactic bacteria ("MTB"). Since the discovery by Blakemore in 1975, a number of MTB species have been known to those skilled in the art (see, eg, Blakemore, R., “Magnetotactic bacteria,” Science 24: 377-379 (1975), all for all purposes. A group of microorganisms (incorporated herein) (Faivre, D. et al., “Magnetotactic bacteria and magnetosomes,” Chem Rev. 108: 4875-4898 (2008), hereby in its entirety for all purposes. Incorporated into the book). MTB has been identified in various subgroups of Proteobacteria and Nitrospira, with most phylotypes belonging to α-proteobacteria. Currently, culturable MTB strains determined to be α-proteobacteria by 16S rRNA sequence similarity include strains originally isolated by Blakemore in 1975; Magnetospirillum magnetotactium (Formerly Aquasprillium magnetotactium), M. gryphiswaldense, M. magneticum strain AMB-1 (“AMB”), Magnetospirillum polymorphum (M. polymorphum), Magnetosprillum species MSM-4 and MSM-6, Magnetococcus marinus, marine vibrio strains MV-1 and MV-2, Marine spirillum strain MMS-1, and Magnetococcus sp., Strain MC-1, and others. Many MTBs, including AMB, are available in pure culture. The doubling time of AMB in pure culture is approximately 8 hours, which is close to that of common mammalian cells.

  [63] Standard MTB growth media uses succinic acid as the main carbon source, but MTB is the only carbon source with fumarate, tartrate, malate, lactate, pyruvate, oxaloacetate, malonate, P-hydroxybutyrate and maleate Can proliferate. These metabolites are present inside eukaryotic cells. Microaerobic, facultative anaerobic, and absolute anaerobic MTB strains have been identified. Oxygen concentrations in the cytosol of eukaryotic cells are low due to capture by proteins such as myoglobin and enrichment in certain cell locations, such as mitochondria, so that the microaerobic or facultative anaerobic environment required for MTB growth is true. It already exists in the nucleus cell.

[64] MTBs can also be classified by the magnetic particles they synthesize, magnetite (Fe ₃ O ₄ ) or grayite (Fe ₃ S ₄ ). Magnetite-producing bacteria are microaerobic or facultative anaerobic, require some oxygen source for magnetosome synthesis, and have an optimal growth temperature near physiological temperature.

  [65] In certain embodiments, a unicellular organism of the invention is genetically modified. Molecular biology tools for the genetic manipulation of the MTB of AMB and the M. gryphiswaldense strain MSR-1 have been most thoroughly developed (Jogler, C. and Schtiler, D., “Magnetoreception and Magnetosomes in Bacteria,” p 134-138, New York, Springer (2007), incorporated herein in its entirety for all purposes). Since the AMB genome of all MTBs was first sequenced, all references to MTB gene herein refer to this genome unless otherwise specified. The genomes of the other two Magnetospirillum strains and Magnetococcus sp., Strain MC-1, were also recently sequenced. Genes derived from these strains or other MTB strains that are currently culturable or non-culturable, either sequenced or unsequenced, known or unknown can be used in the present invention.

  [66] A gene involved in magnetosome formation in the MTB cluster of genomic islands known as magnetosome islands (MAI). In M. gryphiswaldense, the 130 kb MAI generally has the following structure: 4 polycistronic operons: the mamAB operon has 17 identified spreads over 16.4 kb. The mamGFDC operon has four identified ORFs 2.1 kb and 15 kb upstream of mamAB; the mms6 operon has six identified ORFs 3.6 kb and 368 bp upstream of mamGFDC; mamXY The operon has four identified ORFs located approximately 30 kb downstream of mamAB; and the monocistronic mamW gene. In Mal, the proteins Mam W, Mgl457, Mgl458, Mgl459, Mms6, Mgl462, MamG, MamF, MamD, MamC, MamH, Maml, MamE, MamJ, MamK, MamL, MaM, MaM, MaM, MaM, MaM, MaM, MaM, MaM, MaP MamR, MamB, MamS, MamT, MamU, and Mgl505 have been identified, many of which have been given specific functions in magnetosome formation. Four genes outside the MAI, mamY, mtxA, mmsF, and mamX are associated with magnetosome formation. Conserved MAIs with slightly different genomic organization and size were found in other MTBs. These genes have also been identified for AMB-1 (see Table 2, in Fukuda, Y. et al., “Dynamic analysis of a genomic island in Magnetospirillum sp. Strain AMB-1 reveals how magnetosome synthesis developed,” FEBS. Lett. 580: 801-812 (2006), incorporated herein in its entirety for all purposes).

  [67] In certain embodiments, genetic modification is performed on a unicellular organism. Such modifications may be directional modifications, random mutagenesis, or a combination thereof. Natural-endosymbiosis is a new potential donor and often obtains the necessary nutrients from the host.

  [68] Host colonization by host symbiosis takes place in seven stages: 1) propagation, 2) invasion, 3) host defense, 4) positioning, 5) provision of benefits to the host, 6) host environment Survival, and 7) regulation.

  [69] In some embodiments, mutual nutrient dependence (bionutrition) may be established between unicellular organisms and eukaryotic cells. In one embodiment, the unicellular organism comprises at least one deletion or inactivation of a gene encoding an enzyme for synthesizing the essential molecule, thereby resulting in the absence of the enzyme or expression of the inactive enzyme Molecules are produced by eukaryotic host cells. Essential molecules can include, but are not limited to, amino acids, vitamins, cofactors, and nucleotides. For example, bionutrition can be achieved by knocking out the ability of a unicellular organism to produce amino acids, which would then be obtained from the host. Glycine is a reasonable choice because it is present in significant amounts in mammalian cells and is the end product in bacterial amino acid biogenesis; there are at least 22 other possibilities. The enzyme serine hydroxymethyltransferase converts serine to glycine at the end of the 3-phosphoglycerate biosynthetic pathway for amino acid production. In one embodiment, the unicellular organism is AMB genetically modified with the gene amb2339 (encoding the enzyme serine hydroxymethyltransferase). There are a number of methods known to those of skill in the art for mutating or knocking out a gene, including: in vitro mutagenesis, targeted DNA insertion into the gene of interest by homologous recombination, or gene (or operon; Deletion (because most of the bacterial genes are clustered within the operon), or the use of endonucleases, if the appropriate site exists only around the target in the genome.

  [70] In other embodiments, the nutritional dependence of unicellular organisms on host cells is also established by eliminating the ability of unicellular organisms to synthesize various metabolites, cofactors, vitamins, nucleotides, or other essential molecules. it can.

  [71] In some embodiments of the invention, the MTB has mutations and / or deletions in genes associated with movement and / or secretion. MTB has flagella, and in certain embodiments of the invention, MTB has a deletion and / or mutation in at least one gene that encodes a molecular mechanism associated with flagella so that magnetic bacteria do not produce functional flagella. . In addition, many MTBs secrete various compounds such as hydroxamate and catechol siderophore that are harmful to the host or induce an immune response from the host. In the sequenced AMB genome, 83 genes out of 4559 ORFs were associated with cell motility and secretion. The flagellar assembly is known to be composed of the following gene products: amb0498, amb0500, amb0501, amb0502, amb0503, amb0504, amb0505, amb0610, amb0614, amb0615, amb0616, amb0616, amb0616, amb0616, amb0616, amb0616, amb0616, amb0616 , Amb1389, amb2558, amb2559, amb2578, amb2579, amb2856, amb3493, amb3494, amb3495, amb3496, amb3498, amb3824, and amb3827. Flagella is controlled by a chemotaxis mechanism composed of at least the following gene products: amb0322, amb0323, amb0324, amb0325, amb0326, ambl806, ambl963, ambl966, amb2333, amb2635, amb2626, amb2648, amb2932, amb3002, amb3003, amb3004, amb3007, amb3102, amb3329, amb3501, amb3502, amb3654, amb3879, and amb3880.

  [72] In one embodiment, a gene encoding antibiotic resistance is inserted into the genome of a unicellular organism. A eukaryotic cell cultured in a medium containing the antibiotic will require its unicellular organism for survival. Neomycin resistance is conferred by either one of two aminoglycoside phosphotransferase genes, which also confer resistance to geneticin (G418) commonly used in eukaryotes. Hygromycin B resistance is conferred by a kinase that inactivates hygromycin B by phosphorylation. Puromycin is an antibiotic commonly used for mammalian cell culture, and resistance is conferred by the pac gene encoding puromycin N-acetyl-transferase. By external control of the antibiotic concentration, the copy number of unicellular organisms can be regulated intracellularly. Any other system in which resistance or tolerance to this factor is achieved by chemical modification of the external factor can be employed. Indirect nutritional benefits for eukaryotic cells can also be established using MTB and magnetic culture methods. In this embodiment, a magnetic field is established to benefit eukaryotic cells containing MTB. This is possible by providing means for attaching to the culture matrix, providing access to the necessary growth or media factors, or by selection between cell passages.

  [73] In other embodiments, genetic modifications are made in the MTB genome to increase intracellular stability against host defense mechanisms for a particular host cell type. Many endosymbiosis and endoparasites of eukaryotic cells, such as insect proteobacterial endosymbiosis, such as Buchnera, Wigglesworthia and Wolhachia; anaerobic ciliates Methanogenesis-Endosymbiotic; Nitrogen fixation symbiosis in the diatom Rhopalodia; Chemical synthesis of intestinal tubeworm (Olavius or Jnanidrillus)-Endosymbiotic aggregates; Cyanobacteria-endosymbiosis; endosymbiosis of all five existing classes of Echinodermata; plant Rhizobia endosymbiosis; various endosymbiotic algae; Legionella of Ameoba proteus (Legionella) -like X bacteria-endosymbiosis; numerous Salmonella species, Mycobacterium tuberculosis, Legionella pneumophila la pneumophila) are present in membrane-bound vacuole, often called symbiosomes, whereas certain species such as Blochmannia, rickettsia, Shigella ( Shigella, enteroinvasive Escherichia coli, and Listeria have the ability to inhabit the cytosol. The Dot-Icm type IV secretion system is utilized by many intracellular bacteria acquired by phagocytosis to escape the endocytotic pathway and persist in the host cell. This system has been well studied in L. pneumophila and consists of the following proteins: DotA to DotP, DotU, DotV, IcmF, IcmQ to IcmT, IcmV, IcmW and IcmX. In Photorhabdus luminescens, a nematode luminescent endosymbiosis, genes encoding RTX-like toxins, proteases, type III secretion systems, and iron acquisition systems provide intracellular stability and replication. It was shown to support. The gene bacA and the regulatory system BvrRS are essential for maintaining the symbiosis between Rhizobia and plants and the survival of Brucella ahortus in mammalian cells. The PrfA regulon allows certain Listeria species to escape the phagosome and inhabit the cytosol. The desired intracellular location of the intracellular MTB (eg, symbiosome or cytosol) requires which genes are expressed in the MTB (directly from the genome or by a stable vector) for survival and propagation in the host environment. Will control what is. The endogenous plasmid pMGT is highly stable in MTB, and many other broad vectors (including those such as lncQ, IncP, pBBRl, etc.) can stably replicate in MTB.

  [74] In other embodiments, the unicellular organism is transformed into a bacterial growth inhibitor gene (s), siderophore gene (s), metabolic requirement gene (s), suicide gene (s), cell cycle regulation The gene is modified by knock-in of the gene, such as gene (s), transporter gene (s), and phagosome gene (s). In other embodiments, unicellular organisms are randomly mutated and then screened for enhanced integration within the host cell. Random mutation can be achieved by treatment with mutagenic compounds, exposure to UV radiation, or other methods known to those skilled in the art.

  [75] In other embodiments, transgenic modification (s) are performed using genes from various parasites or endosymbiosis to counteract eukaryotic defense. In other embodiments, the balance of internal utilization of host mechanisms (nutrient availability, replication control, etc.) and antibiotic concentrations regulate the population of unicellular organisms in eukaryotic host cells.

  [76] In other embodiments, natural endosymbiosis or intracellular parasites are genetically modified to produce magnetosomes. Nitrogen fixation in insect endosymbiosis, for example, Buchnera, Wigglesworthia and Wolhachia; Anaerobic ciliate methanogenesis-endosymbiosis; Diatom Rhopalodia Symbiotics; Chemical synthesis of intestinal tubeworms (Olavius or Jnanidrillus)-Endosymbiotic aggregates; Sponge cyanobacteria-Endosymbiosis; Echinodermata existing 5 Endosymbiosis of all three classes; plant Rhizobia endosymbiosis; various endosymbiotic algae; Legionella-like X bacteria-endosymbiosis of Ameoba proteus; multiple Salmonella ) Species, Mycobacterium tuberculosis, Legionella pneumophila, etc. that belong to α-proteobacteria The magnetosome can be produced. In other embodiments, an artificial organ symbiosis can be produced by genetically modifying an existing organelle to express one or more magnetosome genes. For example, mitochondria, plastids, hydrogenosomes, apicoplasts, or other organelles with their own genetic material can be genetically altered. Bacteria modified to produce magnetosomes include Francisella tularensis, Listeria monocytogenes, Salmonella typhi, Brucella, Legionella, Mycobacterium, Nocardia, Rhodococcus equi, Yersinia, Neisseria meningitidis, Chlamydia, Rickettsia, Coccella The genus (Coxiella) and the like can be included.

