Bacterial effectors as antimicrobial proteins

Abstract

The present disclosure discloses a method for treating or preventing intracellular microbial infections using Legionella pneumophilia effector protein, LegC4, produced intracellularly or delivered to cells from outside the cell, as an antimicrobial agent.

Description

【Technical Field】 【0001】 Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 351,078, filed on June 10, 2022, the disclosure of which is hereby expressly incorporated by reference in its entirety. 【0002】 Incorporation by Reference of Electronically Submitted Materials Incorporated by reference in its entirety is the computer - readable nucleotide / amino acid sequence listing, submitted simultaneously with this specification and identified as follows: a 6 - kilobyte xml file named "390720.xml" created on May 25, 2023. 【Background Art】 【0003】 Legionella pneumophila is widely distributed in freshwater environments and replicates within single - celled protists. However, L. pneumophila is also an opportunistic human pathogen and can cause Legionnaires' disease in immunocompromised individuals due to uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes the Dot / Icm type IV secretion system to directly translocate a large group of over 300 effector proteins into host cells. In mammals, the translocated effectors contribute to the restriction of L. pneumophila by innate immunity. 【0004】 Natural immunity in healthy individuals effectively controls Legionella infection, in part due to the rapid and robust production of inflammatory cytokines resulting from the detection of Dot / Icm-transported substrates, including effectors. More specifically, the effector LegC4 has been reported to be important for replication of L. pneumophila within its natural host protist but detrimental for replication in a Legionella disease model mouse. LegC4 has been shown to enhance the restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-γ). Furthermore, expression of legC4 was sufficient to restrict the replication of Legionella longbeachae within macrophages activated with TNF or IFN-γ. Thus, LegC4 contributes to the elimination of L. pneumophila from healthy hosts by enhancing cytokine-mediated host defense mechanisms. 【0005】 There is a need for alternative or complementary treatments for microbial pathogens other than antibiotics, which are losing effectiveness due to pathogen resistance. As disclosed herein, the use of bacterial effector proteins represents a promising alternative or complementary treatment for combating microbial pathogens. SUMMARY OF THE INVENTION 【0006】 As pathogens acquire antibiotic resistance, currently available antimicrobial treatments are decreasing in effectiveness for treating bacterial diseases. Within the current population of therapeutics that maintain effectiveness, an even smaller subset effectively targets and treats intracellular pathogens. The present disclosure relates to the use of bacterial effector proteins to enhance cytokine-mediated host defense mechanisms and limit the ability of intracellular pathogens that infect and / or replicate in mammalian cells, including human cells. Bacterial effector proteins can be introduced into mammalian cells using any known standard technique for introducing a nucleic acid encoding the effector protein into the cell or introducing the effector protein itself into the cell. 【0007】 In one embodiment, the bacterial effector protein used in accordance with the present disclosure is the LegC4 effector protein of L. pneumophila. LegC4, produced intracellularly or delivered extracellularly, represents a novel therapeutic approach to enhance the host's resistance to infection and / or interfere with pathogen replication or survival. Unlike antibiotics, this approach does not target essential processes of the pathogen. Thus, the use of LegC4 (or other bacterial effector proteins) has the potential to be a long-term antimicrobial agent without the risk of antibiotic resistance. Long-term therapeutic agents that are not subject to pathogen resistance offer significant economic benefits. Intracellular pathogen infections are difficult to treat and have a high recurrence rate. Therefore, therapeutic agents that enhance the host's ability to eliminate intracellular pathogens would lead to cost savings as repeated treatments would not be necessary. Furthermore, this approach is not likely to contribute to the development of antibacterial resistance when used as a therapeutic agent against bacterial pathogens, including but not limited to intracellular bacterial pathogens. 