Methods for controlling cell survival

Abstract

To provide a method for controlling cell survival.SOLUTION: A method for controlling cell survival includes the step of maintaining cells that have lost the ability to produce and transport compounds essential for survival, in the presence of derivatives of the compounds. The compounds are compounds that cannot be metabolized by cells when supplied from outside the cells. The derivatives are derivatives of the compounds that can be metabolized by cells even when supplied from outside the cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for controlling cell viability, and more particularly to a method for controlling cell viability that allows for biological containment, for example, by maintaining viability under specific culture conditions but suppressing viability outside those conditions (such as in the natural environment). [Background technology]

[0002] Since the dawn of history, various microorganisms have been used in a variety of fields, including medicine, food, environmental purification, and biomass utilization, enriching human life. However, there are many microorganisms that are both useful and dangerous. For example, there are many microorganisms that are both useful and dangerous, such as genetically modified organisms whose safety has not been confirmed, pathogens used in research on treatments and live vaccine development, and highly adaptable microorganisms that are invasive to the environment. Such "useful but dangerous" microorganisms must be contained and used within facilities such as laboratories and factories managed by the user.

[0003] Currently, physical containment, which primarily uses physical barriers to prevent microorganisms from escaping into the outside world, is used, such as safety cabinets, air filtration with HEPA filters, and autoclave sterilization. Effective physical containment requires the absence of equipment failures and human error, so the possibility of target microorganisms leaking due to accidents or disasters cannot be eliminated. However, unlike other environmental pollutants such as hazardous chemicals (e.g., petroleum) and radioactive materials, once microorganisms are leaked, they pose the risk of autonomously multiplying and spreading widely throughout the environment. Therefore, physical containment is not an ideal method for containing microorganisms.

[0004] Another principle for containing microorganisms is biological containment. Biological containment is "a technology that genetically programs microorganisms so that they can survive under human control but die in the natural environment." Biological containment is an autonomous system that is genetically built into the target microorganisms, and there is no possibility of it failing due to human error, as there is in physical containment. Therefore, biological containment is a more suitable principle for achieving perfect containment of microorganisms. For this reason, there is a desire to develop superior biological containment methods.

[0005] In biological containment, target microorganisms are permitted to survive only if a survival tolerance factor is provided. Many of the biological containment methods proposed to date have selected the supply of specific chemicals as the survival tolerance factor (Non-Patent Documents 1-3). The most important issue in this regard is what the survival tolerance factor should be. In early biological containment methods, natural chemicals utilized by living organisms, such as essential nutrients or metabolites, were selected as the survival tolerance factor. A biological containment system can be easily constructed by disrupting genes in the pathways that produce the substance or by constructing a synthetic genetic circuit that controls life and death using genetic elements controlled by the substance (Non-Patent Document 4). Natural chemicals that are rarely present freely in the natural environment are typically selected as survival tolerance factors. Despite this, target organisms often have the ability to ingest the natural chemicals. In fact, most such biological containment methods have failed due to the presence of unexpected environmental niches or supply sources (Non-Patent Document 4). While the incidence of such failures can be reduced by using multiple biological containment methods, it cannot be eliminated (Non-Patent Document 2).

[0006] In recent years, several reports have been published on biological containment that relies on artificial chemicals, such as genome-encoded essential proteins or the production and functioning of antitoxins in artificially introduced toxin-antitoxin systems that rely on unnatural amino acids (Non-Patent Documents 5-9). Such biological containment that relies on artificial chemicals is theoretically infallible because unexpected supply sources are unlikely to occur. However, unlike biological containment that relies on natural chemicals, artificially modified genes are required because gene products that recognize artificial chemicals do not typically exist in nature. Therefore, when synthetic chemicals are used as survival tolerance factors in biological containment, the target microorganisms inevitably become genetically modified organisms (GMs) that contain exogenous genes that are significantly different from natural genes. When using such GM organisms, the containment of exogenous genes poses a particularly serious problem, as there is a risk that artificial genetic elements will be introduced into the natural gene pool, causing unpredictable effects on the ecosystem (Non-Patent Document 1). For example, even if biological containment functions ideally and completely kills the target organisms, it cannot prevent the exogenous genes from being released into the environment from dead cells. Methods using non-specific nucleases or CRISPR nucleases to degrade introduced genes have also been proposed, but these methods also rely on the expression of foreign genes (Non-Patent Documents 4, 10). Furthermore, the use of genetically modified organisms is subject to strict legal restrictions (Non-Patent Document 11). Therefore, it is desirable for biological containment to eliminate genetic elements that differ significantly from those in nature, and ultimately to be a method that does not use foreign genes.

[0007] Therefore, a method that satisfies the above conditions, i.e., a strategy of biological containment that relies on artificial chemicals while eliminating genetic elements that differ greatly from those in nature, is considered an ideal principle, but has not been proposed so far. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Stirling,F. & Silver,PAControlling the implementation of transgenic microbes:Are we ready for what synthetic biology has to offer? Cell Mol.78,614–623(2020).

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[0009] The present invention has been made in consideration of the problems associated with the prior art, and aims to provide a method for controlling cell survival that enables biological containment that relies on artificial chemicals. [Means for solving the problem]

[0010] As a result of extensive research to achieve the above object, the present inventors have conceived a system that satisfies the following three conditions in order to perform biological containment that relies on artificial chemicals. (1) Nutrients and metabolites essential for survival are selected as survival factors. However, survival factors must be inaccessible to the target cells even when administered extracellularly because they are membrane-impermeable, lack transporters, or are easily degraded. (2) The target cells must have lost all pathways for synthesizing and transporting factors that allow survival, such as by gene disruption. Gene disruption can be achieved by spontaneous mutation, chemically or physically induced mutation, or by genome editing, which leaves no trace without the use of transgenes. In other words, it is possible to eliminate genetic elements that are significantly different from those in nature. Furthermore, target cells generated in this way are not considered genetically modified organisms, which are subject to legal restrictions in many countries. (3) The survival factor must be artificially modified so that it can be utilized even when supplied from outside the cell. The modified survival factor must not exist in nature.

[0011] We hypothesized that in a system that satisfies the above conditions, target cells would be able to survive only if they were supplied with artificially modified survival factors. We therefore designed a biological containment system that reflects this theoretically ideal principle, embodied it in bacteria, and demonstrated its concept.

[0012] Specifically, we selected deoxythymidylate (dTMP) as a viability factor. dTMP is the only precursor for the production of dTTP, which is essential for DNA synthesis, and is therefore an essential metabolite for survival in most organisms. Nucleoside phosphates, including dTMP, are large and electrically charged, and therefore cannot cross biological membranes without a carrier protein. Therefore, bacteria such as Bacillus subtilis cannot utilize (metabolize) dTMP supplied from outside the cell. Furthermore, while other nutrient starvation simply stops proliferation, dTMP starvation is known to cause cell death (thymine starvation death).

[0013] Therefore, we eliminated the dTMP synthesis system in Bacillus subtilis by genome editing, etc. Furthermore, to enable the non-dTMP-producing Bacillus subtilis to metabolize dTMP even when provided extracellularly, we chemically modified dTMP to confer membrane permeability and prepared two derivatives: bis(pivaloyloxymethyl)deoxythymidylate (POM2-dTMP) and bis(isopropyloxymethylcarbonyl)deoxythymidylate (POC2-dTMP).