  [77] In a preferred embodiment, the unicellular organism is MTB that produces magnetic particles upon eukaryotic cell culture, which may or may not be genetically altered.

Nucleic acid
[78] The nucleic acids of the invention can include expression vectors, such as plasmids, or viral vectors, or linear vectors, or vectors that are integrated into chromosomal DNA. Expression vectors can include nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of cells. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria. In eukaryotic host cells, eg, mammalian cells, the expression vector is integrated into the host cell chromosome and can then replicate with the host chromosome. Similarly, vectors can be integrated into prokaryotic chromosomes.

  [79] Expression vectors generally include a selection gene, ie, also called a selectable marker. For prokaryotic cells and eukaryotic cells, including host cells of the invention, selectable markers are well known in the art. This selection gene encodes a protein that is necessary for the survival or growth of transformed host cells grown in a selective medium. Host cells that are not transformed with the vector containing the selection gene will not survive in the medium. Common selection genes encode the following proteins: (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline; (b) compensate for auxotrophic deficiencies; or (C) A gene that supplies important nutrients that cannot be obtained from the complex medium, for example, a gene encoding D-alanine racemase for Bacilli. One example of a selection scheme utilizes drugs that stop the growth of host cells. Cells that have been successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection. Other selectable markers for use in bacterial or eukaryotic (including mammalian) systems are well known in the art. In certain embodiments, the selectable marker is a target protein or is encoded by a nucleic acid that is secreted into the host cell by a unicellular organism.

  [80] Expression vectors for producing heterologous polypeptides can also contain an inducible promoter, which is recognized by the host RNA polymerase and is operably linked to the nucleic acid encoding the target protein. . Inducible or constitutive promoters (or control regions) with appropriate enhancers, introns, and other regulatory sequences are well known in the art.

  [81] In certain embodiments, it may be desirable to modify a polypeptide of the invention. Those skilled in the art will recognize a number of ways to make changes to a particular nucleic acid construct. Such well-known methods include site-directed mutagenesis, PCR amplification using denatured oligonucleotides, exposure of nucleic acid-containing cells to mutagens or radiation, chemical synthesis of desired oligonucleotides (eg, generating large nucleic acids). In combination with ligation and / or cloning), and other well-known techniques. See, for example, Giliman and Smith, Gene 8: 81-97 (1979) and Roberts et al., Nature 328: 731-734 (1987), incorporated herein by reference.

[82] In certain embodiments, a recombinant nucleic acid encoding a polypeptide of the invention can be modified to obtain preferred codons that enhance translation of the nucleic acid in the selected organism.
[83] The polynucleotide of the present invention also includes a polynucleotide comprising a nucleotide sequence substantially equivalent to the polynucleotide of the present invention. A polynucleotide according to the present invention can have a sequence identity of at least about 80%, more typically at least about 90%, even more typically at least about 95% to a polynucleotide of the present invention. The present invention also includes the complements of those polynucleotides, which are at least about 80%, more usually at least about 90%, and even more generally relative to polynucleotides encoding the polypeptides set forth above. Provides those comprising a nucleotide sequence with at least about 95% sequence identity. The polynucleotide may be DNA (genome, cDNA, amplified or synthesized) or RNA. Methods and other algorithms for obtaining such polynucleotides are well known to those of skill in the art and include, for example, methods for determining hybridization conditions that can routinely isolate polynucleotides having the desired sequence identity. Can do.

  [84] A nucleic acid encoding a protein analog according to the present invention (ie, one or more amino acids designed to differ from the wild-type polypeptide) is a primer for site-directed mutagenesis or a desired point mutation (single) Or a plurality of PCR amplification. For a detailed description of suitable mutagenesis methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and / or Ausubel et al., Editors, Current See Protocols in Molecular Biology, Green Publishers Inc. and Wiley and Sons, NY (1994). Chemical synthesis using the method described by Engels et al., 1989, in Angew. Chem. Intl. Ed., Volume 28, pages 716-734 can also be used to make such nucleic acids.

  [85] "Recombinant variant" refers to any polypeptide that differs from a natural polypeptide by amino acid insertions, deletions, and substitutions, and that has been produced using recombinant DNA methods. Guidance in determining which amino acid residues can be substituted, added or deleted without negating the activity of interest, eg, enzymatic activity or binding activity, is the sequence of the polypeptide of the homologous peptide. It can be found by minimizing the number of amino acid sequence changes in regions of high homology compared to the sequence.

  [86] Preferably, an amino acid “substitution” is the result of replacing one amino acid with another amino acid having similar structure and / or chemical properties, ie, a conservative amino acid substitution. Amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, 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 positively charged (basic) amino acids include arginine, lysine, and histidine; negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

  [87] "Insertions" or "deletions" are generally in the range of about 1 to 5 amino acids. Acceptable variations are determined experimentally by systematically making amino acid insertions, deletions or substitutions in the polypeptide molecule using recombinant DNA methods and assaying the resulting recombinant variants for activity. it can.

  [88] Alternatively, if a change in function is desired, insertions, deletions or non-conservative changes can be engineered to produce altered polypeptides or chimeric polypeptides. Such changes can alter, for example, one or more biological functions or biochemical characteristics of the polypeptides of the invention. For example, such changes can change polypeptide characteristics, such as ligand binding affinity or degradation / turnover rate. Furthermore, such changes can be selected to produce a polypeptide that is more suitable for expression, scale-up, etc. in the host cell selected for expression.

  [89] Alternatively, recombinant variants encoding these same or similar polypeptides can be synthesized or selected using "redundancy" in the genetic code. Various codon substitutions, such as silent changes that create various restriction sites, can be introduced to optimize cloning into plasmids or viral vectors or expression in specific prokaryotic or eukaryotic systems. Mutations in the polynucleotide sequence are reflected in the polypeptide, or in domains of other peptides added to the polypeptide, to alter the properties of any part of the polypeptide, ligand binding affinity or degradation / turnover Features such as speed can be changed.

  [90] In a preferred method, the polynucleotide encoding the novel nucleic acid is altered by site-directed mutagenesis. This method uses an oligonucleotide sequence that encodes the polynucleotide sequence of the desired amino acid variant, and the adjacent nucleotides on either side of the altered amino acid sufficient to form a stable duplex on either side of the site to be altered. In general, site-directed mutagenesis techniques are well known to those skilled in the art and are exemplified in publications such as Edelman et al., DNA 2: 183 (1983). A versatile and effective method for generating site-specific changes in polynucleotide sequences is described in Zoller and Smith, Nucleic Acids Res. 10: 6487-6500 (1982).

  [91] PCR can also be used to generate amino acid sequence variants of nucleic acids. When a small amount of template DNA is used as a starting material, the desired amino acid variant can be prepared with primer (s) slightly different in sequence from the corresponding region in the template DNA. PCR amplification produces a population of product DNA fragments that differ from the polynucleotide template encoding the target at the location specified by the primer. The product DNA fragment replaces the corresponding region in the plasmid, resulting in the desired amino acid variant.

  [92] Additional techniques for generating amino acid variants include cassette mutagenesis described in Wells et al., Gene 34: 315 (1985), and other mutagenesis methods well known in the art, such as Sambrook et al., Supra, and Ausubel et al., Supra.

Eukaryotic cells
[93] In certain embodiments, the present invention provides eukaryotic cells comprising hereditary unicellular organisms within eukaryotic cells, and methods of introducing unicellular organisms into host cells.

  [94] In some embodiments, the eukaryotic cell is a plant cell. In some embodiments, the eukaryotic cell is a monocotyledonous or dicotyledonous cell, including corn, wheat, barley, rye, oats, rice, soybean, groundnut, pea, lentil and alfalfa, cotton, rapeseed, canola (canola), pepper, sunflower, potato, tobacco, tomato, eggplant, eucalyptus, tree, ornamental plant, perennial grass, or forage crop. In other embodiments, the eukaryotic cell is an algae, and includes Chlorella, Chlamydomonas, Scenedesmus, Isochrysis, Dunaliella, Tetraselmis, Nanno This includes, but is not limited to, the genus Chloropsis (Nannochloropsis), or Prototheca. In some embodiments, the eukaryotic cell is a fungal cell, including Saccharomyces, Klyuveromyces, Candida, Pichia, Debaromyces, Hansenula ( Hansenula, Yarrowia, Zygosaccharomyces, or Schizosaccharomyces fungi, including but not limited to.

  [95] In some embodiments, the eukaryotic cell of the invention is an animal cell. In some embodiments, the eukaryotic cell is from a mammal, such as a mouse, rat, rabbit, hamster, human, pig, cow or dog. The mouse functions routinely as a model for other mammals, most specifically for humans (see, eg, Hanna, J. et al., “Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin, ”Science 318: 1920-1923 (2007); Holtzman, DM et al.,“ Expression of human apolipoprotein E reduces amyloid-β deposition in a mouse model of Alzheimer's disease, ”J Clin Invest. 103 (6): R15-R21 (1999); Warren, RS, et al., “Regulation by vascular endothelial growth factor of human colon cancer tumorigenesis in a mouse model of experimental liver metastasis,” J Clin Invest. 95: 1789-1797 (1995); Each of which is incorporated herein in its entirety for all purposes).

  [96] In some embodiments, the eukaryotic cell is a human cancer cell. There are numerous human cancer cell lines well known to those skilled in the art, including common epithelial tumor cell lines such as Coco-2, MDA-MB-231 and MCF7; and non-epithelial tumor cell lines such as HT-1080 and HL60, as well as the NCI60 cell line panel (see, eg, Shoemaker, R., “The NCI60 human tumor cell line anticancer drug screen,” Nat Rev Cancer 6: 813-823 (2006), the book as a whole for all purposes. Incorporated herein by reference). Furthermore, those skilled in the art are familiar with obtaining cancer cells from primary human tumors.

  [97] In other embodiments, the eukaryotic cell is a stem cell. A person skilled in the art has various stem cell types including embryonic stem cells, induced 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, and testicular cells Familiar with

  [98] In some embodiments, the eukaryotic cell is a cell found in the circulatory system of a human host. For example, red blood cells, platelets, plasma cells, T cells, natural killer cells, etc., and their progenitor cells. Collectively, these cells are defined as circulating host cells of the present invention. The present invention can be used with any of these circulating cells. In some embodiments, the eukaryotic host cell is a T cell. In other embodiments, the eukaryotic cell is a B cell. In some embodiments, the eukaryotic cell is a neutrophil. In certain embodiments, the eukaryotic cell is a megakaryocyte.

  [99] In other embodiments, at least one gene derived from a eukaryotic cell is genetically mutated. In certain embodiments, genetic modifications of eukaryotic cells can establish reciprocal dependence (biotrophic) between the artificial endosymbiosis and eukaryotic cells; known to those skilled in the art specific to the target host cell type The molecular biology technique is used to form eukaryotic cell dependence on unicellular organisms for certain essential macromolecules, thus establishing the environmental burden on bionutrients. In other embodiments, the unicellular organism's nutritional dependence on eukaryotic cells by genetically mutating the unicellular organism to eliminate its ability to synthesize various metabolites, cofactors, vitamins, nucleotides, or other essential molecules. Sex can be established. In such embodiments, the unicellular organism can supply the essential molecule. In other embodiments, a eukaryotic gene encoding the enzyme serine hydroxymethyltransferase that converts serine to glycine at the end of the 3-phosphoglycerate biosynthetic pathway for amino acid production can be modified.

Methods for introducing unicellular organisms into eukaryotic cells
[100] The unicellular organisms of the present invention can be introduced into eukaryotic cells by a number of methods known to those skilled in the art, including microinjection, natural-phagocytosis, induction-phagocytosis, macropinocytosis. , Other cell uptake processes, liposome fusion, erythrocyte ghost fusion, electroporation, receptor-mediated methods and the like (see, eg, Microinjection and Organelle Transplantation Techniques, Celis et al. Eds., Academic Press: New York, (1986), and references cited therein; incorporated herein in its entirety for all purposes).

  [101] In one embodiment, a unicellular organism is introduced into a host cell by microinjection into the cytoplasm of the host cell. A variety of microinjection methods are known to those skilled in the art. Microinjection is the most efficient transfer method available (essentially 100%) and has no cell type restrictions (ibid; Xi, Z. et al., “Characterization of Wolbachia transfection efficiency by using microinjection of embryonic cytoplasm and embryo homogenate, ”Appl Environ Microbiol. 71 (6): 3199-3204 (2005); Goetz, M. et al.,“ Microinjection and growth of bacteria in the cytosol of mammalian host cells, ”Proc Natl Acad. Sci USA98: 12221-12226 (2001); all publications are incorporated herein in their entirety for all purposes).

  [102] Natural-phagocytic cells have been shown to take up bacteria including MTB (Burdette, DL et al., Vibrio VopQ induces PI3-kinase independent autophagy and antagonizes phagocytosis, ”Mol Microbiol. 73: 639 ( 2009); Wiedemann, A. et al., “Yersinia enterocolitica invasin triggers phagocytosis via β1 integrins, CDC42Hs and WASp in macrophages,” Cell Microbiol. 3: 693 (2001); Hackam, DJ et al., “Rho is required for the initiation of calcium signaling and phagocytosis by Fcγ receptors in macrophages, ”J Exp Med. 186 (6): 955-966 (1997); Matsunaga, T. et al.,“ Phagocytosis of magnetite by leucocytes, ”Appl Microbiol Biotechnol. 31 (4): 401-405 (1989); all publications are incorporated herein in their entirety for all purposes).