【0008】 According to one embodiment, there is provided a method of treating or preventing intracellular pathogen infection in mammalian cells, the method comprising introducing a LegC4 polypeptide or a fragment thereof into the mammalian cells. According to one embodiment, there is provided a method of treating mammalian cells to inhibit the replication of intracellular pathogens present in the mammalian cells and / or to reduce the number of intracellular pathogens. In one embodiment, the method of treating or preventing intracellular pathogen infection in mammalian cells comprises increasing the intracellular concentration of a polypeptide having a sequence that is 95% identical to SEQ ID NO: 2 (LegC4) in said cells, provided that the intracellular bacterial pathogen is not Legionella pneumophila. In one embodiment, the intracellular concentration of a polypeptide having a sequence that is at least 95% identical to SEQ ID NO: 2 is increased by transfecting the cells with a polynucleotide encoding said polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS 【0009】 【Figure 1A】Figures 1A-1B: Intracellular survival of Salmonella in LegC4-infected cells. Figure 1A is a graph showing the survival of Salmonella over time. Figure 1B is a graph showing the logarithmic decrease of Salmonella in transfected cells versus non-transfected cells. **, p < 0.005, *, p < 0.05 by ANOVA. 【Figure 1B】 Ibid. 【0010】 【Figure 2】 Bar graph showing the intracellular survival of Pseudomonas in LegC4-transfected cells. **, p < 0.005, *, p < 0.05, by ANOVA. 【0011】 【Figure 3】 Bar graph showing the intracellular survival of Listeria in LegC4-transfected cells. 【0012】 【Figure 4】 Graph showing the survival of HCoV-OC43 in LegC4-transfected MRC-5 cells. 【0013】 【Figure 5】 Provide images of the cytotoxic effect (CPE) of HCoV-OC43-infected LegC4-transfected cells versus non-transfected cells. 【0014】 Detailed description Definition "Cell" refers to the basic structural and functional unit of an organism. In higher organisms, such as animals, cells with similar structures and functions generally aggregate into "tissues" that perform specific functions. Thus, tissues include aggregates of similar cells and the surrounding intercellular substances, such as epithelial tissue, connective tissue, muscle, and nerve. 【0015】 "Co - administration" refers to the administration of unit doses of two or more biologically active agents, where the active agents are administered simultaneously or sequentially within a time frame in which the first - administered agent is still therapeutically active when the last co - administered agent is administered. 【0016】 "Effective amount" means an amount sufficient, for example, for the treatment of a disease (such as cancer) or condition, to achieve a desired therapeutic result and / or to achieve the pharmacokinetic or pharmacodynamic effects of a treatment in a subject, in the required dosage and period. A therapeutically effective amount can be administered in one or more doses. A therapeutically effective amount can vary depending on factors such as the disease state of the subject, age, gender, and weight. 【0017】 "Pharmaceutically acceptable excipient" includes, but is not limited to, any adjuvant, carrier, excipient, lubricant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifying agent that has been approved by the U.S. Food and Drug Administration as being acceptable for use in humans or domestic animals. 【0018】 "Subject" refers to any mammal for which diagnosis, treatment, or therapy is desired, such as humans, laboratory animals (e.g., primates, rats, mice, rabbits, and guinea pigs), domestic animals (e.g., cows, sheep, goats, and pigs), household pets (e.g., dogs, cats, and rodents), and horses. 【0019】 "Treat", "treating", or "treatment" refers to an act for obtaining beneficial or desired clinical results, including, but not limited to, alleviation or recovery (e.g., partial or complete regression) of one or more symptoms or signs of a disease or condition, reduction in the extent of a disease or condition, stabilization of the condition of a disease or condition (i.e., not getting worse, achieving stability of the disease), restoration or remission of a disease state or condition, reduction in the rate or time of progression, and remission (whether partial or complete). For example, treatment of an intracellular pathogen includes reducing the ability of the pathogen to infect, replicate, or survive in host cells. 