[0014] Furthermore, by testing non-dTMP-producing Bacillus subtilis with dTMP or its derivatives, it was found that thymine-starvation death was induced in the Bacillus subtilis when dTMP was provided extracellularly, whereas cell death was suppressed when the derivative was provided extracellularly. Thus, the present inventors found that a system that satisfies the above three conditions is extremely effective in biological containment, leading to the completion of the present invention.

[0015] That is, the present invention provides the following aspects.

[0016] [1] A method for controlling cell viability, comprising the step of maintaining cells that have lost the ability to produce and transport a compound essential for survival in the presence of a derivative of the compound, wherein the compound is a compound that the cell cannot metabolize when provided extracellularly, and the derivative is a derivative of the compound that the cell can metabolize even when provided extracellularly.

[0017] [2] The method according to [1], wherein the compound is a nucleotide.

[0018] [3] The method according to [1], wherein the compound is deoxythymidylic acid (dTMP).

[0019] [4] The method according to [3], wherein the derivative is at least one deoxythymidylic acid derivative selected from bis(pivaloyloxymethyl)deoxythymidylic acid (POM2-dTMP) and bis(isopropyloxymethylcarbonyl)deoxythymidylic acid (POC2-dTMP).

[0020] [5] A system for controlling cell survival, comprising a cell that has lost the ability to produce and transport a compound essential for survival, and a derivative of the compound, wherein the compound is a compound that the cell cannot metabolize when provided extracellularly, and the derivative is a derivative of the compound that the cell can metabolize even when provided extracellularly.

[0021] [6] A drug for controlling cell viability, comprising as an active ingredient a derivative of a compound essential for cell viability, wherein the cell has lost the ability to produce and transport the compound, the compound is a compound that the cell cannot metabolize when provided extracellularly, and the derivative is a derivative of the compound that the cell can metabolize even when provided extracellularly. [Effects of the Invention]

[0022] According to the present invention, it is possible to control cell viability. Consequently, viability is maintained in the presence of the derivative, but is suppressed in the absence of the derivative (such as in a natural environment), enabling biological containment. Furthermore, by losing the ability of the cells to produce and transport compounds essential for survival through spontaneous mutation, chemically or physically induced mutation, or traceless genome editing without the use of transgenes, it is possible to eliminate genetic elements that differ significantly from those found in nature using the methods of the present invention. In other words, it is possible to contain non-recombinant organisms without converting them to recombinant forms. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 is a diagram showing an overview of one embodiment of the present invention. [Figure 2A] 1 is a diagram showing an outline of a specific embodiment of the present invention, illustrating the use of a membrane-permeable dTMP derivative (POM2-dTMP) as an artificial survival-permitting factor. [Figure 2B] This figure shows an outline of the scheme for constructing a temperature-sensitive dTMP-non-producing Bacillus subtilis strain (a B. subtilis tdk-thyA-thyB triple mutant). In the figure, "EmR" and "KmR" represent an erythromycin resistance gene cassette and a kanamycin resistance gene cassette, respectively. tdk and thyA were inactivated by replacing the ORFs of the target genes (tdk and thyA) with these antibiotic resistance gene cassettes. ThyB is a temperature-sensitive enzyme and can be inactivated at 46°C. [Figure 2C] This graph shows the growth rates of the tdk-thyA double-gene disruptant, the tdk-gene disruptant, and the wild-type strain. In the graph, "wt," "tdk," and "tdkthyA" represent the growth rates of the wild-type strain, the tdk-gene disruptant, and the tdk-thyA double-gene disruptant, respectively (number of tests: 3, growth rates are shown as mean ± standard deviation (sd)). For each item, the left side shows the growth rate at 37°C, and the right side shows the growth rate at 46°C. [Figure 2D]This graph shows the results of analyzing nucleotide metabolism in the tdk-thyA double gene disruptant. In the graph, "37," "46," and "46P" represent the quantification results (relative area) of each nucleotide after incubation of the tdk-thyA double gene disruptant for 30 minutes at 37°C in the absence of POM2-dTMP, 46°C in the absence of 3 mM POM2-dTMP, and 46°C in the presence of 3 mM POM2-dTMP. Quantitative analysis was performed on three deoxynucleotides: dATP, dUMP, and dTMP, which have been reported to change significantly during thymine-starvation death due to loss of Thy function. ADP was also analyzed as a control. [Figure 3A] This is a graph showing the time course of survival rates of the tdk-thyA double gene disruptant at 46°C. In the figure, under color display, the red line indicates the results of culturing in the presence of 8 mM POC2-dTMP (solvent: 2% ethanol), and the black line indicates the negative control (culturing in the presence of 2% ethanol (solvent only)). [Figure 3B] This graph shows the results of an analysis of the dose effect of POM2-dTMP or dTMP. The tdk-thyA double gene disruptant was incubated at 46°C for 4 hours in the presence of POM2-dTMP or dTMP, and then the survival rate was measured (number of experiments = 3, each growth rate is shown as the mean ± standard deviation (sd)). 2% ethanol was used as the solvent for POM2-dTMP and dTMP. [Figure 3C] This is a graph showing the time course of the survival rate of the tdk-thyA double gene disruptant at 46°C. In the figure, the red line under the color representation indicates the results of culturing in the presence of 4 mM POM2-dTMP (solvent: 2% ethanol). [Figure 3D]This graph shows the results of an analysis of the dose effect of POM2-dTMP on a tdk-thyA double gene disruptant or a tdk-gene disruptant. The tdk-thyA double gene disruptant was incubated in the presence of POM2-dTMP at 46°C for 45 minutes, and then the survival rate was measured (three experiments per group, and each growth rate is expressed as the mean ± standard deviation (sd)). Note that <1% DMSO was used as the solvent for POM2-dTMP. [Figure 3E] This graph shows the time course of the survival rate of the tdk-thyA double gene disruptant at 46°C. In the figure, the red line under the color display indicates the results of culturing in the presence of 4 mM POM2-dTMP (solvent: 2% ethanol). In the figure, "POC2-dTMP addition" indicates the results of culturing without further supplementation after adding 3 mM POC2-dTMP (solvent: <1% DMSO) at T=0. "POC2-dTMP continuous supply" indicates the results of culturing with fresh 3 mM POM2-dTMP (solvent: <1% DMSO) supplemented every 30 minutes. "Negative control" indicates the results of culturing in the presence of 0.375% DMSO (solvent only). DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention relates to a method for controlling cell survival, more particularly maintaining cells that have lost the ability to produce and transport a compound essential for survival in the presence of a derivative of said compound; The present invention provides a method for controlling cell viability, wherein the compound is a compound that the cell cannot metabolize when provided from outside the cell, and the derivative is a derivative of the compound that the cell can metabolize even when provided from outside the cell.

[0025] The "cells" that are the subject of the present invention may be prokaryotic or eukaryotic cells, and examples thereof include microorganisms, plant cells, insect cells, and animal cells. The "microorganisms" that are the subject of the present invention may be prokaryotes (eubacteria, archaea) or eukaryotes (algae, protists, fungi, slime molds). More specifically, examples of prokaryotes include bacteria belonging to the genus Bacillus, such as Bacillus subtilis, the genus Escherichia, such as Escherichia coli, the genus Pseudomonas, such as Pseudomonas putida, and the genus Rhizobium, such as Rhizobium meliloti. Furthermore, examples of eukaryotes include yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe.