  [103] The method can be scaled up, but may be limited to specific cell types (eg, macrophages). However, recent studies have shown that non-phagocytic cells can endocytose bacteria when co-cultured with various factors: media and chemical and biological factors (eg, baculovirus, protein factors, gene knock-ins, etc.) It has been shown that the type can be induced (eg, Salminen, M., et al., “Improvement in nuclear entry and transgene expression of baculoviruses by disintegration of microtubules in human hepatocytes,” J Virol. 79 (5): 2720-2728 (2005); Modalsli, KR et al., “Microinjection of HEp-2 cells with coxsackie B1 virus RNA enhances invasiveness of Shigella flexneri only after prestimulation with UV-inactivated virus,” APMIS 101: 602-606 (1993); Hayward, RD et al., “Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella,” EMBO J. 18: 4926-4934 (1999); Yoshida, S. et al., “Shigella deliver an effector protein to trigger host microtubul e destabilization, which promotes Rac1 activity and efficient bacterial internalization, ”EMBO J. 21: 2923-2935 (2002); Bigildeev et al. J Exp Hematol. 39: 187 (2011); Finlay, BB et al., Common themes in microbial pathogenicity revisited, ”Microbiol Mol Biol Rev. 61: 136-169 (1997); all publications are incorporated herein in their entirety for all purposes).

  [104] A related method, macroropinocytosis or “cell drinking”, is the method used by many bacteria and viruses for intracellular entry (Zhang (2004): Molecular Imaging and Contrast Agent Database (MICAD) (database online); Bethesda (MD): National Library of Medicine (US), NCBI; 2004-2011; both publications are incorporated herein in their entirety for all purposes). There are a variety of protocols that can be used to induce cells to take up bacteria. Several agents such as nucleic acids, proteins, drugs and organelles have been encapsulated in liposomes and delivered to cells (Ben-Haim, N. et al., “Cell-specific integration of artificial organelles based on functionalized polymer vesicles, ”Nano Lett. 8 (5): 1368-1373 (2008); Lian, W. 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. 313 (1): 53-64 (2007); Heng, BC et al., “Immunoliposome-mediated delivery of neomycin phosphotransferase for the lineage-specific selection of 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, Vol. 1 54: 198 (1990); all publications Incorporated herein in its entirety for purposes). This method is inexpensive, relatively simple and can be scaled up. In addition, liposome uptake can be enhanced by manipulation of incubation conditions, changing liposome loading, receptor-mediated, and magnetic enhancement (see, eg, Pan et al. Int J Pharm. 358: 263 (2008); Sarbolouki, MN). et al., “Storage stability of stabilized MLV and REV liposomes containing sodium methotrexate (aqueous & lyophilized),” J Pharm Sci Techno. 52 (10): 23-27 (1998); Elorza, B., et al., “ Comparison of particle size and encapsulation parameters of three liposomal preparations, ”J Microencapsul. 10 (2): 237-248 (1993); Mykhaylyk, O. et al.,“ Liposomal Magnetofection, ”Methods Mol Bio. 605: 487-525 (2010); all publications are incorporated herein in their entirety for all purposes).

  [105] Erythrocyte-mediated transmission is similar to liposome fusion and has been shown to be highly efficient and effective across all cell types tested (Microinjection and Organelle Transplantation Techniques, Celis et al. Eds .; Academic Press: New York (1986), incorporated in its entirety for all purposes). In general, erythrocytes are loaded by osmotic shock or electroporation (Schoen, P. et al.,: Gene transfer mediated by fusion protein hemagglutinin reconstituted in reactive lipid vesicles, ”Gene Ther. 6: 823-832 (1999 ); Li, LH et al., “Electrofusion between heterogeneous-sized mammalian cells in a pellet: potential applications in drug delivery and hybridoma formation,” Biophys J. 71: 479-486 (1996); Carruthers, A. et al. , “A rapid method of reconstituting human erythrocyte sugar transport proteins,” Biochem. 23: 2712-2718 (1984); each publication is incorporated herein by reference in its entirety for all purposes. Red blood cells can be loaded indirectly by loading organisms and inducing them to differentiate and expand into red blood cells containing single cell organisms.

  [106] Electroporation is a commonly used low-cost method for delivering factors to cells (Potrykus, I., “Gene transfer methods for plants and cell cultures,” Ciba Found Symp. 154: 198-208, discussion 208-12 (1990); Wolbank, S. 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 is incorporated herein in its entirety for all purposes).

[107] In other embodiments, eukaryotic cells (eg, Chinese hamster ovary (CHO)) that naturally endocytose bacteria are used. In one embodiment, the modified unicellular bacteria are added directly to the CHO culture. Prior to MTB infection, CHO cells are cultured by standard methods, for example in Ham's F-12 medium containing 10% fetal calf serum medium. After infection, the medium is further supplemented with iron (40-80 μM) as either iron malate or FeCl ₃ . Many other cell types internalize bacteria by endocytosis, or more specifically phagocytosis; endosymbiosis or parasites have their own methods for cell invasion, and these natural processes Can be used for internalization of artificial endosymbiosis to form so-called symbiosomes. In other embodiments, microinjection, organelle transplantation, and chimera methods can be used to transplant symbiosomes derived from one cell into another cell type (ie, those that cannot endocytose an artificial endosymbiosis). it can. These host cells are cultured in a general medium in a manner specific to the cell type.

  [108] In one embodiment, a unicellular organism is introduced into a host cell by a liposome-mediated method. Mitochondria and chloroplasts larger than MTB were efficiently introduced into eukaryotic cells when encapsulated in liposomes (Bonnett, HT Planta 131: 229 (1976); Giles, K. et al., “Liposome-mediated uptake of Vitro Cellular & Developmental Biology-Plant 16 (7): 581-584 (1980); each publication is incorporated herein in its entirety for all purposes). Numerous liposome fusion protocols and agents are available and can be used by one skilled in the art without undue experimentation (see, eg, Ben-Haim, N. et al., “Cell-specific integration of artificial organelles based on functionalized polymer vesicles, ”Nano Lett. 8 (5): 1368-1373 (2008); Lian, W. 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. 313 (1): 53-64 (2007); Heng, BC et al.,“ Immunoliposome-mediated delivery of neomycin phosphotransferase for the lineage-specific selection of 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, 1 (54): 198 (1990); Is incorporated herein in its entirety for all purposes).

Methods of using eukaryotic cells, including unicellular organisms
[109] In another aspect, the present invention provides a method of using a phenotype introduced into a eukaryotic cell by a unicellular organism of the present invention. In some embodiments, the phenotype used is a heritable function that is not otherwise present in the eukaryotic cell. In some embodiments, eukaryotic cells with a magnetic phenotype can be magnetically manipulated.

[110] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype can be detected and monitored using magnetic detection or imaging methods; for example, magnetic resonance imaging (MRI), magnetic particle imaging (MPI) , Magnetic relaxation switching (MRS), magnetic resonance, superconducting quantum interference (SQUID), magnetometer, nuclear magnetic resonance (NMR), Mossbauer spectrometer, electron Electron paramagnetic resonance (EPR) and magnetic circular dichroism. MRI is non-invasive, can see inside optically opaque objects (including mice, humans, and other mammals), tends to have low spatial resolution and limited depth of penetration It is a widely used clinical diagnostic tool because it provides contrast between soft tissues with reasonably high spatial resolution compared to non-magnetic imaging (eg, optical probes). MPI is a non-invasive diagnostic method similar to MRI; however, it specifically detects the magnetic field generated by superparamagnetic iron oxide nanoparticles and produces an image with very low background. General MRI almost exclusively focuses on anatomical visualization and has no specificity for any particular cell type. The 'probe' used in general MRI is proton ¹ H in a widely existing mobile water molecule. Contrast agents can be used to obtain cell type specificity, but contrast agents are diluted or have toxicity issues and can only be used for short-term testing. Certain aspects of the present invention facilitate cell specific MRI, MPI or other magnetic detection imaging for long term testing in living subjects.

  [111] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype are mammalian cancer cells, including human cancer cell lines, such as human cancer cell line NCI 60; mouse cancer cell lines; and canine cancers. Cell lines are included. These magnetic cancer cells can be injected into immunocompromised mammals, such as mice, and then monitored by magnetic imaging to follow tumor progression over time. In certain embodiments, an immunocompromised mammal can be administered anti-cancer treatment or hypothetical treatment during a period in which tumor progression is followed in real time. In certain embodiments, the viability of eukaryotic cells of the invention with a magnetic phenotype is monitored by MRI, MPI or other magnetic detection means to assess in vivo cellular responses to a variety of conditions, including drug treatment.

  [112] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype are metastatic cancer cells, which are introduced into experimental animals by methods including injection. MRI, MPI or other magnetic detection can then be used to monitor the metastatic cancer cell metastasis and migration process throughout the experimental animal. In certain embodiments, magnetic eukaryotic cancer cells of the invention are injected into tumor-bearing animals, such as mice, and MRI, MPI or other magnetic detection is used to track metastatic cells circulating throughout the mammal. Can do.

  [113] In one embodiment, the eukaryotic cell of the invention with a magnetic phenotype is a macrophage and is injected into a laboratory animal. Magnetic imaging is used to detect any aggregation of macrophages in the animal body. Macrophages aggregate at sites of inflammation that may have been caused by malignant lesions including metastases.

  [114] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype are ES cells obtained from mammalian species including, but not limited to, humans, mice, rats and pigs, iPS Cells, or stem cells, including adult stem cells, or derived from those stem cells. Stem cells can be introduced directly into the target organism or can be first differentiated in vitro and then introduced into the target organism. In vivo fate, including the location of the introduced cells, growth rate and viability, can be assayed by magnetic imaging.

  [115] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype are therapeutic T cells. The therapeutic T cells can be introduced directly into the target organism and the in vivo kinetics including the location, proliferation rate and viability of the introduced cells can be assayed by magnetic imaging.

  [116] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype are hematopoietic stem cells or progenitor cells, which are then introduced into a mammal. When hematopoietic stem or progenitor cells are introduced into a mammal, these cells will stay in the bone marrow. The magnetic hematopoietic stem or progenitor cells can be monitored for their behavior, including the location, proliferation, and migration into the bloodstream when subjected to various stimuli.

  [117] In certain embodiments, the magnetic-artificial endosymbiosis divides at a slower rate than the stem cell host cell in which they reside. Over time, stem cells that divide generally at a slower rate than more differentiated progenitor cells retain a magnetic phenotype for a longer time than more differentiated progenitor cells, and these two types of cells differ measurable when magnetically imaged. It will be like that. In one embodiment, a chimeric cell is produced by fusing a eukaryotic cell of the invention with a magnetic phenotype to a eukaryotic system of the desired cell type. Chimeric cells can be introduced into animals and followed by magnetic imaging.

  [118] In some embodiments, the eukaryotic cell of the invention with a magnetic phenotype is an embryonic cell. In some embodiments, the embryonic cell is a pregnant animal, such as a mouse or rat egg. In certain embodiments, when an embryo is implanted into a female animal and developed, an animal is born that includes a unicellular organism throughout the body. Magnetic tissues can be collected from the organisms born and magnetic cell lines can be obtained from them. In certain embodiments, these animals are bred and the magnetic phenotype is maternally inherited. In certain embodiments, eukaryotic cells of the invention with a magnetic phenotype are introduced into a multicellular embryo. The magnetic cell lineage can be followed by magnetic imaging as the embryo develops. In some embodiments, the artificial endosymbiosis is not retained in an adult animal, but due to their presence early in development, the animal's immune system causes the artificial endosymbiosis, or a host cell comprising an artificial endosymbiosis Is not recognized as a foreign object.

  [119] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype move by magnetically attracting eukaryotic cells. In some embodiments, this motion is achieved using an externally generated magnetic field and magnetic field gradient. Various devices for magnetic targeting have been reported, such as those described below; US Patent No. 8,159,224, and Riegler J. et al., “Superparamagnetic iron oxide nanoparticle targeting of MSCs in vascular injury,” Biomaterials 34 ( 8): 1987-94 (2013). In certain embodiments, eukaryotic cells of the invention with a magnetic phenotype are isolated in vitro or in vivo (after being introduced into an organism) from a heterologous population of non-magnetic cells by attracting the magnetic cells with a magnet. ).

  [120] In some embodiments, eukaryotic cells of the invention with a magnetic phenotype are introduced into the bloodstream or other bodily fluid of the target organism. Eukaryotic cells can be directed to this region and localized there by a magnet placed adjacent to the target region of the organism. In some embodiments, the eukaryotic cells may be stem cells, which hold them directed to a region of the mammalian body where they can have a therapeutic effect. In certain embodiments, eukaryotic cells may be immune cells, which can be directed to a specific location in the mammalian body including the tumor or injury site. In certain embodiments, eukaryotic cells can be loaded with a therapeutic agent and released after magnetic attraction to the desired area.