【0020】 As used herein, the term "intracellular pathogen inhibitor" defines any agent or condition that, either as a direct causative agent or by inducing the production of a substance or condition unfavorable to the intracellular pathogen, has an adverse effect on the viability or the ability of the intracellular pathogen to infect or replicate. Thus, the inhibitors used herein can act directly or indirectly and can act either alone or in combination with another active moiety or condition. Inhibiting an intracellular pathogen defines a process of directly or by inducing the production of an intracellular pathogen inhibitor to reduce the viability of the intracellular pathogen or its ability to infect or replicate. 【0021】 As used herein, the term "microbial pathogen" refers to any microorganism capable of causing disease, including bacteria, fungi, viruses, and protists. 【0022】 An "intracellular pathogen" is an organism that can grow and multiply within a host cell. These pathogens can be divided into facultative intracellular pathogens and obligate intracellular pathogens, and facultative intracellular pathogens can grow and multiply both inside and outside host cells. 【0023】 An "antimicrobial agent" refers to any compound or condition that has an adverse effect on the ability of a microorganism to grow, replicate, or infect a host cell. 【0024】 As used herein, the term "bacterial effector" defines a protein secreted by a pathogenic bacterium that helps the pathogen invade host tissue, suppress its immune system, or otherwise aids pathogen survival. 【0025】 As used herein, the term "heterologous" with respect to a nucleic acid refers to a nucleic acid sequence derived from a foreign species, or, in the case of a species derived from the same species, a recombinant construct comprising a first heterologous nucleic acid sequence linked to a second nucleic acid sequence. For example, a "heterologous" promoter is a promoter that is operably linked to a coding sequence by intentional human intervention to form a recombinant chimeric gene, and the promoter and the coding sequence are not naturally related to each other. 【0026】 Embodiments The present disclosure relates to compositions and methods using bacterial effector proteins to enhance cytokine-mediated host defense mechanisms and limit the ability of intracellular pathogens that infect and / or replicate in mammalian cells, including human cells. This method can be used to treat and / or prevent infections by pathogens selected from fungi, bacteria, and viruses, but excluding the bacterial pathogen Legionella pneumophilia. 【0027】 The therapeutic bacterial effector proteins of the present disclosure can be introduced into mammalian cells using any of the known standard techniques for introducing nucleic acids and polypeptides into cells. Introduction or increased concentration of the bacterial effector protein in the host cell can be accomplished either by introducing an exogenous source of the polypeptide into the cell or by expressing a nucleic acid sequence encoding the bacterial effector protein intracellularly. In one embodiment, a nucleic acid sequence encoding the effector protein or a polypeptide fragment thereof is introduced into the cell. Alternatively, the effector polypeptide itself, or a polypeptide fragment thereof, is introduced directly into the cell. According to one embodiment, a nucleic acid sequence encoding one or more bacterial effector proteins or fragments thereof is introduced into mammalian cells via a delivery vehicle, optionally, the delivery vehicle is targeted to a preselected cell population. 【0028】 In one embodiment, the encoded bacterial effector protein is a protein produced by a bacterium of the genus Legionella, and in one embodiment, the effector protein is LegC4, a Legionella pneumophila protein. According to one embodiment, a nucleic acid sequence encoding a polypeptide having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2, or a polypeptide having at least 85%, 90%, 95%, or 99% sequence identity to a peptide fragment of SEQ ID NO: 2 is provided. In one embodiment, the nucleic acid sequence encoding the LegC4 protein is operably linked to a heterologous promoter that is functional in mammalian cells. In one embodiment, the promoter is an inducible promoter. In one embodiment, an expression vector is provided that can be packaged in a delivery vehicle capable of delivering the expression vector into mammalian cells for transient expression or for integration into the genome of the cell. In one embodiment, the expression vector comprises a nucleic acid sequence encoding a polypeptide having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2 or a polypeptide having at least 85%, 90%, 95%, or 99% sequence identity to a peptide fragment of SEQ ID NO: 2, the peptide fragment being a contiguous 10, 15, 20, 50, 100, 300, 500, 800, or 1000 amino acid fragment of SEQ ID NO: 2, operably linked to a heterologous promoter. 