[0026] In the present invention, "control of cell viability" means controlling the maintenance or suppression of cell viability depending on the presence or absence of a derivative of a compound essential for survival, as described below. "Maintaining viability" means, for example, that the cell growth rate or survival rate is 0.2 times or more, preferably 0.5 times or more, and more preferably 0.8 times or more, compared to normal rates.

[0027] "Inhibition of viability" means killing cells and / or inhibiting cell proliferation. For example, the cell proliferation rate can be reduced to at least one-tenth, preferably at least one-hundredth, and more preferably at least one-thousandth of that of normal cells (e.g., cells (parent strain, etc.) before they lose the ability to produce and transport compounds essential for survival). Also, the cell death rate can be reduced to at least 10 times, preferably at least 100 times, and more preferably at least 1000 times that of normal cells.

[0028] In the present invention, a "compound essential for cell viability" refers to a compound that cannot be metabolized by the cell when provided from outside the cell, and more specifically, refers to a compound whose cell viability is suppressed in its absence or under conditions in which it cannot be produced and transported. Examples of such compounds include metabolites (substances before being metabolized, intermediate products of metabolic processes, or end products of metabolism) that are essential for cell viability, and nutrients that are essential for cell viability. More specifically, examples include nucleosides, proteins (amino acids, peptides, etc.), carbohydrates, lipids, organic acids, vitamins, minerals, and coenzymes that are essential for cell viability. "Metabolism" refers to obtaining energy by breaking down substances (catabolism) or synthesizing substances using energy (anabolism).

[0029] In the present invention, "nucleoside" includes not only nucleosides themselves but also their phosphate ester compounds (phosphorylated nucleosides, nucleotides) and polynucleotides, such as adenosine, guanosine, cytidine, thymidine, uridine, inosine, xanthosine, orotidine, deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxycytidine monophosphate (dCMP), deoxythymidylate (dTMP), deoxyuridine monophosphate (dUMP), deoxyinosinate (dIMP), DNA, and RNA. Among these, compounds essential for cell viability according to the present invention are preferably nucleotides, more preferably dAMP, dGMP, dCMP, dTMP, dUMP, and even more preferably dTMP.

[0030] In the present invention, the "ability to produce a compound essential for survival" means, as the term is used literally, the ability to produce the compound by a synthesis reaction and / or a degradation reaction. Furthermore, the "ability to transport a compound essential for survival" means the ability to be involved in the delivery of the compound to an intracellular organelle that can metabolize the compound, and includes, for example, the ability to take up the compound essential for survival into the cell and the ability to translocate the compound essential for survival to the intracellular organelle.

[0031] In the present invention, the "loss of the ability to produce and transport compounds essential for survival" is not particularly limited as long as the ability can be lost, and examples include inhibition of synthesis, promotion of degradation, and inactivation of proteins involved in the ability (for example, enzymes such as synthetases and degradative enzymes, and transport proteins such as membrane transporters). Furthermore, "inhibition of synthesis" includes not only inhibition of protein synthesis (translation) based on mRNA, but also inhibition of mRNA synthesis (transcription) based on genomic DNA (gene), and mutations in the genes (deletion of all or part of the genes).

[0032] Those skilled in the art can introduce mutations into the gene by known mutagenesis methods, including, but not limited to, genome editing, physical mutagenesis, chemical mutagens, transposon introduction into genomic DNA, and transcription product targeting using sRNA, siRNA, shRNA, antisense RNA, and RNA with ribozyme activity.

[0033] Genome editing is a method for modifying target genes using site-specific nucleases (e.g., zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), DNA double-strand break enzymes such as CRISPR-Cas9). For example, fusion proteins such as ZFNs (U.S. Patent Nos. 6,265,196, 8,524,500, 7,888,121, European Patent No. 1,720,995), TALENs (U.S. Patent Nos. 8,470,973, 8,586,363), and nuclease domain-fused PPR (pentatricopeptide repeat) (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (U.S. Patent No. 8,697,359, International Publication No. 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)), and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and Examples include methods that use guide RNA and protein complexes such as those described in "Vertebrate Adaptive Immune Systems, Science, DOI:10.1126 / science.aaf8729,(2016)."

[0034] Physical mutagenesis methods include, for example, heavy ion beam (HIB) irradiation, fast neutron irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0035] Examples of methods using chemical mutagens include treatment with chemical mutagens (see, for example, Zwar and Chandler, Planta, 1995, vol. 197, pp. 39-48). Chemical mutagens are not particularly limited, but include ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), N-methyl-N-nitrosourea (MNU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, formic acid, and nucleotide analogs.

[0036] Methods for introducing transposons or the like into genomic DNA include, for example, the method described in H. Mori et al., Research in Microbiology, 2000, Vol. 151, No. 2, pp. 121-128, and the method described in S.Y. Gerdes et al., J. Bacteriol., 2003, Vol. 185, No. 19, pp. 5673-5684.

[0037] For cells into which a mutation has been introduced by the above-described method, the introduction of a mutation into the gene can be confirmed by known methods. Examples of such known methods include DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, Southern blotting, and Northern blotting. Using these methods, whether or not a mutation has been introduced into the gene can be determined by comparing the sequence or length of the gene before and after the introduction of the mutation. Furthermore, by using Northern blotting, RT-PCR, Western blotting, ELISA, microarray analysis, etc., if a decrease in the expression level of the transcription or translation product of the gene (preferably, the expression level is substantially absent) is observed in a cell into which a mutation has been introduced, the cell can be confirmed as a cell into which a mutation has been introduced into the gene.

[0038] Another method for confirming that a mutation has been introduced into the gene is TILLING (Targeting Induced Local Lesions IN Genomes) (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when a non-selective mutation is introduced into the genome using the aforementioned heavy ion beam irradiation or chemical mutagen, the gene or a portion thereof can be amplified by PCR, and then cells having a mutation in the amplification product can be selected by the TILLING method.

[0039] Furthermore, the gene can also be deleted by homologous recombination, which can be performed using, for example, the lambda red recombination system (T. Baba et al., Mol. Syst. Biol., 2006, 2, 20060008), Cre / Lox, attB / attP, or other integrase systems.

[0040] In the present invention, the loss of the ability to produce and transport compounds essential for survival may be due to not only the introduction of artificial mutations as described above, but also natural mutations. In addition, from the viewpoint that the loss in the present invention can eliminate genetic elements that are significantly different from those in nature, genome editing methods, physical mutagenesis methods, methods using chemical mutagens, and natural mutations are preferred, genome editing methods are more preferred, and genome editing methods that do not use transgenes and leave no trace are even more preferred.

[0041] In the present invention, it is necessary for cells to lose all of the abilities to produce and transport compounds essential for survival, but some types of cells inherently lack some of these abilities. In such cells, it is not necessary to lose both the ability to produce and the ability to transport compounds essential for survival. For example, in the Examples described below, in Bacillus subtilis, which does not inherently have the ability to transport dTMP, it is sufficient to lose only the inherent ability.