  [121] In one embodiment, for a technique called Magnetic Hyperthermia Technique (MHT), the eukaryotic cell of the present invention with a magnetic phenotype is replaced with an alternating magnetic field or an alternating magnetic field (alternative magnetic field, alternative magnetic field). Place in magnetic field (referred to as AMF). AMF and MHT are described in US Publication No. US20120302819 (US Application No. 13 / 510,416); and Silva AC et al., “Application of hyperthermia induced by superparamagnetic iron oxide nanoparticles in glioma treatment,” Int J Nanomedicine 6: 591-603. (2011); each publication is incorporated herein in its entirety for all purposes). Hyperthermia is a therapy that promotes an increase in body tissue temperature to change the functionality of cellular structures. Its activity is based on the fact that elevated temperature can induce cell damage including cell lysis and cell death. In certain embodiments, the eukaryotic cells of the invention are subjected to an alternating magnetic field for 1, 5, 10, 20, 30, 40, 50 or 60 minutes. In one embodiment, the magnetic field frequency of the applied AMF is 50 kHz to 1 MHz. In some embodiments, the magnetic field amplitude of the applied AMF remains below 100 mT. In certain embodiments, eukaryotic cells of the present invention that are subjected to MHT are tumor cells, which are less tolerated than normal surrounding cells against sudden increases in temperature. In certain embodiments, eukaryotic cells of the invention are tumor cells or are adjacent to tumor cells and subjected to an alternating magnetic field until the internal temperature of the tumor reaches 43 ° C to 47 ° C.

  [122] In some embodiments, eukaryotic cells of the present invention with a magnetic phenotype are placed in a rotating magnetic field, causing cell damage, including rotation of intracellular single cell organisms and cell death. In certain embodiments, applying an alternating or rotating magnetic field to the entire cell population selectively damages eukaryotic cells of the invention with a magnetic phenotype within a heterologous population of non-magnetic cells. In some embodiments, a eukaryotic cell of the invention with a magnetic phenotype within a heterologous population of nonmagnetic cells is placed in an alternating or rotating magnetic field and is in proximity to the eukaryotic cell of the invention and the eukaryotic cell of the invention. Causes damage to both non-magnetic cells. In certain embodiments, eukaryotic cells of the invention with a magnetic phenotype are introduced into an animal and targeted to a location within the animal by magnetic manipulation or other forms of cell targeting known in the art. An alternating or rotating magnetic field can then be applied at that location in the animal to cause damage to the cells surrounding the magnetic cells. In certain embodiments, eukaryotic cells of the invention with a magnetic phenotype are introduced into an animal and targeted to a tumor site within the animal by magnetic manipulation or other forms of cell targeting known in the art. The tumor can be subjected to an alternating or rotating magnetic field to cause damage to the tumor cells surrounding the magnetic cells. In some embodiments, eukaryotic cells of the invention with a magnetic phenotype are stem cells, including ES cells obtained from mammalian species including, but not limited to, humans, mice, rats and pigs. , IPS cells, or adult stem cells. Stem cells can be introduced directly into the target organism or can be first differentiated in vitro and then introduced into the target organism. After the introduction, the animal can be subjected to an alternating magnetic field or a rotating magnetic field to kill the introduced stem cells and the lines resulting from these stem cells. In vivo kinetics including the location of introduced cells, growth rate and viability can be assayed by magnetic imaging.

Multimodal detection
[123] In another aspect, the invention relates to a eukaryotic cell that is engineered in a unicellular organism, eg, an artificial endosymbiosis. A unicellular organism can confer at least one desired phenotype to a eukaryotic cell. In certain embodiments, the unicellular organism is a polypeptide (s), nucleic acid (s), lipid (s), carbohydrate (s), amino acid (s), or other factor (s). Or) can be secreted into and / or transported from the host cell. This communication between the unicellular organism and the host cell can give rise to the desired phenotype or phenotypes for the eukaryotic cell. Such desired phenotype or desired multiple phenotypes may allow multimodal detection or observation of eukaryotic cells. Thus, in certain embodiments, a unicellular organism can be used as a multimodal probe for multimodal detection or observation of eukaryotic cells.

  [124] In some embodiments, the multimodal probe comprises a magnetic bacterium that expresses one or more reporters for multimodal detection. In certain embodiments, the magnetic bacterium comprises a magnetotactic bacterium that expresses one or more reporters for multimodal detection. In some embodiments, a heterologous gene encoding a reporter protein is introduced into a magnetic bacterium for use as a multimodal probe, whereby the genetically modified magnetic bacterium expresses the reporter. In certain embodiments, the magnetic bacterium is engineered to express a single reporter. In certain embodiments, different magnetic bacteria, each expressing a different reporter, are introduced into eukaryotic cells for multimodal detection. In certain embodiments, a magnetic bacterium is engineered to express two or more reporter products, for example by use of a single vector construct encoding two or more reporters. In certain embodiments, the reporter comprises a heterologous reporter and is expressed in a functional form for detection. Reporter gene expression provides additional imaging modalities in addition to the magnetic phenotype of magnetic bacteria.

  [125] For example, the present disclosure demonstrates reporter gene expression effective for two modalities using the magnetotactic bacterial strain Magnetospirillum magneticum (AMB-1). In one embodiment for expressing a reporter gene, a plasmid containing a reporter gene and an appropriate resistance gene (eg, one that confers antibiotic resistance) is introduced into an Escherichia coli cell and the E. coli cell is targeted Mating with magnetotactic bacterial cells and then selecting for magnetotactic bacteria. Appropriate assays can then be performed on the transformed magnetotactic bacteria to confirm the presence and positive expression of the reporter. In certain embodiments, when selecting a reporter gene for uptake, care is taken to ensure that it can still be expressed in a functional form using prokaryotic transcription and translation machinery. These modified bacteria have a magnetic phenotype and a reporter gene phenotype, thereby allowing multimodal detection of eukaryotic cells carrying genetically modified magnetotactic bacteria. Introduction and expression of additional reporter genes provides additional imaging possibilities as long as each reporter gene can be detected using different imaging modalities.

  [126] In some embodiments, the genetically modified magnetic bacterial magnetosome comprises an MRI system, a magnetic particle imaging (MPI) system, a magnetic relaxation switching (MRS) system, a magnetic resonance spectrometer, a superconducting quantum interference device (SQUID) , Magnetometers, nuclear magnetic resonance (NMR) systems, Mossbauer spectrometers, electron paramagnetic resonance (EPR) systems, and magnetic circular dichroism systems. The product of the reporter gene can be detected by any suitable detection method and device, depending on the type of reporter product expressed from the reporter gene. For example, a representative reporter gene encodes a photoprotein (eg, luciferase or eGFP) and this phenotype can be detected by optical imaging, which can be non-invasive (for BLI and FLI) or to some degree invasive, eg Can be performed with biomicroscopy and fluorescence laparoscopy (see, eg, Gahlen, J. et al., “Laparoscopic fluorescence diagnosis for intraabdominal fluorescence targeting of peritoneal carcinosis,” Ann Surg. 235: 252-260 (2002), Incorporated herein in its entirety for all purposes). As used herein, reporter expression is intended to include expression of the corresponding reporter gene that results in expression of the encoded reporter or reporter molecule.

  [127] In some embodiments, the multimodal probe comprises a positron emission tomography (PET) reporter, a single photon emission computed tomography (SPECT) reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasonograph It includes magnetic (eg, magnetotactic) bacterial cells that express one or more reporters selected from sonic reporters.

  [128] In some embodiments, the multimodal probe further expresses a fluorescent or bioluminescent reporter in addition to the reporter. In one embodiment, the multimodal probe further expresses a fluorescent reporter and a bioluminescent reporter in addition to the reporter.

[129] In some embodiments, the multimodal probe expresses at least a positron emission tomography (PET) reporter. Various PET reporters can be expressed and used for multimodal probes. In certain embodiments, the PET reporter comprises thymidine kinase. In some embodiments, the thymidine kinase is selected from herpes simplex virus-thymidine kinase, varicella-zoster virus-thymidine kinase, human-mitochondrial thymidine kinase, or an active variant thereof (see, eg, Campbell et al., J Biol Chem 287 (1): 446-54 (2012)). PET detection of thymidine kinase is generally accomplished using a PET specific reporter probe. A typical PET reporter probe for HSV thymidine kinase is [ ¹⁸ F] 9- (4- [18F] -fluoro-3-hydroxymethylbutyl) -guanine, a fluorine-18 labeled penciclovir analog. Contained and becomes retained in the cell when phosphorylated by thymidine kinase (TK). Another thymidine kinase reporter probe is 5- (76) Br-bromo-2′-fluoro-2′-deoxyuridine. In certain embodiments, a thymidine kinase reporter probe that is preferentially acted upon by heterologous-thymidine kinase compared to any endogenous thymidine kinase is used.

[130] Other PET reporters that can be used for multimodal probes include, among others, dopamine D2 (D2R) receptor, sodium iodide transporter (NIS), deoxycytidine kinase, somatostatin receptor subtype 2, norepinephrine transporter ( NET), cannaboid receptor, glucose transporter (Glut1), tyrosinase, and active variants thereof. Related reporter probes for each PET reporter are well known to those skilled in the art. An exemplary reporter probe for the dopamine D2 (D2R) receptor is 3- (2 ′-[ ¹⁸ F] fluoroethyl) spiperone (FESP) (MacLaren et al., Gene Ther. 6 (5): 785 -91 (1999)). A typical reporter probe for the sodium iodide transporter is ¹²⁴ I, which is retained intracellularly after transport by the transporter. An exemplary reporter probe for deoxycytidine kinase is 2′-deoxy-2′- ¹⁸ F-5-ethyl-1-β-d-arabinofuranosyluracil ( ¹⁸ F-FEAU). Representative reporter probes for somatostatin receptor subtype 2 are ¹¹ In-, ^99m / ^94m Tc-, ⁹⁰ Y-, or ¹⁷⁷ Lu-labeled octreotide analogs, such as ⁹⁰ Y-, or ¹⁷⁷ Lu- Labeled DOTATOC (Zhang et al., J Nucl Med. 50 (suppl 2): 323 (2009)); ⁶⁸ Ga-DOTATE; and ¹¹¹ In-DOTABASS (see, eg, Brader et al., J Nucl Med. 54 (2): 167-172 (2013), incorporated herein by reference). An exemplary reporter probe for the norepinephrine transporter is ¹¹ Cm-hydroxyephedrine (Buursma et al., J Nucl Med. 46: 2068-2075 (2005)). Representative reporter probe for Kan'naboido ^receptors, 11 C-labeled a CB ₂ ligand ^{11 C-GW405833 (Vandeputte et al} , J Nucl Med 52 (7):.. 1102-1109 (2011)) is . An exemplary reporter probe for the glucose transporter is [ ¹⁸ F] fluoro-2-deoxy-d-glucose (Herschman, HR, Crit Rev Oncology / Hematology 51: 191-204 (2004)). Representative reporter probe for tyrosinase, N- (2- (diethylamino) ethyl) - ^{18 F-5-fluoro-picolinic} amide (Qin et al, Sci Rep 3 :.. 1490 (2013)) is. Other reporter probes are described, for example, in Yaghoubi et al., Theranostics 2 (4): 374-391 (2012), which is incorporated herein by reference.

  [131] In some embodiments, the multimodal probe expresses at least a single photon emission computed tomography (SPECT) reporter. Exemplary SPECT reporters include sodium iodide transporter, dopamine D2 (D2R) receptor, and active variants thereof. In some embodiments, the SPECT reporter comprises a modified haloalkane dehalogenase capable of covalently binding a SPECT detectable cycloalkane substrate (see, eg, Hong et al., Am J Transl Res. 5 (3): 291- 302 (2013), incorporated herein by reference).

  [132] In some embodiments, the multimodal probe expresses at least a photoacoustic reporter. Exemplary photoacoustic reporters include, among others, tyrosinase and β-galactosidase (see, eg, Krumholz et al., J Biomed Optics. 16 (8): 1-3 (2011)). Reporter probes for tyrosinase are described above. An exemplary reporter probe for β-galactosidase is 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) (Li et al., J Biomed Opt. 12 (2) : 020504 (2007)).

  [133] In some embodiments, the multimodal probe expresses at least an X-ray reporter. Exemplary x-ray reporters include, among others, somatostatin receptor 2, or other types of receptor-based binding agents. The reporter probe can have a radiopaque label moiety attached to the reporter probe and is imaged, for example, by x-ray or computed tomography. Representative radiopaque labels are iodine, especially polyiodinated chemical groups (see, eg, US Patent No. 5,141,739), and paramagnetic labels (eg, gadolinium), which are reported by conventional means. It can be attached to the probe.

  [134] In some embodiments, the multimodal probe expresses at least an ultrasonic reporter. Exemplary ultrasound reporters include, inter alia, binders that can bind ultrasound contrast agents, such as microbubble contrast agents. For example, a binding agent can include an antibody that specifically directs a peptide expressed in a magnetotactic bacterium, which peptide is bound to a microbubble contrast agent (see, eg, Kiessling et al., J Nucl. Med. 53: 345-348 (2012)).

  [135] In embodiments of the invention in which the reporter is a fluorescent reporter, any number of fluorescent reporters can be used. These include, for example, green fluorescent proteins derived from Aequorea victoria or Renilla reniformis, and active variants thereof (eg, blue fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, etc.); Fluorescent proteins derived from jellyfish, copepods, Ctenophora, Anthrozoas, and Entacmaea quadricolor, and their active variants; and phycobiliproteins (phycobiliprotein), and active variants thereof.