【0029】 In one embodiment, the bacterial effector composition disclosed herein is introduced into mammalian cells of a subject to stimulate the cytokine-mediated host defense mechanism. In one embodiment, a nucleic acid sequence encoding one or more polypeptides having at least 95% sequence identity to SEQ ID NO: 2, or a nucleic acid encoding a peptide fragment of SEQ ID NO: 2, is introduced into the cell to increase the intracellular concentration of the encoded one or more polypeptides and stimulate the cytokine-mediated host defense mechanism. In one embodiment, the nucleic acid to be introduced encodes at least a contiguous 10, 15, 20, 50, 100, 300, 500, 800, or 1000 amino acid fragment of SEQ ID NO: 2. In one embodiment, the nucleic acid to be introduced has at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1 or a fragment thereof. 【0030】 According to one embodiment, one or more polypeptides having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2, or polypeptides having at least 85%, 90%, 95%, or 99% sequence identity to a peptide fragment of SEQ ID NO: 2, optionally wherein the fragment is at least 10, 15, 20, 50, 100, 300, 500, 800, or 1000 amino acids in length, are introduced into mammalian cells of a subject to stimulate the cytokine-mediated host defense mechanism. The polypeptide can be introduced into mammalian cells via a controlled and / or targeted delivery mechanism, including, for example, liposomes or other membrane-bound vesicles, nanoparticles, or cell-penetrating peptide (CPP)-mediated delivery. 【0031】 According to the present disclosure, delivery of a bacterial effector protein such as LegC4 (delivered either by intracellular expression of the gene or by extracellular delivery of the protein) to mammalian cells that are potential host targets represents a novel therapy for enhancing the ability of the potential host to resist infection and / or interfering with pathogen replication or survival. The administered bacterial effector composition can be used with or in combination with any standard antimicrobial treatment. Since this approach does not target essential processes of pathogens like antibiotics, it has the potential to be a long-term antimicrobial agent that bacteria and other pathogens cannot evolve to overcome. A long-term therapeutic agent that is not subject to pathogen resistance has significant economic benefits. Intracellular pathogen infections are difficult to treat and have a high recurrence rate. Therefore, a therapeutic agent that enhances the host's ability to remove intracellular pathogens will lead to cost savings because repeated treatment is not necessary. Furthermore, this approach is not likely to contribute to the progression of antimicrobial resistance when used as a therapeutic agent against bacterial pathogens including, but not limited to, intracellular bacterial pathogens. 【0032】 According to one embodiment, a method for treating or preventing intracellular pathogen infection in mammalian cells is provided, the method comprising introducing a LegC4 polypeptide or a fragment thereof into the mammalian cells. In one embodiment, the LegC4 polypeptide or a fragment thereof is introduced into the cells by transfecting the cells with a nucleic acid encoding the LegC4 polypeptide or a fragment thereof. In one embodiment, the cells are transfected with a polynucleotide encoding a polypeptide or a peptide fragment thereof having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2 that enhances the cytokine-mediated host defense mechanism. In one embodiment, the polynucleotide encoding the bacterial effector protein is operably linked to a non-native heterologous constitutive promoter that functions in mammalian cells, and optionally, the promoter is a cytomegalovirus promoter. 【0033】 In one embodiment, the introduced nucleic acid sequence encoding the bacterial effector protein is transiently expressed in mammalian cells. In one embodiment, the expression of the bacterial effector protein (e.g., LegC4 polypeptide) is under the control of an inducible promoter. In one embodiment, the mammalian cells are stably transfected with a nucleic acid encoding the bacterial effector protein (e.g., LegC4 polypeptide). According to one embodiment, the nucleic acid encoding LegC4 has a size similar or identical to SEQ ID NO: 2 and encodes a polypeptide having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 2, or a polypeptide having at least 85%, 90%, 95%, or 99% sequence identity to a peptide fragment of SEQ ID NO: 2. According to one embodiment, the nucleic acid encoding LegC4 has a size similar or identical to SEQ ID NO: 2 and encodes a polypeptide having at least 95% sequence identity to SEQ ID NO: 2, or a polypeptide having at least 95% sequence identity to a peptide fragment of SEQ ID NO: 2, wherein optionally the peptide fragment is a contiguous amino acid fragment of SEQ ID NO: 2 having a length of at least 10, 15, 20, 50, 100, 300, 500, 800, or 1000 amino acids. In one embodiment, the nucleic acid encoding LegC4 encodes a polypeptide comprising the sequence of SEQ ID NO: 2. 