[0042] In the present invention, the term "derivative of a compound essential for survival" refers to a derivative of the compound that can be metabolized by cells even when provided from outside the cell. Note that the term "derivative" in the present invention refers to a compound that has undergone the introduction of a functional group, modification of a functional group, oxidation, reduction, atom substitution, etc., but in which the structure and properties of the compound (parent compound) have not been substantially changed, and includes modified, altered, derived, and analogous compounds.

[0043] Such derivatives are not particularly limited, but examples include compounds essential for survival that have been introduced with a protecting group that enables transport into cells but is removed inside the cells, as shown in the Examples below.

[0044] Examples of such protecting groups include hydrophobic protecting groups that are removed by intracellular enzymes (such as esterases), and more specifically, pivaloyloxymethyl (POM) groups, isopropyloxymethylcarbonyl (POC) groups, S-acyl-2-thioethyl (SATE) groups, phosphoramidite groups, cyclosaligenyl groups, and alkyloxyalkyl groups. Protecting groups that are removed intracellularly in response to environmental signals such as light, temperature, or chemical cofactors may also be used. More specifically, such protecting groups include photolabile protecting groups such as coumarin-type caging groups, BODIPY-type caging groups, cyanine-type caging groups, and ortho-nitrobenzyl-type caging groups (see, e.g., Bardhan, A. & Deiters, A. Development of photolabile protecting groups and their application to the optochemical control of cell signaling Curr. Opin. Struct. Biol. 57, 164-175 (2019)), and pH-labile protecting groups such as 2-nitroarylamide groups (see, e.g., Larroque-Lombard, A.-L. et al. Design and mechanism of action of a new prototype of combi-molecule “programmed” to release bioactive species at a pH range akin to that of the tumor microenvironment. Pharmaceuticals 14, 160 (2021)).

[0045] The derivatives of the present invention are as described above, but are preferably nucleotides having a POM group or a POC group introduced therein, more preferably dTMP having a POM group or a POC group introduced therein, even more preferably bis(pivaloyloxymethyl)deoxythymidylic acid (POM2-dTMP) and bis(isopropyloxymethylcarbonyl)deoxythymidylic acid (POC2-dTMP), and more preferably POM2-dTMP.

[0046] Furthermore, as shown in the Examples below, a person skilled in the art can synthesize the derivatives of the present invention using known techniques while appropriately selecting reaction raw materials, reaction reagents, reaction conditions (e.g., solvent, reaction temperature, catalyst, reaction time), etc., based on the structure of a compound essential for survival. Furthermore, the derivatives synthesized in this manner can be separated and purified using methods commonly used for isolating and purifying general compounds (e.g., reverse phase chromatography, ion exchange chromatography, adsorption chromatography, recrystallization, etc.), either alone or in combination.

[0047] In the present invention, the cells may be maintained in the presence of the derivative in any state so long as they are in contact with the derivative so that the cells can take up the derivative. For example, the derivative may be included (dissolved, etc.) in a culture system for the cells (such as a culture medium containing the cells). The amount (concentration) of the derivative at this time is not particularly limited, and for example, the amount (concentration) of the derivative may be a concentration that can maintain the viability of the cells while suppressing the toxicity of the derivative, as shown in the examples below.

[0048] Although the preferred embodiments of the method for controlling cell survival of the present invention have been described above, the present invention is not limited to the above embodiments.

[0049] For example, the present invention can provide a drug for controlling cell viability, which comprises as an active ingredient a derivative of a compound essential for cell viability, wherein the cell has lost the ability to produce the compound, the compound is a compound that the cell cannot metabolize when provided extracellularly, and the derivative is a derivative of the compound that the cell can metabolize even when provided extracellularly.

[0050] The agent of the present invention, also referred to as a reagent, may contain, in addition to the derivative, a pharmacologically acceptable carrier or vehicle, such as a solvent (e.g., ethanol, DMSO), culture medium, sterile water, physiological saline, buffer solution, vegetable oil, suspending agent, surfactant, stabilizer, emulsifier, diluent, isotonic agent, bulking agent, or other additive.

[0051] The present invention can also provide a system for controlling cell survival, comprising a cell that has lost the ability to produce a compound essential for survival, and a derivative of the compound, wherein the compound is a compound that the cell cannot metabolize when provided extracellularly, and the derivative is a derivative of the compound that the cell can metabolize even when provided extracellularly.

[0052] The system of the present invention may be in the form of a kit, and may include, in addition to the cells and derivatives, compounds essential for the survival of the cells, the pharmacologically acceptable carrier or medium (solvent, culture medium, etc.), instructions for use, etc. as its constituent items. [Example]

[0053] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The examples were carried out using the following methods.

[0054] (Fungal culture) All Bacillus subtilis strains were cultured in LB medium (1% BactoTriptone, 0.5% Bacto Yeast Extract, 1% NaCl). Antibiotics (erythromycin 1 mg / L or kanamycin 25 mg / L) were added as necessary. Liquid cultures were performed in an air incubator with shaking (200 rpm). Solid medium was prepared by adding 2% agar to the LB medium. Bacteria were cultured at 37°C unless otherwise noted.

[0055] (Construction of a temperature-sensitive tdk-thyA-thyB triple mutant) The tdk gene disruptant (BKE37060) in B. subtilis 168 strain, constructed by ORF replacement with an erythromycin resistance cassette, was distributed by the National BioResource Project Bacillus subtilis (NIG) (https: / / shigen.nig.ac.jp / bsub / ).

[0056] The thyA gene was deleted by replacing it with a kanamycin resistance cassette [Koo, BM et al. Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Syst. 4, 291-305, e7 (2017)]. Specifically, a kanamycin resistance cassette containing the 5' and 3' flanking regions of the hyA gene (approximately 1 kb each) was synthesized by gene synthesis and amplified by PCR. The resulting PCR product was used directly for transformation. Colonies exhibiting resistance to both erythromycin and kanamycin were then selected on solid medium. Furthermore, strains that were confirmed to be lethal at 46°C were selected from the selected antibiotic-resistant colonies. Genomic DNA was extracted from the resulting strains and used as a template for PCR to confirm the replacement of the thyA gene with the kanamycin resistance cassette. Genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's protocol. PCR amplification was performed by nested PCR using GoTaq green master mix (Promega). The PCR products were analyzed by agarose gel (1%) electrophoresis.

[0057] Genome sequencing using Bacillus subtilis strain 168 as a reference confirmed that the tdk and thyA genes had been replaced with antibiotic resistance cassettes, as well as other off-target mutations.

[0058] The integrity of the extracted DNA was confirmed using Genomic DNA Screen Tapes on a TapeStation 2200 (Agilent), and gDNA was quantified using Quant-IT PicoGreen (Invitrogen, Grand Island, NY) and a VICTOR Nivo (Perkin Elmer, Waltham, MA, USA) multimode plate reader.