[136] In certain embodiments of the invention in which the reporter is a bioluminescent reporter, any number of bioluminescent reporters can be used as the reporter. This includes, but is not limited to, aequorin (and other Ca ⁺² regulatory-photoproteins), luciferase based on a luciferin substrate, luciferase based on a coelenterazine substrate (eg, Renilla ), Gaussia, Metridina), and Cypridina luciferases, and active variants thereof.

  [137] In some embodiments, the multimodal probe is at least two or more reporters selected from a fluorescent reporter, a bioluminescent reporter, a PET reporter, a SPECT reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasound reporter, Express at least 3 or more reporters, at least 4 or more reporters, or at least 5 or more reporters.

  [138] In some embodiments, the multimodal probe expresses at least two reporters selected from a fluorescent reporter, a bioluminescent reporter, a PET reporter, a SPECT reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasound reporter.

  [139] In certain embodiments, the multimodal probe expresses at least three reporters selected from a fluorescent reporter, a bioluminescent reporter, a PET reporter, a SPECT reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasound reporter.

  [140] In certain embodiments, a reporter gene for a multimodal probe generates a reporter that can be detected by multiple imaging modalities, such as photoacoustic imaging (PAI), MRI, and PET (using an appropriate radio tracer) signal Encodes the indicated tyrosinase (see, eg, Qin, C. et al., “Tyrosinase as a multifunctional reporter gene for photoacoustic / MRI / PET triple modality molecular imaging,” Scientific Rep. 3: 1490 (2013), Incorporated herein in its entirety for all purposes). Alternatively, the reporter gene may be a fusion protein comprising two or more reporters linked together (eg, luciferase-GFP-thymidine kinase triple fusion reporter) (Ray P. et al., “Imaging tri-fusion multimodality reported gene expression in living subjects, ”Cancer Res. 64: 1323-1330 (2004), incorporated herein in its entirety for all purposes). In certain embodiments, each reporter comprises a different reporter, and each reporter can be detected by a different imaging modality.

  [141] In certain embodiments, a gene that expresses a heterologous reporter may be extrachromosomal, eg, as part of a self-replicating plasmid, cosmid, or bacmid. In certain embodiments, a gene that expresses a reporter is transformed into bacteria by, for example, homologous recombination, or recombinases such as Cre-loxP, Dre-rox, yeast flippase (FLP) mediated integration, and attTn7 based integration. It can be integrated into the chromosome (Choi et al., Appl Environ Microbiol. 72 (1): 753-758 (2008)). In certain embodiments, the reporter gene is constructed to express a functional reporter that is not fused to another protein. In certain embodiments, the reporter gene is constructed to express a functional reporter as a fusion, ie, a fusion to another heterologous protein or protein of a magnetotactic bacterium.

  [142] In certain embodiments, genetically modified magnetic bacteria are introduced into eukaryotic cells for multimodal detection. Once inside the eukaryotic cell, the genetically modified magnetic bacteria have the ability to self-replicate and thus maintain their levels even after a cell division event, which allows them to maintain long-term cell tracking and other Become a powerful tool for imaging possibilities. The magnetite-based iron core of magnetotactic bacteria can generate strong in vivo MRI signals. Furthermore, genetically modified magnetic bacteria allow for imaging by multimodal imaging of, for example, genetically modified magnetic bacteria and cells labeled with genetically modified magnetic bacteria before being taken up by eukaryotic cells Additional functionality can be provided by adding reporter genes. A eukaryotic cell containing a multimodal probe can include any of the aforementioned cells, including plant cells and animal cells. In certain embodiments, the eukaryotic cell comprising the multimodal probe is a mammalian cell. In some embodiments, mammalian cells can include mouse (murine), rat, rabbit, hamster, human, porcine, bovine, or canine cells. In certain embodiments, the mammalian cell is a cancer cell or stem cell, including the specific cancer cells, stem cells, and embryonic cells described in this disclosure.

  [143] Thus, in certain embodiments, a eukaryotic cell can comprise a membrane-encapsulated magnetosome and at least one expressed reporter. In some embodiments, the eukaryotic cell can comprise a magnetic bacterium, which can express or express one or more reporters. In certain embodiments, the eukaryotic cell can comprise a magnetotactic bacterium, which can express or express one or more reporters. In certain embodiments, the reporter is selected from: a fluorescent reporter, a bioluminescent reporter, a positron emission tomography (PET) reporter, a single photon emission computed tomography (SPECT) reporter, an X-ray as described herein. Reporters, photoacoustic reporters, and ultrasonic reporters.

  [144] In some embodiments, the eukaryotic cell comprises a magnetic bacterium that expresses at least two or more reporters. In certain embodiments, the two or more reporters are a fluorescent reporter, a bioluminescent reporter, a positron emission tomography (PET) reporter, a single photon emission computed tomography (SPECT) reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasound reporter Selected from.

  [145] In some embodiments, the eukaryotic cell comprises a magnetic bacterium that expresses at least three or more reporters. In certain embodiments, the three or more reporters are a fluorescent reporter, a bioluminescent reporter, a positron emission tomography (PET) reporter, a single photon emission computed tomography (SPECT) reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasound reporter Selected from.

  [146] In some embodiments, the eukaryotic cell comprises a magnetic bacterium that expresses at least one reporter selected from: positron emission tomography (PET) reporter, single photon emission computed tomography Imaging (SPECT) reporter, X-ray reporter, photoacoustic reporter, and ultrasound reporter. In certain embodiments, a magnetotactic bacterium that expresses one or more of the above reporters can also express a fluorescent reporter, a bioluminescent reporter, or both a fluorescent reporter and a bioluminescent reporter.

  [147] In some embodiments, the eukaryotic cell comprises a unicellular organism, wherein the eukaryotic cell can be detected by multiple imaging modalities. Imaging modalities are ultrasound imaging, computed tomography imaging, optical imaging, magnetic resonance imaging, optical coherence tomography imaging, radiography imaging, nuclear medicine imaging, positron emission tomography imaging, tomography imaging, photoacoustic tomography imaging, X You can choose from line imaging, thermal imaging, fluoroscopic imaging, bioluminescence imaging, and fluorescence imaging, magnetic particle imaging, and magnetic resonance spectroscopy. In some embodiments, eukaryotic cells can be detected by at least 2 or more, 3 or more, 4 or more, or 5 or more of the imaging modalities.

  [148] In some embodiments, eukaryotic cells can be detected by multiple modalities, wherein at least one modality is magnetic resonance imaging. That is, eukaryotic cells can be detected by the first modality, in which case the first modality is magnetic resonance imaging. In some embodiments, the eukaryotic cell is ultrasound imaging, computed tomography imaging, optical imaging, optical coherence tomography imaging, radiography imaging, nuclear medicine imaging, positron emission tomography imaging, single photon emission computed tomography, tomography It can be detected by a second modality selected from imaging, photoacoustic tomography imaging, X-ray imaging, thermal imaging, fluoroscopic imaging, bioluminescence imaging, and fluorescence imaging, magnetic particle imaging, and magnetic resonance spectroscopy.

  [149] In some embodiments, eukaryotic cells can be detected by multiple modalities, wherein at least one modality is magnetic particle imaging. In other words, eukaryotic cells can be detected by the first modality, where the first modality is magnetic particle imaging. In some embodiments, the eukaryotic cell is ultrasound imaging, computed tomography imaging, optical imaging, optical coherence tomography imaging, radiography imaging, nuclear medicine imaging, positron emission tomography imaging, single photon emission computed tomography, tomography Detection is possible with a second modality selected from imaging, photoacoustic tomography imaging, X-ray imaging, thermal imaging, fluoroscopic imaging, bioluminescence imaging, fluorescence imaging, and magnetic resonance spectroscopy.

  [150] In certain embodiments, when eukaryotic cells can be detected by magnetic resonance imaging or magnetic particle imaging, the second modality is fluorescence imaging. In certain embodiments, the second modality is bioluminescence imaging. In certain embodiments, the second modality is PET imaging. In certain embodiments, the second modality is photoacoustic imaging. In certain embodiments, the second modality is x-ray imaging. In certain embodiments, the second modality is ultrasound imaging. In some embodiments, eukaryotic cells can be detected by a third modality, a fourth modality, a fifth modality, a sixth modality, or more, each modality being different.

  [151] Although the use of magnetic bacteria that express one or more reporter genes provides flexibility, in some embodiments, eukaryotic cells themselves can express one or more reporters. In particular, eukaryotic cells contain magnetic bacteria, and the eukaryotic cells express one or more reporters. Any of the reporters described herein can be expressed in eukaryotic cells, including magnetic bacteria. In some embodiments, the eukaryotic cell is from a fluorescent reporter, bioluminescent reporter, positron emission tomography (PET) reporter, single photon emission computed tomography (SPECT) reporter, X-ray reporter, photoacoustic reporter, and ultrasound reporter. The selected reporter can be expressed.

  [152] In certain embodiments of eukaryotic cells comprising a magnetotactic bacterium, the eukaryotic cell can express one or more reporters, and the magnetotactic bacterium can express one or more reporters. In some embodiments, the set of reporters expressed by eukaryotic cells is different from the set of reporters expressed by magnetic bacteria. In certain embodiments, expression of different reporters by eukaryotic cells and magnetotactic bacteria provides a method of distinguishing eukaryotic cells from eukaryotic cells harboring magnetotactic bacteria.

  [153] In an embodiment of the present invention, a multimodal probe can be used to image any cell, tissue or organ containing the multimodal probe. In particular, a multimodal probe can be introduced into a eukaryotic cell, eg, as an artificial endosymbiosis, and the eukaryotic cell can be imaged by any of the methods described herein. Eukaryotic cells containing multimodal probes can be imaged in an isolated state or as part of a tissue or organ.

  [154] Accordingly, in certain embodiments, the composition can be used in a multimodal imaging method comprising: (a) magnetic imaging of a magnetotactic bacterium expressing one or more heterologous reporters; and (b) ) Imaging the reporter using a non-magnetic imaging modality.

  [155] In some embodiments, the multimodal imaging method can include: (a) magnetically imaging a eukaryotic cell comprising a magnetotactic bacterium, and (b) using at least one non-magnetic imaging modality, Eukaryotic cells are imaged by imaging one or more expressed reporters. Any reporter described in this disclosure can be used in these imaging methods. These include reporters selected from fluorescent reporters, bioluminescent reporters, positron emission tomography (PET) reporters, single photon emission computed tomography (SPECT) reporters, X-ray reporters, photoacoustic reporters, and ultrasound reporters. included.

  [156] In certain embodiments, eukaryotic cells can be detected multimodally using magnetic imaging, such as magnetic resonance imaging or magnetic particle imaging, and appropriate optical imaging methods. In certain embodiments, the optical imaging method is based on the expression of a fluorescent reporter, such as green fluorescent protein or an active variant thereof.

  [157] In certain embodiments, eukaryotic cells can be detected multimodally using magnetic imaging, such as magnetic resonance imaging or magnetic particle imaging, and appropriate PET imaging methods. In certain embodiments, the PET imaging method is based on the expression of a PET reporter, such as thymidine kinase or cytidine kinase. In this embodiment, eukaryotic cells can be detected multimodally using MRI and PET imaging.

  [158] In certain embodiments, eukaryotic cells can be detected multimodally using magnetic imaging, such as magnetic resonance imaging or magnetic particle imaging, and appropriate photoacoustic imaging methods. In certain embodiments, the photoacoustic imaging method is based on the expression of a photoacoustic reporter, such as tyrosinase or β-galactosidase. In this embodiment, eukaryotic cells can be detected multimodally using MRI and photoacoustic imaging.

  [159] As discussed herein, eukaryotic cells can be imaged using multimodal probes and imaging methods, including eukaryotic cells that are part of a tissue or multicellular organism. It is. In some embodiments, a multimodal imaging method for imaging a tissue or multicellular organism comprises: (a) imaging a eukaryotic cell comprising a magnetotactic bacterium with a first imaging modality; wherein the eukaryotic cell is A component of a tissue or multicellular organism, the magnetotactic bacterium expresses one or more heterologous reporters, and the first imaging modality is a magnetic modality; (b) one or more using a second imaging modality The eukaryotic cells are optically imaged by imaging the expressed reporter of ii; wherein the second modality is a non-magnetic modality; and (c) the tissue or organism is imaged with a third imaging modality. In certain embodiments, eukaryotic cells are imaged according to step (b). As will be apparent to those skilled in the art, in certain embodiments, in view of multiple reporters that can be expressed in magnetotactic bacteria, the second imaging modality comprises one or more imaging methods, ie, imaging modalities.

  [160] In some embodiments of the method of imaging a tissue or multicellular organism, the first imaging modality is magnetic particle imaging or magnetic resonance imaging. In some embodiments, the second imaging modality is ultrasound imaging, computed tomography imaging, optical imaging, optical coherence tomography imaging, radiography imaging, nuclear medicine imaging, positron emission tomography imaging, tomography imaging, photoacoustic tomography Non-magnetic imaging modalities selected from imaging, X-ray imaging, thermal imaging, fluoroscopic imaging, bioluminescence imaging, and fluorescence imaging.