【0034】 In one embodiment, the LegC4 polypeptide or a fragment thereof is introduced into cells by transfecting the cells with a nucleic acid encoding a peptide comprising at least a contiguous 10, 15, 20, 50, 100, 300, 500, 800, or 1000 amino acid fragment of SEQ ID NO: 2. In one embodiment, the nucleic acid encoding the LegC4 polypeptide is a nucleic acid encoding a polypeptide having at least 85%, 90%, 95%, or 99% sequence identity to a peptide fragment of SEQ ID NO: 2. In one embodiment, the nucleic acid encoding the LegC4 polypeptide is a nucleic acid having at least 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 1. In one embodiment, the nucleic acid encoding the LegC4 polypeptide comprises the nucleic acid sequence of SEQ ID NO: 1. 【0035】 According to one embodiment, there is provided a method of treating mammalian cells to inhibit the replication of intracellular pathogens present in the mammalian cells and / or to reduce the number of intracellular pathogens, provided that the intracellular bacterial pathogen is not Legionella pneumophilia. In one embodiment, there is provided a method for reducing the number of intracellular pathogens present in mammalian cells contacted with the intracellular pathogen as compared to untreated mammalian cells contacted with the intracellular pathogen, the method comprising increasing the intracellular concentration of a polypeptide having a sequence 95% identical to SEQ ID NO: 2 (LegC4) in the treated cells, provided that the intracellular bacterial pathogen is not Legionella pneumophilia. In one embodiment, the mammalian cells are human cells, and more particularly, the mammalian cells to be treated are in vivo. 【0036】 According to one embodiment, a method for treating intracellular pathogens in mammalian cells is provided, where the intracellular pathogen is a fungus, virus, or bacterial pathogen, the bacterial pathogen is other than Legionella pneumophilia, and the method includes introducing or increasing the concentration of a bacterial effector protein in the host cell. In one embodiment, the viral pathogen to be treated according to the present disclosure is a member of the viral family selected from the group consisting of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Filoviridae, Orthomyxoviridae, and Retroviridae. In one embodiment, the viral pathogen is a member of the Coronaviridae family and includes, but is not limited to, Human coronavirus 229E, Human coronavirus NL63, Human coronavirus OC43, Human coronavirus HKU1, Middle East respiratory syndrome-related coronavirus, Severe acute respiratory syndrome coronavirus, and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In one embodiment, a method for treating intracellular bacterial infection is provided, where the bacterial pathogen is other than Legionella pneumophilia, and the method includes the step of introducing or increasing the concentration of a bacterial effector protein into the host cells of an infected individual. 【0037】 In one embodiment, the intracellular bacterial pathogen to be treated in accordance with the present disclosure is selected from the group consisting of Yersinia pestis, Burkholderia pseudomallei, Burkholderia mallei, Listeria monocytogenes, Pseudomonas aeruginosa, Brucella abortus, Chlamydia trachomatis, Coxiella burnetii, Mycobacterium tuberculosis, Salmonella enterica, and Shigella flexneri. In one embodiment, the intracellular bacterial pathogen to be treated in accordance with the present disclosure is Listeria monocytogenes, Pseudomonas aeruginosa, or Salmonella enterica. 【0038】 According to one embodiment, there is provided a method of treating mammalian cells to inhibit and / or reduce the replication of intracellular pathogens, the method comprising introducing or increasing the concentration of a bacterial effector protein in the mammalian cells. In one embodiment, a method of treating or preventing intracellular pathogen infection in mammalian cells comprises increasing the intracellular concentration of a polypeptide having a sequence of 95% relative to SEQ ID NO: 2 (LegC4), provided that the intracellular bacterial pathogen is not Legionella pneumophilia. In one embodiment, the intracellular concentration of a polypeptide having a sequence of at least 95% relative to SEQ ID NO: 2 is increased by transfecting the cells with a polynucleotide encoding the polypeptide, the polynucleotide being operably linked to regulatory elements and enabling the expression of the encoded polypeptide in mammalian cells including human cells. In one embodiment, the mammalian cells are transfected with a polynucleotide encoding the polypeptide of SEQ ID NO: 2 or a fragment thereof. In one embodiment, the mammalian cells are transfected with a nucleic acid coding sequence comprising SEQ ID NO: 1, the coding sequence being operably linked to regulatory elements and enabling the expression of a protein by SEQ ID NO: 1. 