[0059] Sequencing libraries were prepared using the TruSeq DNA PCR-free Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA) according to the manufacturer's instructions. Briefly, 1 μg of genomic DNA was fragmented using adaptive focused ultrasound (AFA; Covaris), and the fragmented DNA was end-repaired to generate 5'-phosphorylated blunt-end dsDNA molecules. After end-repair, DNA was size-selected using a bead-based method. These DNA fragments were then ligated with a single "A" base and TruSeq indexing adapters to obtain purified libraries. The purified libraries were quantified by qPCR according to the qPCR Quantification Protocol Guide (KAPA Library Quantification kits for Illumina Sequencing platforms) and qualified on a high-sensitivity DNA chip (Agilent Technologies, Waldbronn, Germany). Paired-end (2 × 150 bp) sequencing was performed by Macrogen using the NovaSeq6000 platform. Adapter sequences and low-quality reads were removed using Trimmomatic v0.38 (http: / / www.usadellab.org / cms / ?page=trimmomatic), and read quality control was performed using FastQC v0.11.6 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ). Trimmed reads were mapped to the B. subtilis 168 reference genome (GenBank accession number AL009126.3) using BWA-MEM v0.7.17. Samtools (http: / / samtools.sourceforge.net / ) was used to convert SAM to BAM format and sort the mapped sequences. Picard v2.17.2 (http: / / broadinstitute.github.io / picard / ) was used to check the quality of the BAM files and flag duplicate reads.Variants (SNPs and small INDELs) were called using Samtools, and functional annotation of variants was performed using SnpEff v5.0e (https: / / pcingola.github.io / SnpEff / ).

[0060] (Synthesis of membrane-permeable dTMP derivative (POC2-dTMP)) Bis(isopropyloxymethylcarbonyl)deoxythymidylic acid (POC2-dTMP) was synthesized according to the reaction scheme shown below.

[0061] [ka]

[0062] Specifically, thymidine (10.0 g, 41.3 mmol) was added to trimethyl phosphate (100 mL) under a nitrogen atmosphere. The resulting white suspension was cooled to 3 °C, and phosphorus oxychloride (5.75 mL, 61.9 mmol) was carefully added dropwise over 1.5 h, maintaining the internal temperature below 5 °C. The reaction mixture was stirred at 3 °C for 6.5 h and then at room temperature for 16 h. The resulting pale yellow solution was poured into vigorously stirred ice-water (1 L), and 1 M KOHA (250 mL) was added. The pH of this solution was >10 as determined by pH paper. The solvent was removed under reduced pressure, and the residue was purified by flash column chromatography (ARG silica gel 200 g, acetonitrile / water = 90 / 10 to 70 / 30) to obtain potassium salt 1 as a pale yellow oil (35.6 g). The potassium salt of compound 1 (approximately 13.4 g) was passed through a cation exchange resin (Dowex 50WX8-200, 100 mL) to obtain crude compound 1 (10.6 g, ca. 15.5 mmol) as a purple oil after lyophilization. 1 H NMR (270MHz, D2O): δ7.71(1H),6.32(dd,J=7.0Hz,1H),4.60-4.49(m,1H),4.20-4.00(m,3H),2.34(dd,J=7.0,4.6Hz,1H),1.88(s,3H).

[0063] The crude product of 1 (10.6 g, approximately 15 mmol) was dissolved in 1-methyl-2-pyrrolidone (200 mL) under a nitrogen atmosphere. Next, triethylamine (21.1 mL, 152 mmol) was added and the mixture was stirred at 60 °C for 30 min. After cooling to room temperature, chloromethyl isopropyl carbonate (20.0 mL, 150 mmol) was added to the resulting pale black solution and the mixture was stirred at 60 °C for 7 h. Brine (200 mL) was added to the resulting black solution, which was then extracted with ethyl acetate (50 mL x 9). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the sodium sulfate, and evaporated at 65 °C to remove the 1-methyl-2-pyrrolidone, yielding the crude product (8.88 g). This was purified by flash column chromatography (100 g silica gel, dichloromethane / methanol = 99 / 1 to 97 / 3) to obtain POC2-dTMP (2.08 g, UPLC purity 79.5%) as a brown oil. This was further purified by flash column chromatography (120 g DIOL silica gel, dichloromethane / methanol = 100 / 0 to 98 / 2) to obtain POC2-dTMP (1.56 g, UPLC purity ca. 85%). Furthermore, this POC2-dTMP (1.56 g) was combined with separately synthesized low-purity POC2-dTMP (557 mg), and purified by flash column chromatography (30 g silica gel, dichloromethane / methanol = 99 / 1 to 97 / 3) and flash column chromatography (30 g silica gel, toluene / ethyl acetate = 80 / 20 to 20 / 80) to obtain POC2-dTMP (1.11 g, UPLC purity ca. 96%). was obtained as a white amorphous substance. 1H NMR (270MHz, CDCl3): δ8.65(brs,1H),7.37(s,1H),6.33(dd,J=6.8Hz,1H),5.75-5.60(m,4H),5.00-4.83(m,2H),4.61-4.61(m,1H),4.51(m,1H) ),5.51(m,1H),4.40-4.30(m,2H),4.11-4.02(m,1H),3.03(brs,1H),2 .43-2.31(m,1H),2.26-2.12(m,1H),1.93(s,3H),1.35-1.25(m,12H). LCMS(ESI+):m / z calcd.for[M]+=555.4.

[0064] (Synthesis of membrane-permeable dTMP derivative (POM2-dTMP)) Bis(pivaloyloxymethyl)deoxythymidylic acid (POM2-dTMP) was synthesized according to the reaction scheme shown below.

[0065] [ka]

[0066] Specifically, Compound 1 (8.0 g, 22 mmol) was dissolved in ultrapure water and passed through a cation exchange resin (Dowex 50WX8-200, 130 mL). After lyophilization, Compound 2 (6.6 g, 20 mmol) was obtained as a white solid. 1 H NMR (270MHz, D2O): δ7.71(1H),6.32(dd,J=7.0Hz,1H),4.60-4.49(m,1H),4.20-4.00(m,3H),2.34(dd,J=7.0,4.6Hz,1H),1.88(s,3H).

[0067] Compound 2 (6.6 g, 20 mmol) was dissolved in 1-methyl-2-pyrrolidone (136 mL) under a nitrogen atmosphere. Next, triethylamine (14 mL, 100 mmol) was added and the mixture was stirred at 60 °C for 30 min. After cooling to room temperature, chloromethyl pivalate (14 mL, 98 mmol) was added and the mixture was stirred at 60 °C for 18 h. After cooling to room temperature, the reaction mixture was poured into water (300 mL) and extracted with ethyl acetate (200 mL x 3). The aqueous phase was concentrated to approximately 1 / 4 volume under reduced pressure. The resulting brown slurry was filtered and concentrated under reduced pressure to give a solution containing compound 4. Meanwhile, the organic phase was evaporated, dissolved in ethyl acetate / hexane = 80 / 20 (100 mL), and washed with water (100 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and evaporated to give crude POM2-dTMP (crude A).