  [161] In some embodiments of the method of imaging a tissue or multicellular organism, the third imaging modality is different from the first imaging modality. In certain embodiments, the third imaging modality is a non-magnetic imaging modality. In some embodiments, the third imaging modality is ultrasound imaging, computed tomography imaging, optical imaging, optical coherence tomography imaging, radiography imaging, nuclear medicine imaging, positron emission tomography imaging, tomography imaging, photoacoustic tomography imaging , X-ray imaging, thermal imaging, fluoroscopic imaging, bioluminescence imaging, and fluorescence imaging.

  [162] In some embodiments, multimodality is used for non-invasive in vivo imaging. In general, imaging modalities can be described as either anatomical or functional with respect to the information they provide. Anatomical modalities include X-ray computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US), which include various aspects of the anatomical structure of interest (eg, for CT Density variation, proton environment for MRI, and acoustic reflectivity for US). Functional modalities include positron emission tomography (PET), single photon emission computed tomography (SPECT), fluorescence imaging (FLI) and bioluminescence imaging (BLI), certain types of MRI (eg, diffusion-weighted (Diffusion) Weighted (DW) or Dynamic Contrast Enhanced (DCE)), magnetic particle imaging (MPI), photoacoustic imaging (PAI), contrast enhanced ultrasound (ceUS), Raman imaging (RI), contrast enhancement CT (ceCT) and X-ray fluorescence CT (XFCT) are included (Kuang, Y. et al., “First demonstration of multiplexed X-ray fluorescence computed tomography (XFCT) imaging,” IEEE Trans Med Imaging 32: 262-267 (2012), which is incorporated herein in its entirety for all purposes), within a host cell (containing a unicellular organism). (James, ML et al., “A molecular imaging primer: modalities, imaging agents, and applications,” Physiol Rev. 92: 897-965 (2012); Molecular Imaging : Principles and Practice, Eds. Weissleder, R., Ross, BD, Rehemtulla A. and Gambhir, SS, People's Medical Publishing House, USA, (2010); each publication is incorporated herein in its entirety for all purposes. Do). This is done because the signal to be measured is generated by a specific probe molecule, which may be a chemical (contrast agent) or a protein (reporter protein). The contrast agent is exogenous and is administered to the subject prior to scanning, whereas the reporter protein is produced by a reporter gene that is gene-encoded into a host cell or unicellular organism.

  [163] In certain embodiments, multi-modal imaging of the present disclosure is used to track eukaryotic cells in vivo. In order to track cells for multiple generations of eukaryotic cells, the probe must be able to maintain a detectable concentration as the eukaryotic cells divide. Contrast agent methods, including the nanoparticle methods described above, generally do not meet this requirement because they rapidly dilute to undetectable levels after several rounds of cell division. A reporter gene that expresses a detectable reporter can maintain a sustained detectable level, but requires extensive genetic engineering of the eukaryotic cell. Furthermore, there are few reporter gene options useful for long-term cell imaging by MRI. Researchers used MRI reporter genes using ferritin heavy chain genes (see, eg, Cohen, B. et al., “Ferritin as an endogeneous MRI reporter for noninvasive imaging of gene expression in C6 glioma tumors,” Neoplasia 7: 109 -117 (2005), incorporated herein in its entirety for all purposes), and genes derived from magnetotactic bacteria such as MagA (Goldhawk DE et al., “Magnetic resonance imaging of cells overexpressing MagA, an endogeneous contrast agent for live cell imaging, ”Mol Imaging 8: 129-139 (2009); Zurkiya, O. et al.,“ MagA is sufficient for producing magnetic nanoparticles in mammalian cells, making it an MRI reporter, ”Magnet Reson Med. 59: 1225 -1231 (2008), each of which is herein incorporated by reference in its entirety for all purposes), the contrast that can be obtained from this method is generally weak and the detectability obtained is poor. The advantage of a magnetic bacterium expressing a multimodal probe, such as one or more reporters described herein, is that the genetically modified magnetic bacterium can be easily introduced into a eukaryotic cell, and the magnetic bacterium in a eukaryotic cell Is able to inherit to daughter cells and thus maintain reporter levels.

  [164] In certain embodiments for multimodal imaging, multimodal probes, or eukaryotic cells comprising multimodal probes, are present in the presence of corresponding expressed reporters, such as PET, SPECT, photoacoustic, X-ray or ultrasound reporters. Is contacted with an appropriate reporter probe under conditions suitable for detecting. Where cell tracking or imaging is in a tissue or animal in vivo, an appropriate reporter probe can be administered to the tissue or animal to detect and / or image the expression of the corresponding reporter. Representative reporter probes and imaging methods are described above for the corresponding reporters. Reporter probes can be administered to a tissue or animal host by various routes. In certain embodiments, a suitable reporter probe can be administered orally or parenterally. Parenteral administration in this respect includes administration by: intravenous, intramuscular, subcutaneous, ophthalmic, intrasynovial, transepithelial (including transdermal), ocular, sublingual And oral; inhalation, topical administration by aerosol: including intraocular, cutaneous, ocular, rectal and nasal inhalation; and systemic rectal. The reporter probe is administered in a variety of forms, including formulations containing pharmaceutically acceptable excipients or carriers. Pharmaceutically acceptable excipients and carriers are substantially non-toxic and of pharmaceutically acceptable purity. In certain embodiments, forms suitable for injection can include, for example, sterile solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In certain embodiments, the reporter probe can be prepared for oral administration, for example as a liquid, tablet, capsule or powder for oral administration.

  [165] In certain embodiments, as described above, multimodal imaging can be performed in several ways. The first is to image the object or target using different imaging modalities, thus obtaining multiple types of images, and then aligning them so that different images spanning each imaged portion of the object can be compared. In certain embodiments, a representative combination is an anatomical scan (eg, CT) and a functional scan (eg, PET); this allows functional data to enter anatomical concepts. . In some embodiments, a multimodal scanner can be used to acquire two or more image types simultaneously. Two commonly used examples are combined PET / CT scanners, FLI, BLI and optical imagers capable of acquiring wide area images, and US scanners that also perform photoacoustic imaging (PAI). It should be understood that a wide variety of combinations of image types can be obtained.

  [166] In one aspect, multimodal imaging can be performed using a probe that generates a signal observable with two or more imaging modalities. This has the advantage that the image creator can image the probe using a wide range of imaging methods, allowing greater flexibility when designing imaging trials; using a multimodal probe to achieve the desired phenotype This is because it can be introduced into an object or target, thereby using the desired imaging modality of the object or target. To this end, in some embodiments, the multimodal probe can express at least two reporters, eg, by using a dual fusion reporter construct that expresses two reporter genes, thereby using at least two imaging modalities. Is possible. In certain embodiments, the multimodal probe can express at least three reporters, eg, by using a triple fusion reporter construct that expresses three reporter genes, thereby allowing cells engineered to express them. Imaging can be performed using at least three imaging modalities such as FLI, BLI and PET. In certain embodiments, the probe may be a single probe that produces a signal observable with more than one imaging modality. A typical probe that can be imaged with more than one imaging modality is the reporter tyrosinase, which can be imaged using PET, MRI and photoacoustic imaging methods. Many modifications can be made to this method in view of the guidelines of the present invention, which are explicitly described in this disclosure.

  [167] In some embodiments, multimodal images are collected, which include at least two of the following types of images: one or more images corresponding to light emitted from a cell, corresponding to light transmitted by the cell And one or more images corresponding to the light scattered by the cells. Such multimode imaging can include any of the following types of images or combinations: (1) one or more fluorescent images and at least one bright field image; (2) one or more fluorescent images and at least one (3) one or more fluorescent images, at least one bright field image and at least one dark field image; and (4) a bright field image. Simultaneous collection of multiple different fluorescent images (separated by spectrum) and simultaneous collection of multiple different bright field images (using transmitted light by two different spectral filters) may also be beneficial. Preferably, multi-mode images are collected simultaneously.

  [168] In some embodiments, multimodal imaging can compensate for very low signals by combining low signals with anatomical imaging data spatially matched to low signals from other detection modalities. In certain embodiments, different imaging methods (or modalities) can visualize different aspects of a eukaryotic cell and its environment, and by combining these multiple modalities, more about that eukaryotic cell and its environment. I can know.

  [169] In one embodiment, an image acquired by any one modality in any of the above methods can be processed in various ways, for example, the image data can be compared and reconstructed. In some embodiments, one image obtained using one modality can be compared to an image obtained using different modalities. In certain embodiments, the comparison and / or reconstruction can be performed by superimposing an image acquired from one modality on an image acquired from a different modality. The comparison and / or reconstruction can be performed on two or more images, three or more images, four or more images, five or more images, or six or more images, where each image is obtained from a different modality. In general, the image is in the form of a pixel image. For example, in a multimodal imaging method comprising (a) magnetic imaging of a magnetotactic bacterium expressing one or more heterologous reporters, and (b) imaging the reporter with a non-magnetic modality, the method comprises: Further, it may include superimposing the image acquired / captured from (a) and the image acquired / captured from (b). In some embodiments, the image in (a) and the image in (b) include a pixel image. Various methods for image processing, including comparing or reconstructing images obtained with different modalities, are described, for example, in U.S. patent publication No. 20110262016 and 20130177224.

  [170] In addition to the heritability of magnetic bacteria in eukaryotic cells to overcome the need to construct eukaryotic cells that express reporters while maintaining the level of reporter for detection, multimodal probes The use of multi-modal imaging based on magnetic bacteria can enhance in-situ imaging and eukaryotic cell tracking, such as imaging changes in cell dynamics within tissues containing eukaryotic cells. included. This added attribute enhances the ability to track cells and tissues containing such cells.

  [171] For example, in certain embodiments, a multimodal probe can be introduced into a cancer cell and the modified cancer cell can be introduced into an experimental animal, for example, to follow cancer cell metastasis and migration in that animal. Cancer cells can also be tracked and imaged after various therapeutic treatments to assess the effectiveness of the treatment and to examine the survival and migration kinetics of cancer cells before and after treatment.

  [172] In certain embodiments, multimodal probes can be introduced into stem cells to follow in vivo kinetics, including the location, proliferation rate, and viability of the stem cells. When the stem cell is a hematopoietic stem cell or progenitor cell, the stem cell containing a multimodal probe is introduced into a mammal and the magnetic hematopoietic stem cell or progenitor cell is located, proliferated, and into the bloodstream when subjected to different stimuli Their behavior, including movements, is monitored by multimodal imaging. This can be coupled with various types of treatment means to promote stem cell transplantation and homing. Other uses of the multimodal imaging method will be apparent from the guidance presented in this disclosure.

  [173] The invention disclosed herein will be better understood from the following experimental details. However, one of ordinary skill in the art will readily recognize that the specific methods and results discussed are merely illustrative of the invention which are more fully described in the following claims.

Example 1: Microinjection of gfp ⁺ AMB-1 into mouse cells Construction of gfp ⁺ AMB-1
[174] Expression vectors for eGFP sequences, ie those containing a Shine-Dalgarno sequence upstream of the gfp gene and those not containing a Shine-Dalgarno sequence, were cloned into the cryptic broad-range host vector pBBR1MCS-2 (Kovach, ME et al., “Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes,” Gene 166, 175-176, (1995), incorporated herein in its entirety for all purposes. ). AMB-1 (ATCC 7000026) was transformed with this construct (see, eg, Matsunaga, T. et al., “Complete genome sequence of the facultative anaerobic magnetotactic bacterium Magnetospirillum sp. Strain AMB-1,” DNA Res. 12, 157-166 (2005); Burgess JG, et al., “Evolutionary relationships among Magnetospirillum strains inferred from phylogenetic analysis of 16S rDNA sequences,” J Bacteriol. 175: 6689-6694 (1993); Matsunaga T, et al. , “Gene transfer in magnetic bacteria: transposon mutagenesis and cloning of genomic DNA fragments required for magnetosome synthesis,” J Bacteriol. 174: 2748-2753 (1992); Kawaguchi R, et al., “Phylogeny and 16s rRNA sequence of Magnetospirillum sp AMB-1, an aerobic magnetic bacteria, ”Nucleic Acids Res. 20: 1140 (1992); each publication is incorporated herein in its entirety for all purposes).

  [175] Transformation was achieved by conjugation with a donor E. coli strain. (Ref: Goulian, M. et al., “A simple system for converting lacZ to gfp reporter fusions in diverse bacteria,” Gene 372: 219-226 (2006); Scheffel, A. Schueler, D., “The Acidic Repetitive Domain of the Magnetospirillum gryphiswaldense MamJ Protein Displays Hypervariability but Is Not Required for Magnetosome Chain Assembly, ”J Bacteriol. 189 (17): 6437-6446 (2007); Each publication is incorporated herein in its entirety for all purposes. ). These mating reactions were cultured for 10 days in the absence of DAP under defined microaerobic conditions to select positive transformants.

[176] After conjugation, gfp ⁺ AMB-1 transformants containing and without the Shine-Dalgarno sequence succeeded in showing GFP fluorescence. Transformants that contained the Shine-Dalgarno sequence showed higher levels of GFP fluorescence than transformants that did not contain this sequence. The resulting fluorescence did not leave gfp ⁺ AMB-1 cells when excited at 488 nm and observed at 100 × magnification.

[177] The magnetic properties of gfp ⁺ AMB-1 were analyzed by MRI. gfp ⁺ AMB-1 was suspended in an agar plug and the imaging properties were optimized and elucidated using a 1.5T instrument. FIG. 1 shows the positive contrast generated with a T ₁ pulse sequence over a long scale concentration ranging about 10 ⁸ MTB / mL for gfp ⁺ AMB-1. Signal intensity was related to concentration.

B. Microinjection into mouse embryo cells
[178] gfp ⁺ AMB-1 was microinjected into one cell of each of 170 mouse embryos at the 2-cell stage. Six concentrations were injected over a long scale concentration ranging from about 10 ⁵ gfp ⁺ AMB-1 per cell as estimated by optical density at 565 nm. Cell mortality after microinjection was constant over the various concentrations injected. Images of the fluorescence image of the cells injected with the highest concentration (10 ⁵ MTB / cell) and the image overlaid with the differential interference contrast (DIC) image were compared. Controls that were not injected showed low levels of autofluorescence. Sections in different horizontal planes of 8-cell stage embryos at specific time points were compared. In each embryo, all four cells derived from the injected cells showed significant fluorescence, whereas none of the cells from the internal controls that were not injected showed significant fluorescence.

  [179] Embryos were developed for 3 days after injection. At each concentration level, embryos survived for a total of 3 days, developed to the blastocyst stage of 256 cells, and looked healthy enough to be transplanted. Numerous cells within each blastula show significant fluorescence, which is transmitted to daughter cells through multiple cell divisions by the artificial endosymbiosis as embryos containing eukaryotic host cells develop to the blastocyst stage. Prove that has been done. One such blastocyst is shown in FIG. 2; panel A shows the differential interference contrast (DIC) image of the blastocyst, and panel B) shows the same image of grayscale fluorescence capture, and fluoresces a large number of cells throughout the blastocyst. Show.

  [180] Using confocal microscopy, the total expression of GFP across four individual embryos was quantified by measuring the GFP fluorescence of the whole embryo at various time points starting at the 8-cell stage of the embryo. FIG. 3 shows the time course of embryo fluorescence. This is because the artificial endosymbiont copy number was maintained in daughter cells for at least 7 generations, thereby maintaining the host cell's fluorescent phenotype as the embryo passed from the 2-cell stage to the 256-cell blastocyst stage. It shows that.

[181] These results indicate that gfp ⁺ AMB-1 did not disappear or degrade immediately when delivered by microinjection, nor was it toxic to developing embryos over the course of the three day experiment. Prove that. Microinjected embryos divide normally, suggesting that gfp ⁺ AMB-1 does not present virulence markers or secrete toxic compounds. They are transmitted to daughter cells over many cell divisions, are encapsulated in the cytoplasm, are well distributed in punctiformity, and maintain copy number within the daughter host cell, thereby causing the fluorescence phenotype of the eukaryotic host cell to Maintained in cells for at least 7 generations. These results demonstrate that AMB-1 can be stably maintained in the cell and is transmitted to daughter cells over at least 7 cell divisions.

Example 2: AMB-1 phagocytic invasion
[182] Receptor-mediated: The inlAB gene is amplified from L. monocytogenes genomic DNA (ATCC 19114) and pBBR1MCS-5, the gentamicin homologue of pBBR1MCS-2 (see Kovach, ME, et al., “Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes,” Gene 166, 175-176, (1995)) and inserting gfp ⁺ inlAB ⁺ AMB-1 Make it. This gfp ⁺ inlAB ⁺ AMB-1 is a true epithelial tumor cell line Coco-2, MDA-MB-231 and MCF7, non-epithelial tumor cell lines such as HT-1080 and HL60, and mouse stem cells. Co-culture with nuclear host cells. Monitor and quantify internalization and intracellular location using fluorescence microscopy and FACS.

[183] Pore-forming haemolysin (hlyA) expression in AMB-1 is achieved by amplification of hlyA from Listeria monocytogenes genomic DNA (ATCC 19114). The amplified hlyA is inserted into pBBR1MCS-3 (a tetracycline analog of pBBR1MCS-2), which is then used to transform gfp ⁺ AMB-1. The resulting AMB-1 strain is exposed to a mouse macrophage cell line J774 capable of natural-phagocytosis. Gentamycin treatment is used to remove uninternalized bacteria and hlyA ^- AMB-1 is used as a negative control. Fluorescence microscopy is used to monitor the kinetics and location of AMB-1 in cells.

  [184] If the bacterium remains confined within the phagosome, it introduces two genes, plcA and plcB, which have been shown to be involved in the escape of Listeria monocytogenes into the cytosol (Smith, GA et al. al., Infection and immunity 63: 4231 (1995); Camilli, A. et al., J Exp Med. 173: 751 (1991); each publication is incorporated herein in its entirety for all purposes). If the bacteria are successfully escaped but are unable to grow, introduce hpt (see Goetz, M. et al., Proc Natl Acad Sci USA 98: 12221 (2001); Chico-Calero, I. et al., Proc Natl Acad Sci USA 99: 431 (2002); each publication is incorporated herein in its entirety for all purposes). In Listeria monocytogenes, hpt encodes a transporter involved in the uptake of glucose-6-phosphate from the cytosol. Other genes from Listeria monocytogenes (glnA and gltAB and argD) have been shown to be involved in sustaining growth in the host and are introduced systematically as needed (Joseph , B. et al., J Bacteriol. 188: 556 (2006), incorporated herein in its entirety for all purposes).

Example 3: Regulation of AMB-1 proliferation
[185] Regulation of AMB-1 proliferation in embryonic stem cells can be regulated as follows. Choleoptericin-A (ColA) is amplified from the total cDNA of Sitophilus oryzae. The expression of ColA in beetles of the genus Sitophilus is a gamma protobacteria class (γ-Protobacterium) that develops a close symbiotic relationship with these beetles in nature and stays in special cells called bacteriocytes. (Login, FH et al., “Antimicrobial peptides keep insect endosymbionts under control,” Science 334 (6054): 362-365 (2011), incorporated herein in its entirety for all purposes) .

[186] Mouse embryonic stem cells containing gfp ⁺ AMB-1 are processed by a neural differentiation protocol. MTB expression levels are quantified by qPCR and fluorescence microscopy. Amplified colA is then expressed in gfp ⁺ AMB-1 embryonic stem cells. The promoter is selected to obtain the optimal ColA expression level.

Example 4: Magnetic phenotype of mouse cells containing gfp ⁺ AMB-1
[187] Cells from a macrophage cell line J774.2 derived from a mouse ascites solid tumor into which gfp ⁺ AMB-1 had been introduced were applied to a magnetic column and retained on the column. Since J774.2 mouse cells were magnetically enriched and magnetically harvested after the introduction of gfp ⁺ AMB-1, these results demonstrate that they were detected magnetically and manipulated magnetically.

Example 5: Gfp ⁺ AMB-1 in human breast cancer cell line MDA-MB-231
[188] Gfp ⁺ AMB-1, Gfp ⁺ InlA / B ⁺ AMB-1, and Gfp ⁺ Pla1 ⁺ AMB-1 were each introduced into the human breast cancer cell line MDA-MB-231. In more than 90% of MDA-MB-231 cells, GFP fluorescence was detected for 48 hours after introduction of Gfp ⁺ AMB-1, Gfp ⁺ InlA / B ⁺ AMB-1, and Gfp ⁺ Pla1 ⁺ AMB-1 respectively. . GFP fluorescence in Gfp ⁺ AMB-1 was observed in these MDA-MB-231 cells at least 13 days after gfp ⁺ AMB-1 introduction, the fourth passage MDA-MB-231 cells after introduction, ie between each passage. Observed in a MDA-MB-231 cell population doubling 3-4 times. GFP fluorescence was observed in both daughter cells of MDA-MB-231 cells, including gfp ⁺ AMB-1 introduced during cell division.

[189] ^gfp + after the introduction of the AMB-1, stained with MDA-MB-231 cells with Lysotracker (TM) Red DND-99 dye (purchased from Life Technologies) and Hoechst 33342 nuclear staining (specific for lysosomes). Green GFP fluorescence is observed in individual MDA-MB-231 cells in a localized state apart from red lysosome staining and blue nuclear staining, which is due to the localization of AMB-1 in the cytoplasm and through the lysosomal pathway. Suggests not digested.

[190] Plated MDA-MB-231 cells and plated control MDA-MB-231 cells were fixed in formalin and glutaraldehyde after washing with PBS at 24 and 72 hours after introduction. The cells were then stained with Prussian blue and observed by microscopy at a magnification of 40x. gfp ⁺ AMB-1 iron staining in some of MDA-MB-231 cells were introduced was ^observed, in the control MDA-MB-231 cells not introduced the gfp + AMB-1 was observed. The proportion of cells that showed iron staining was between MDA-MB-231 cells 72 hours after introduction of gfp ⁺ AMB-1 cells and MDA-MB-231 cells 24 hours after introduction of gfp ⁺ AMB-1 cells. It was similar.

[191] 48 hours after introduction of gfp ⁺ AMB-1 cells, MDA-MB-231 cells were trypsinized and resuspended in PBS. One sample of these cells was placed in a slide glass chamber. A magnet was placed on the side of the chamber and cell movement was observed under a 20 × magnification microscope. MDA-MB-231 cells introduced with gfp ⁺ AMB-1 showed movement in the direction of the magnet, but no control MDA-MB-231 cells not introduced with gfp ⁺ AMB-1. Other samples of these cells were placed in small tubes and allowed to stick to the magnet for 1 hour. Control MDA-MB-231 cells not transfected with gfp ⁺ AMB-1 settled to the bottom of the tube. However, MDA-MB-231 cells introduced with gfp ⁺ AMB-1 were aligned on the magnet side of the tube.

[192] These results indicate that gfp ⁺ AMB-1 did not disappear immediately from human breast cancer MDA-MB-231 cells. They were transmitted to daughter cells over at least 12 cell divisions and stayed outside both the lysosome and nucleus in MDA-MB-231 cells. These results also demonstrate that MDA-MB-231 cells containing the presented gfp ⁺ AMB-1 were magnetically detected and manipulated for at least 48 hours after introduction of gfp ⁺ AMB-1 cells; Is moved by magnetism, targeted to a certain position by magnetism, concentrated by magnetism, and collected by magnetism. Furthermore, MDA-MB-231 cells contained an observable amount of iron at least 72 hours after introduction of gfp ⁺ AMB-1 cells.

Example 6: Gfp ⁺ AMB-1 in human induced pluripotent stem cells
[193] In human induced pluripotent stem ("IPS") cells, GFP fluorescence was observed at the second passage of IPS cells for at least 8 days after introduction of gfp ⁺ AMB-1 into IPS. These results indicate that gfp ⁺ AMB-1 was not immediately lost from human IPS cells but was transmitted to daughter cells.

Example 7: Cell imaging in mouse tumors
[194] Cell visualization was tested in mice bearing two subcutaneous tumors, one on its left flank and one on its right flank. 1.5 × 10 ⁶ MDA-MB-231 cells containing the introduced gfp ⁺ AMB-1 were directly injected into the tumor on the left flank of the mouse. The same number of control MDA-MB-231 cells without cells were injected into the right flank of the mice. Mice were imaged using a desktop 1T MRI with a T2w pulse sequence. The image obtained showed a dark area at the injection site of the right mouse tumor, ie, MDA-MB-231 cells containing the introduced gfp ⁺ AMB-1, but the MDA-MB-231 cells were There was no signal in the tumor on the right side of the mice injected into the tumor.

Example 8: Monitoring mouse tumors
[195] Gfp ⁺ AMB-1 cells are introduced into MDA-MB-231 human cancer cells. The resulting magnetic cells and their daughter cells are injected into the mammary fat pad of a group of immunocompromised mice. Tumor growth is monitored by MRI imaging at regular intervals. Mice with established tumors were assigned to either experimental or control groups. The experimental group of mice is treated with an effective anti-tumor therapeutic compound, whereas the control group of mice is treated with an inert vehicle. Following treatment, tumor size and growth are monitored non-invasively using MRI to assess the effectiveness of the test compound in combating the tumor.

Example 9: Enhancement of cell retention by magnetism
[196] Gfp ⁺ AMB-1 cells are introduced into rat heart-derived stem cells (CDC). The resulting magnetic CDC cells are used in an ischemia / reperfusion model. Rat Cheng K. et al., “Magnetic enhancement of cell retention, engraftment, and functional benefit after intracoronary delivery of cardiac-derived stem cells in a rat model of ischemia / reperfusion,” Cell Transplant. 21 (6): 1121- Treat as described in 35 (2012). The magnetic CDC cells are then introduced into the left ventricular cavity of the treated rat. A 1.3T magnet is placed above the heart during and after the injection. The animal's chest is closed and allowed to recover. The short and long term behavior of labeled CDC in rats is regularly monitored by MRI imaging.

Example 10: Treatment of AMF tumor
[197] Gfp ⁺ AMB-1 cells are introduced into MDA-MB-231 cells. The obtained cells are injected into subcutaneous tumors formed in nude mice by 4T1 cells. Untreated control MDA-MB-231 cells are injected into the contralateral flank tumor of each animal. After treatment, each animal is placed in an alternating magnetic field (30.6 kA / m, 118 kHz) for 30 minutes and allowed to recover after this operation. Animals are sacrificed at regular intervals and histological analysis is performed on tumors from both mice injected with magnetic MDA-MB-231 and mice injected with control MDA-MB-231. In experimental mice, the labeled cells and surrounding tumor are damaged, resulting in tumor damage over time.