[0068] A solution containing compound 4 and triethylamine (14 mL, 100 mmol) was stirred at 60 °C for 30 min under a nitrogen atmosphere. After cooling to room temperature, chloromethyl pivalate (14 mL, 98 mmol) was added to the reaction mixture, and the mixture was stirred at 60 °C for 2.5 h and at room temperature for 18 h. The dark brown reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic phases were evaporated, and the residue was redissolved in ethyl acetate / hexane = 80 / 20 (100 mL), washed with water (100 mL x 2), and washed with water / brine = 100 / 10 (110 mL). The organic phase was dried over anhydrous sodium sulfate, filtered to remove sodium sulfate, and evaporated to give crude POM2-dTMP (crude B). Crude A and crude B were purified by flash column chromatography (dichloromethane / methanol = 99 / 1 to 95 / 5) to give POM2-dTMP (1.7 g, 15%, UPLC purity 100%) as a white solid. 1H NMR (270MHz, CDCl3): δ9.10(brs,1H),7.37(s,1H),6.32(dd,J=6.8Hz,1H)5.75-5.58 (m,4H),4.58-4.48(m,1H),4.40-4.25(m,2H),4.11-4.50(m,2H),5.50-4.50(m,1H)00(m,1H),3.53(dJ =2.7Hz,1H),2.49-2.30(m,1H),2.24-2.09(m,1H),1.92(s,3H),1.22(s,9Hx2).LCMS(ESI+):m / z calcd.for [M]+=551.7.

[0069] (Live cell count) Bacteria were grown overnight at 33°C in erythromycin-containing medium. To obtain bacteria in the logarithmic growth phase, the overnight culture was diluted 1 / 100 and grown at 37°C for 2-3 hours (OD ). 590 = 0.1-0.3). 1 mL of the 50-fold diluted re-culture was used for the experiment. After the experiment, 20 μL of the culture was taken and adjusted to 1 mL with antibiotic-free medium, and then diluted 50-fold. 250 μL of the diluted culture was inoculated onto solid medium containing erythromycin in a 9 cm diameter dish. After overnight incubation at 37°C, the resulting colonies were counted.

[0070] (Determination of dose-effect curve) For POC2-dTMP, a 400 mM solution was prepared in 50% ethanol. For POM2-dTMP, a 400 mM solution was prepared in 50% ethanol, and an 800 mM solution was prepared in 100% DMSO. A 2- or 3-fold dilution series was also prepared in the same solvent. This dilution series was then diluted again with medium to prepare a medium in which the final concentration of each dTMP derivative was further halved. This medium was shaken at 46°C for approximately 2 hours to completely dissolve the dTMP derivative.

[0071] The medium containing the dTMP derivative was mixed with an equal volume of diluted logarithmic-phase bacterial suspension. To allow the bacteria to incorporate the dTMP derivative, the bacterial culture was incubated at 37°C for 10 minutes. 20 μL was removed and plated for bacterial count at time 0. The bacterial culture was then incubated at 46°C and plated using the same procedure. The plates were incubated overnight at 37°C, and surviving bacteria were detected as colonies. The effect of the dTMP derivative was evaluated by comparing the number of viable bacteria before and after incubation at 46°C.

[0072] (Penetrance of temperature-sensitive phenotypes) The tdk-thyA double gene disruptant strain in the logarithmic growth phase was diluted in antibiotic-free medium and analyzed at OD 590 =0.1. Furthermore, 10 -1 Double to 10 -5 Ten-fold serial dilutions were prepared. The diluted bacterial suspension (250 μL) was inoculated onto solid medium containing erythromycin. After overnight incubation at 46°C, surviving bacteria were detected as colonies. The total number of inoculated bacteria was calculated from the number of colonies formed on the plate incubated at 37°C. The surviving colonies were diluted again and inoculated onto two plates. The plates were incubated separately at 37°C and 46°C to test the temperature sensitivity of the surviving bacteria.

[0073] (Growth inhibition by solvent) tdk in logarithmic growth phase - The OD was measured using liquid media containing erythromycin and various concentrations of ethanol or DMSO. 590 The bacterial cell suspension was cultured at 46°C for 3 hours, then cooled to 0°C to stop growth. After diluting 3-fold with fresh medium, the OD 590 The growth inhibition rate was evaluated as a relative value to growth in a medium containing no ethanol or DMSO.

[0074] (Quantification of nucleotides) Microbial cells were harvested by centrifugation at 5,800 × g and 4°C for 5 minutes and then washed twice with 10 mL of Milli-Q water. They were then immersed in 1,600 μL of methanol and sonicated. The cell extract was then treated with 1,100 μL of Milli-Q water containing an internal standard (H3304-1002, Human Metabolome Technologies, Inc. (HMT), Tsuruoka, Yamagata Prefecture) for 30 seconds at room temperature, cooled on ice, and centrifuged at 2,300 × g and 4°C for 5 minutes. Seven hundred μL of the supernatant was then centrifuged at 9,100 × g and 4°C for 120 minutes using a Millipore 5 kDa cutoff filter (UltrafreeMC-PLHCC, HMT) to remove macromolecules. The filtrate was evaporated to dryness under vacuum and reconstituted with 50 μL of Milli-Q water for metabolomic analysis using HMT.

[0075] Nucleotide analysis was performed using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS) according to previously published methods [Soga, T. et al., "Simultaneous determination of anionic intermediates for Bacillus subtilis metabolic pathways by capillary electrophoresis electrospray ionization mass spectrometry," Anal. Chem. 74, 2233-2239 (2002); Ohashi, Y. et al., "Depiction of metabolome changes in histidine-starved Escherichia coli by CE-TOFMS," Mol. Biosyst. 4, 135-147 (2008)]. Briefly, CE-TOFMS analysis was performed using an Agilent CE capillary electrophoresis system equipped with an Agilent 6210 time-of-flight mass spectrometer (Agilent Technologies, Inc., Santa Clara, CA, USA). The system was controlled by Agilent G2201AA ChemStation software version B.03.01 (Agilent Technologies) and connected to a fused silica capillary (50 μm id × 80 cm total length) using commercially available electrophoresis buffers (H3301-1001 and I3302-1023, HMT, for cation and anion analysis, respectively) as electrolytes. The areas of the target nucleotide peaks were then normalized to the internal standard and sample volume.

[0076] (Morphological analysis) Morphological analysis of live bacteria was performed using a Nomarski differential interference microscope Axioskop2 (Zeiss, Jena, Germany) equipped with a CoolSnap ver. 1.1 imaging device (Roper Industries, Sarasota, Florida, USA).

[0077] (Sustained proliferation with POM2-dTMP supplementation) The tdk-thyA double gene disruptant strain in logarithmic growth phase was inoculated into 1 ml of medium containing 3 mM POM2-dTMP and erythromycin. To count the viable cell count at T = 0, 20 μL of the bacterial suspension was removed and diluted with 1 ml of antibiotic-free medium, and 250 μL was inoculated onto solid medium containing erythromycin. The bacteria were incubated at 46°C for 30 minutes, after which the same procedure was repeated. The mixture was then mixed with an equal volume of fresh medium containing POM2-dTMP preheated to 46°C. 1 ml of the mixture was dispensed and re-incubated. The viable cell count was measured before and after incubation using the same procedure. The re-incubation was repeated once more. The total viable cell count was calculated from the dilution rate.

[0078] Below we present the results obtained using the above method.

[0079] <Realization of the theoretically ideal containment> As a result of extensive research to achieve the above-mentioned objective, the inventors have conceived a system that satisfies the following three conditions in order to perform biological containment that relies on artificial chemicals while eliminating genetic elements that differ greatly from those found in nature: (1) Nutrients and metabolites essential for survival are selected as survival factors. However, survival factors must be inaccessible to the target cells even when administered extracellularly because they are membrane-impermeable, lack transporters, or are easily degraded. (2) The target cells must have lost all pathways for synthesizing and transporting survival factors through gene disruption or other means. (3) The survival factor must be artificially modified so that it can be utilized even when supplied from outside the cell. The modified survival factor must not exist in nature.