Example 11: Bioluminescent AMB-1
[198] The gene sequence of the Photorhabdus luminescens luxCDABE operon was PCR amplified from the pXen-13 plasmid (Perkin Elmer Hopkinton, Mass.), And the In-Fusion® HD cloning kit (Clontech, California, USA) Was cloned into pBBR1-MCS2. The positive clone was confirmed by showing that it was an insertion sequence of the correct size by restriction digestion and detecting luminescence with a luminescence reader. The purified plasmid was introduced into Escherichia coli WM3064 which is a mating strain. AMB-1 and WM3064 strain were crossed for plasmid transfer, and kanamycin resistant AMB-1 strain was selected. Luminescence positive strains were cultured in liquid MG medium. Only lux ⁺ AMB-1 was checked for fluorescence using a desktop luminometer. Magnetism was confirmed using CMag measurement. Strains were introduced into eukaryotic cells by co-centrifugation or incubation with a strong magnet adjacent to the cell container. The magnetism was confirmed by MRI phantom, by observation of magnetic sensitivity with a microscope, or by trapping on a magnetic column.

Example 12: Preparation of fluorescent AMB-1
[199] In one embodiment, a genetically modified magnetotactic bacterium capable of detection by FLI is produced using enhanced green fluorescent protein (eGFP) inserted into a suicide plasmid pJQ200 and introduced into a diaminopimelate auxotrophic E. coli strain And used for mating with AMB-1. Gentamicin resistant strains were selected and fluorescence was confirmed by fluorescence microscopy. After 3 passages in gentamicin medium, cells were grown for 3 additional passages without antibiotic selection. They were then grown for 6 days in the presence of 1% sucrose and single colonies were inoculated on MGB agar plates. Single colonies were examined for GFP. Detection was performed using a Nikon Ti-S inverted epifluorescence microscope using a FITC filter and a 40 × objective lens. Fluorescent helical bacteria can be observed at a magnification of 40 ×.

[200] GFP positive AMB-1 was inserted into MDA-MB-231, 4T1, hAMSC, J774.2, MCF-7 (and others) cells. gfp ⁺ AMB-1 labeled and unlabeled cells were observed by fluorescence microscopy, and for gfp ⁺ AMB-1 labeled cells, a clear enhancement of cell fluorescence, often accompanied by a punctate pattern, was observed, which was localized in intracellular vesicles Suggests

[201] FIGS. 6A-L show the following confocal microscopy and fluorescence images: iPS cells containing gfp ⁺ AMB-1 (pluripotent stem cells), MDA-MB containing gfp ⁺ AMB-1 -231 cells (human breast ^carcinoma), J774.2 cells containing the gfp + AMB-1 (murine ^macrophage), BJ cells (human ^fibroblasts) which accommodates the gfp + ^AMB-1, containing the gfp + AMB-1 HEP1 cells (human liver adenocarcinoma) and MCF-7 cells (human epithelial breast adenocarcinoma) containing gfp ⁺ AMB-1. gfp ⁺ AMB-1 was inserted into these cell lines and microscopy images were acquired using a Nikon Ti-S epifluorescence microscope equipped with a phase contrast optical element or FITC fluorescent filter and equipped with a 40 × magnification lens. . 6A-B show phase contrast images and fluorescence images of iPS cells (pluripotent stem cells) containing gfp ⁺ AMB-1. 6C-D show phase contrast and fluorescence images of MDA-MB-231 cells (human breast cancer) containing gfp ⁺ AMB-1. FIGS. 6E-F show phase contrast and fluorescence images of J774.2 cells (mouse macrophages) containing gfp ⁺ AMB-1. FIGS. 6G-H show phase contrast and fluorescence images of BJ cells (human fibroblasts) containing gfp ⁺ AMB-1. FIGS. 6I-J show phase contrast and fluorescence images of HEP1 cells (human liver adenocarcinoma) containing gfp ⁺ AMB-1. FIG. 6K-L shows phase contrast and fluorescence images of MCF-7 cells (human epithelial breast adenocarcinoma) containing gfp ⁺ AMB-1. All these cell types showed enhanced fluorescence when cells contained gfp ⁺ AMB-1.

Example 13: Production of PET-detectable AMB-1
[202] In order to produce PET-modified genetically modified magnetotactic bacteria, the human herpes simplex virus-thymidine kinase (HSV1-Tk) gene sequence is selected and a selected gene (eg, for kanamycin or gentamicin antibiotic resistance). It can be introduced into WM3064 via an appropriate plasmid, which can then be crossed with AMB-1 for plasmid delivery. Antibiotic resistant AMB-1 strains can then be selected. Positive expression of the reporter can be assessed by adding ³ H-PCV (penciclovir), a radiolabeled HSV1-Tk substrate, and quantifying AMB-1 uptake with a scintillation counter.

[203] PET reporter positive AMB-1 can be inserted into MDA-MB-231, 4T1, hAMSC, J774.2, MCF-7 (and other) cells. AMB-1 labeled and unlabeled cells can be injected into a living subject, and then the subject can be injected with a radioactive substrate for the HSV1-TK reporter gene (eg, ¹⁸ F-FHBG). After 1 hour, the subject's PET scan shows significant uptake in the area of AMB-1 labeled cells.

Example 14: Detection of eukaryotic cells containing AMB-1 by fluorescence imaging and MRI
[204] MDA-MB-231 cells containing gfp ⁺ AMB-1 were grown to confluence. gfp ⁺ and imaged with AMB-1 accommodated MDA-MB-231 cells and ^gfp + AMB-1 to MDA-MB-231 cells confocal microscopy and fluorescence imaging without the. Phase contrast and fluorescence images were acquired using a Nikon Ti-S epifluorescence microscope equipped with a phase contrast optical element or a FITC fluorescence filter and equipped with a 40 × magnification lens. The fluorescent image was exposed for 0.5 seconds. 4A and 4D show the phase contrast images of MDA-MB-231 with and without gfp ⁺ AMB-1. 4B and 4E show fluorescence imaging of MDA-MB-231 with and without gfp ⁺ AMB-1. FIG. 4B demonstrates the fluorescence signal from gfp ⁺ AMB-1 in MDA-MB-231 cells.

[205] MDA-MB-231 cells with and without gfp ⁺ AMB-1 were prepared for imaging by MRI according to the following. MDA-MB-231 cells with and without gfp ⁺ AMB-1 were trypsinized and 2 million cells were resuspended in 100 μl PBS. The cell suspension was pipetted into a PCR tube containing 100 μl of 1% agarose previously coagulated. The PCR tube was sealed and left for 10 minutes to allow the cells to settle at the agarose / PBS boundary. A Varian 7T 300 MHz Horizontal Bore preclinical MR system was used. The scanner was in the form of a 40 G / cm 120 mm high duty cycle gradient coil. Images were acquired with vnmrj 4.0 image acquisition software. A 2D T2w MEMS sequence (TR 3000 ms, TE Min 7 ms, NE 48, NEX 1,128 × 128, 40 × 40 mm FOV, 1 mm cross section thickness, coronal plane image was used to image a phantom tube (Rapid F19). figure .MRI images imaged the tube using a volume coil ^{(volume coil) 4C (MDA-} MB-231 containing gfp + AMB-1) and 4F ^(MDA-MB-231 without the gfp + AMB-1) A black band confirming the position of MDA-MB-231 containing gfp ⁺ AMB-1 is visible in Figure 4C, and no such band is visible in Figure 4F.

[206] FIGS. 5A-C show examples of low magnification MRI, fluorescence and visual inspection imaging of MDA-MB-231 cells containing gfp ⁺ AMB-1. MDA-MB-231 cells were labeled with gfp ⁺ AMB-1, washed 24 hours later, treated with trypsin, collected at 48 hours, centrifuged in a centrifuge, and the resulting pellet was placed in a cylindrical 100 cc tube. Of 1% agarose gel. Fluorescence images were obtained with an IVIS 49 fluorescence imager using a Cy5.5 filter and a 1 second acquisition time. MRI image is T2w RARE pulse sequence (TR 1500ms, TE 96ms, FA 180 degrees, acquisition time 5 minutes, FOV 3.5cm, 128x128, section thickness 1.25mm, gap between sections 1.00mm), diameter Obtained with a Bruker ICON 1T preclinical MRI scanner equipped with a 2.5 cm rat coil. FIGS. 5A-C show the detection of different modalities of the same MDA-MB-231 cells containing gfp ⁺ AMB-1 suspended in agarose. FIG. 5A shows MDA-MB-231 cells viewed by visual inspection of plastic tubes. FIG. 5B shows an MRI image of MDA-MB-231 cells containing gfp ⁺ AMB-1 in the same tube. FIG. 5C shows a fluorescence image of MDA-MB-231 cells in the same tube. MDA-MB-231 cells containing gfp ⁺ AMB-1 can be detected using MRI and fluorescence imaging.

Example 15: Detection of AMB-1 by fluorescence and MRI
[207] AMB-1 was transformed with the pBBR1-MCS2 lux plasmid to produce luminescent AMB-1. These luminescent AMB-1s were introduced into MDA-MB-231 cells. MDA-MB-231 cells containing lux ⁺ AMB-1 were grown to confluence and 1 million cells were photometrically measured with a TD-20 / 20 luminometer using the manufacturer's protocol. Measurements were taken for 1 minute and the results were normalized to OD400 = 1. FIG. 7A shows a chart comparing the luminescence signal from MDA-MB-231 cells containing AMB-1 with MDA-MB-231 cells not containing AMB-1. MDA-MB-231 cells containing AMB-1 showed a significant luminescence signal compared to MDA-MB-231 cells not containing AMB-1.

[208] MRI images of MDA-MB-231 cells containing luminescent AMB-1 were obtained as follows. MDA-MB-231 cells containing luminescent AMB-1 were trypsinized and 2 million cells were resuspended in 100 μl PBS. The cell suspension was pipetted into a PCR tube containing 100 μl of 1% agarose previously coagulated. The PCR tube was sealed and left for 10 minutes to allow the cells to settle at the agarose / PBS boundary. Images were acquired with an actively shielded Discovery MR901 preclinical 7T MRI; bore diameter -310 mm, gradient: (120 mm ID, 600 mT / m, 6000 T / m / s), 8 receive channels; 16 bits per channel 1MHz. A T2 ^* w GRE sequence was used as an imaging sample (TR 300ms, TE 30ms, NEX 2,256x256, 30mm FOV). FIG. 7B shows an MRI image of MDA-MB-231 cells containing luminescent AMB-1. MDA-MB-231 cells containing luminescent AMB-1 appear as a black band in the middle of the tube.

  [209] All publications, patents and patent applications discussed and cited herein are hereby incorporated by reference in their entirety. It should be understood that the disclosed invention is not limited to the particular methods, protocols and materials described, as these may vary. Since the scope of the invention is limited only by the claims, the names used herein are for the purpose of describing particular embodiments only and are intended to limit the scope of the invention. It should also be understood that it was not done.

  [210] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents of the specific embodiments described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (16)

The following steps:
Preparing a eukaryotic cell comprising a magnetotactic bacterium, wherein the magnetotactic bacterium comprises a heterologous reporter;
Magnetically detect magnetotactic bacteria in eukaryotic cells; and detect heterologous reporters;
Including a multimodal detection method. The following steps:
Preparing a eukaryotic cell comprising a magnetotactic bacterium, wherein the magnetotactic bacterium comprises a heterologous reporter;
Magnetic imaging of magnetotactic bacteria in eukaryotic cells; and imaging heterologous reporters;
A multimodal imaging method.   The method according to claim 1 or 2, wherein the eukaryotic cell is a mammalian cell.   4. The method according to claim 3, wherein the mammalian cell is a stem cell, cancer cell or circulating cell.   The method according to claim 4, wherein the mammalian cell is a human cell.   The method according to claim 4, wherein the stem cell is a neural stem cell.   The method according to claim 4, wherein the mammalian cell is a mouse cell.   The method according to claim 4, wherein the circulating cells are T cells or B cells.   4. The method of claim 3, wherein the reporter is an optical reporter.   10. The method of claim 9, wherein the optical reporter is a fluorescent reporter or a bioluminescent reporter.   Eukaryotic cells including unicellular organisms, ultrasound imaging, computed tomography imaging, bioluminescence imaging, magnetic resonance imaging, optical coherence tomography imaging, radiography imaging, nuclear medicine imaging, positron emission tomography imaging, tomography A eukaryotic cell that can be detected by at least two modalities selected from the group consisting of imaging, photoacoustic tomography imaging, X-ray imaging, thermal imaging, and magnetic particle imaging.   The eukaryotic cell according to claim 11, wherein the eukaryotic cell is a mammalian cell.   The eukaryotic cell according to claim 12, wherein the mammalian cell is a stem cell, cancer cell or circulating cell.   The eukaryotic cell according to claim 13, wherein the stem cell is a neural stem cell.   The eukaryotic cell according to claim 13, wherein the circulating cell is a T cell or a B cell.   A multimodal probe comprising a magnetotactic bacterium, wherein the magnetotactic bacterium is selected from the group consisting of a positron emission tomography reporter, a single photon emission computed tomography reporter, an X-ray reporter, a photoacoustic reporter, and an ultrasound reporter The multimodal probe expressing one or more heterologous reporters.