[0080] We hypothesized that in a system that satisfies the above conditions, such as that shown in Figure 1, target cells would be able to survive only if they were supplied with artificially modified survival factors. We therefore attempted to realize this theoretically ideal biological containment system.

[0081] We first investigated chemicals suitable for the survival factor and selected 2'-deoxythymidine 5'-monophosphate (dTMP). 2'-deoxythymidine 5'-triphosphate (dTTP), essential for DNA synthesis, is produced only through a pathway that uses dTMP as a precursor. dTMP is an essential metabolite for most organisms [Ahmad, SI, Kirk, SH & Eisenstark, A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 52, 591-625 (1998)]. On the other hand, nucleoside phosphates, including dTMP, cannot cross biological membranes without a carrier protein due to their large size and charge [Winkler, HH & Neuhaus, E. Non-mitochondrial ATP transport. Trends Biochem. Sci. 24, 64-68 (1999). Di Noia, MA et al. The human SLC25A33 and SLC25A36 genes of solute carrier family 25 encode two mitochondrial pyrimidine nucleotide transporters. J. Biol. Chem. 289, 33137-33148 (2014). Alonzo, JR, Venkataraman, C., Field, MS & Stover, PJ. The mitochondrial inner membrane protein MPV17 prevents uracil accumulation in mitochondrial DNA. J. Biol. Chem. 293, 20285-20294 (2018)]. Therefore, bacteria such as Bacillus subtilis cannot utilize dTMP supplied from outside the cell.Furthermore, whereas other nutrient starvation simply stops growth, dTMP starvation induces cell death (thymine-deficient death), making it advantageous for biological containment [Ahmad, SI, Kirk, SH & Eisenstark, A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 52, 591-625 (1998)., Steidler, L. et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 21, 785-789 (2003).]

[0082] Next, we investigated how to enable target bacteria that do not produce dTMP to acquire dTMP. Chemical modification is required for cellular uptake of dTMP. Chemical modifications that confer membrane permeability to nucleoside phosphates have been widely reported for the intracellular delivery of prodrugs used as antiviral and anticancer drugs [Pradere, U., Garnier-Amblard, EC, Coats, SJ, Amblard, F. & Schinazi, R.F. Synthesis of nucleoside phosphate and phosphonate prodrugs. Chem. Rev. 114, 9154-9218 (2014)]. A common strategy is to neutralize the negative charge of the phosphate, thereby increasing cell permeability. Once inside the cell, the mask is enzymatically removed to form the free nucleoside phosphate.

[0083] Finally, we selected Bacillus subtilis, a well-known Gram-positive bacterium, as the microorganism to test this biological containment strategy, because information on thymine-starved death has been accumulated, the details of physiological responses and the genes involved have been elucidated, and gene disruption methods have been established [Ahmad, SI, Kirk, SH & Eisenstark, A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 52, 591-625 (1998). Farmer, JL & Rotham, F. Transformable thymine-requiring mutant of Bacillus subtilis. J. Bacteriol. 89, 262-263 (1965). Koo, BM et al. Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Syst. 4, 291-305. e7 (2017). Nikaido, H. Molecular Basis of Bacterial Outer Membrane Permeability Revisited.Microbiol.Mol.Biol.Rev.67,593-656(2003)., Winkler,HH & Neuhaus,E.Non-mitochondrial ATP transport.Trends Biochem.Sci.24,64-68(1999).,Morono,Y.et al.Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate.Biotechnol.Lett.26,379-383(2004)., Antonczak,AK,Simova,Z. & Tippmann,EMA critical examination of Escherichia coli esterase activity.J.Biol.Chem.284,28795 - 28800(2009).].

[0084] <Construction of a dTMP-non-producing Bacillus subtilis strain> We constructed a Bacillus subtilis strain in which all pathways for dTMP synthesis were blocked. B. subtilis possesses two dTMP synthesis pathways: a de novo synthesis pathway, in which 2'-deoxyuridine 5'-monophosphate (dUMP) is converted to dTMP catalyzed by thymidylate synthases ThyA and ThyB, and a salvage pathway, in which thymidine is converted to dTMP catalyzed by thymidine kinase Tdk (Figure 2A) [Ahmad, SI, Kirk, SH & Eisenstark, A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 52, 591-625 (1998)]. Because B. subtilis can survive and grow in the presence of any of the enzymes ThyA, ThyB, and Tdk, we determined that the function of all three genes must be lost to inhibit dTMP production in B. subtilis. It is noteworthy that ThyA is thermostable, whereas ThyB is not [Morono, Y. et al. Application of glutaraldehyde for the staining of esterase-active cells with carboxyfluorescein diacetate. Biotechnol. Lett. 26, 379-383 (2004)]. Therefore, for experimental convenience, we generated a thermosensitive strain lacking dTMP synthesis by disrupting only the tdk and thyA genes and leaving the thyB gene intact, as shown in Figure 2B. This strain grows at the permissive temperature of 37°C, but is expected to die aseptically at the high temperature of 46°C due to the inactivation of ThyB. Although it is possible to disrupt the target gene without transgenicity, we used an antibiotic resistance marker gene here for ease of screening and maintenance.The tdk gene was disrupted by replacing it with an erythromycin resistance gene cassette from the National BioResource Project (NIG) [Yamazaki, Y. et al. NBRP databases: databases of biological resources in Japan. Nucleic Acids Res. 38, D26-D32 (2010)]. The thyA gene was then disrupted by replacing it with a kanamycin resistance gene in the Tdk gene disruptant [Koo, B. M. et al. Construction and analysis of two genome-scale deletion libraries for Bacillus subtilis. Cell Syst. 4, 291-305. e7 (2017)].

[0085] As a result, a strain was isolated that was resistant to both erythromycin and kanamycin and died at 46°C. Furthermore, PCR confirmed that the thyA gene had been replaced with a kanamycin resistance gene cassette. Finally, to confirm the tdk-thyA double gene disruption, the genome was sequenced. The results confirmed that the tdk gene and thyA gene had been replaced with erythromycin resistance and kanamycin resistance gene cassettes, respectively. Although several small mutations (off-target mutations) were detected, no clear loss-of-function mutations were predicted.

[0086] Furthermore, as shown in Figure 2C, the tdk-thyA double gene disruptant, unlike its wild-type and parent strain, showed a decrease in viable cell count at 46°C. More specifically, the tdk-thyA double gene disruptant grew at 37°C, albeit at a reduced growth rate due to de novo synthesis of dTMP by the intact thyB gene. On the other hand, the tdk-thyA double gene disruptant rapidly died within 30–45 min at 46°C, where ThyB is inactivated, whereas the wild-type and parent tdk gene disruptant grew normally.

[0087] Although not shown in the figure, the cellular morphology of the tdk-thyA double gene disruption strain was indistinguishable from that of wild-type and tdk gene disruption strains at 37°C, whereas at 46°C, elongated morphology and filamentation, which are seen in sterile cells, were clearly observed.

[0088] Furthermore, as shown in Figure 2D, a decrease in dTMP levels and accumulation of dATP and dUMP, which are observed upon inactivation of thymidylate synthase, were detected, suggesting that the tdk-thyA double gene disruptant died due to thymine starvation at 46°C.

[0089] Therefore, we concluded that the tdk-thyA double gene disruptant we constructed was a temperature-sensitive tdk-thyA-thyB triple mutant.

[0090] <Rescue by synthetic dTMP derivatives> By esterifying the phosphate group with a highly hydrophobic protecting group, two highly membrane-permeable dTMP derivatives were synthesized as shown in the formula below.

[0091] [ka]

[0092] We selected the isopropyloxymethyl carbonate (POC) and pivaloyloxymethyl (POM) protecting groups, which have been used in the prodrug development of several anticancer and antiviral drugs [Pradere, U., Garnier-Amblard, EC, Coats, SJ, Amblard, F. & Schinazi, R.F. Synthesis of nucleoside phosphate and phosphonate prodrugs. Chem. Rev. 114, 9154-9218 (2014)]. These dTMP derivatives, POC2-dTMP and POM2-dTMP, are expected to cross the cell membrane without transporters and be degraded by nonspecific esterases abundant in B. subtilis, resulting in their conversion to free dTMP within the cell (Figure 2A). Both POC and POM produce degradation products during excretion from dTMP, but POC is less toxic [Wiemer, AJ & Wiemer, D.F. Prodrugs of phosphonates and phosphates: crossing the membrane barrier. Top Curr Chem. 360, 115-160 (2015)]. On the other hand, POM is more stable and less susceptible to pH changes than POC [Wiemer, AJ & Wiemer, D.F. Prodrugs of phosphonates and phosphates: crossing the membrane barrier. Top Curr Chem. 360, 115-160 (2015)]. Yuan L.-C., Dahl, T.C. & Oliyai, R. Degradation kinetics of oxycarbonyloxymethyl prodrugs of phosphonates in solution. Pharm. Res. 18, 234-237 (2001)].

[0093] These dTMP derivatives were soluble in 50% ethanol and 100% dimethyl sulfoxide (DMSO) at concentrations exceeding 100 mM. Initially, ethanol was selected as the solvent [Freed, J.J., Farquhar, D. & Hampton, A., Evidence for acyloxymethyl esters of pyrimidine 5'-deoxyribonucleotides as extracellular sources of active 5'-deoxyribonucleotides in cultured cells. Biochem. Pharm. 38, 3193-3198 (1989)]. In our experimental conditions, 2% ethanol was ultimately included in the bacterial culture, but it was later found to inhibit Bacillus subtilis growth. This growth inhibition is due to the entry into stationary phase induced by SigB, a sigma factor that regulates the transcription of many genes [Boylan, SA, Redfield, AR, Brody, MS & Price, CW Stress-induced activation of the σB transcription factor of Bacillus subtilis. J. Bacteriol. 175, 7931-7937 (1993)]. Stationary-phase bacteria are expected to survive longer because they are more resistant to thymine starvation death [Ahmad, SI, Kirk, SH & Eisenstark, A. Thymine metabolism and thymineless death in prokaryotes and eukaryotes. Annu. Rev. Microbiol. 52, 591-625 (1998)].

[0094] We first investigated whether the less toxic POC2-dTMP could delay thymine starvation death in the presence of ethanol. Preliminary studies showed a delaying effect at concentrations above 2.7 mM POC2-dTMP. Time course experiments, as shown in Figure 3A, clearly demonstrated that 8 mM POC2-dTMP delayed thymine starvation death after 2–4 h of incubation at 46°C.

[0095] Next, we tested the more chemically stable POM2-dTMP. As observed with POC2-dTMP, thymine starvation death was inhibited at concentrations of several mM or higher, but the bacteria died at 8 mM (Fig. 3B). On the other hand, free dTMP supplied in the medium did not exhibit such an inhibitory effect (Fig. 3B).

[0096] Because the effect of POM2-dTMP was clearly greater than that of POC2-dTMP, subsequent experiments were performed using only POM2-dTMP (Fig. 3C). Furthermore, because <4% DMSO does not inhibit B. subtilis growth, in contrast to ethanol, we attempted to restore growth using POM2-dTMP in DMSO. As shown in Fig. 3D, we observed a more than two-fold increase in growth for the first time in experiments using POM2-dTMP dissolved in DMSO.

[0097] As with the ethanol solution, 8 mM POM2-dTMP kills the bacteria, so it is effective only within a narrow concentration range (2-4 mM). Furthermore, as shown in Figure 3D, growth inhibition at 4 mM and death at 8 mM were observed in both the parent and tdk gene disruptants, suggesting that POM2-dTMP is toxic at high concentrations.

[0098] On the other hand, when 3 mM POM2-dTMP was used, the growth recovery at that concentration was lost within 30-60 minutes, as shown in Figure 3E, and the viable cell count then rapidly decreased. This is presumably because POM2-dTMP was decomposed and consumed, dropping below the effective concentration. Therefore, fresh medium containing POM2-dTMP was supplied every 30 minutes to maintain the required concentration, ultimately resulting in sustained growth (Figure 3E).

[0099] Although not shown in the figure, B. subtilis showed the same elongated morphology as observed in the absence of POM2-dTMP, but filamentous formation was hardly observed. Furthermore, as shown in Figure 2D, the abnormal accumulation of dATP and dUMP was resolved, and the amount of dTMP was restored.

[0100] These results demonstrate that the survival and growth of the B. subtilis tdk-thyA-thyB triple mutant, which is unable to produce dTMP, can be restored by providing POM2-dTMP. [Industrial Applicability]

[0101] As described above, the present invention makes it possible to control cell viability. Consequently, viability is maintained in the presence of the derivative, but is suppressed in the absence of the derivative (such as in a natural environment), enabling biological containment. Furthermore, by losing the ability of the cells to produce and transport compounds essential for survival through spontaneous mutation, chemically or physically induced mutation, or traceless genome editing without the use of transgenes, it is possible to eliminate genetic elements that differ significantly from those found in nature using the methods of the present invention. In other words, it is possible to contain non-recombinant organisms without converting them to recombinant forms.

[0102] In this way, the present invention is useful in the production of biopharmaceuticals, etc., because it is possible to biologically contain non-genetically modified microorganisms, including research pathogens and live vaccines.

Claims

1. A method for controlling the maintenance and suppression of cell viability, comprising: maintaining viability of cells that have completely lost the ability to produce a compound essential for survival in the presence of a derivative of said compound, while inhibiting viability in the absence of said derivative; the compound is deoxythymidylic acid (dTMP), In the presence of the derivative, 2-4 mM bis(pivaloyloxymethyl)deoxythymidylic acid (POM ２ -dTMP) or 2.7-8 mM bis(isopropyloxymethylcarbonyl)deoxythymidylic acid (POC ２ -dTMP), and The cell is a Bacillus subtilis cell in which tdk, thyA, and thyB have been inactivated. method.

2. A system for performing the method according to claim 1, comprising: the cell and the derivative, The derivative is POM ２ -dTMP or POC 2 -dTMP, and The cell is a Bacillus subtilis cell in which tdk, thyA, and thyB have been inactivated. system.

3. A drug for carrying out the method according to claim 1, comprising: containing the derivative as an active ingredient, and The derivative is POM ２ -dTMP or POC 2 -dTMP.

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