【0039】 Exemplary embodiments According to Embodiment 1, there is provided a method for treating mammalian cells to prevent intracellular pathogen infection, or to suppress or reduce the number of intracellular pathogens present in mammalian cells after said intracellular pathogen infection, or to reduce the total number of infected cells in a subject, wherein the intracellular concentration of a bacterial effector protein, optionally a polypeptide of a size similar to SEQ ID NO: 2 (e.g., at least 90% or 95% the length of SEQ ID NO: 2) or the same size and having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2 (LegC4), or a peptide fragment thereof having at least 95% sequence identity to a contiguous fragment of SEQ ID NO: 2, said fragment being at least 10, 15, 20, 50, 100, 300, 500, or 1,000 amino acids in length, is increased in said cells, provided that the intracellular bacterial pathogen is not Legionella pneumophilia. 【0040】 According to Embodiment 2, there is provided the method according to Embodiment 1, wherein the step of increasing the intracellular concentration of the polypeptide comprises delivering an exogenous source of the polypeptide, or a polynucleotide encoding the polypeptide, into the interior of the mammalian cells. 【0041】 According to Embodiment 3, there is provided the method according to Embodiment 1 or 2, wherein the intracellular concentration of the polypeptide is increased by transfecting the cells with a polynucleotide encoding the polypeptide, optionally wherein the polynucleotide is of a size similar to SEQ ID NO: 2 (e.g., at least 90% or 95% the length of SEQ ID NO: 2) or the same size and having at least 95% sequence identity to SEQ ID NO: 2, or a polypeptide fragment of SEQ ID NO: 2, said fragment encoding a polypeptide fragment having at least 95% sequence identity to a contiguous 10, 15, 20, 50, 100, 300, 500, or 1,000 amino acid fragment of SEQ ID NO: 2. 【0042】 According to Embodiment 4, the cell is transfected with a polynucleotide encoding the polypeptide, wherein the polynucleotide is operably linked to a non-native heterologous regulatory element (promoter) that enables expression of the polypeptide in mammalian cells, and optionally, the promoter is an inducible promoter, and the method according to any one of Embodiments 1 to 3 is provided. 【0043】 According to Embodiment 5, the method according to Embodiment 4 is provided, wherein the cell is transiently transfected with the polynucleotide and the promoter is a constitutive promoter that functions in mammalian cells. 【0044】 According to Embodiment 6, the method according to any one of Embodiments 1 to 5 is provided, wherein the intracellular concentration of the polypeptide is increased in the target population of cells. 【0045】 According to Embodiment 7, the method according to any one of Embodiments 1 to 6 is provided, wherein the cell is transfected with a polynucleotide having at least 95% sequence identity to SEQ ID NO: 1, and the sequence of SEQ ID NO: 1 is operably linked to a heterologous promoter that functions in mammalian cells. 【0046】 According to Embodiment 8, the method according to any one of Embodiments 2 to 7 is provided, wherein the cell is stably transfected with the polynucleotide, and optionally, the polynucleotide is operably linked to an inducible promoter. 【0047】 According to Embodiment 9, the method according to any one of Embodiments 1 to 8 is provided, wherein the mammalian cell is a human cell. 【0048】 According to Embodiment 10, the method according to any one of Embodiments 1 to 9 is provided, wherein the intracellular pathogen is an intracellular bacterial pathogen. 【0049】 According to Embodiment 11, the method according to Embodiment 10 is provided, wherein the intracellular pathogen is selected from the group consisting of Yersinia pestis, Burkholderia pseudomallei, Burkholderia mallei, Listeria monocytogenes, Pseudomonas aeruginosa, Brucella abortus, Chlamydia trachomatis, Coxiella burnetii, Mycobacterium tuberculosis, Salmonella enterica, and Shigella flexneri. 【0050】 According to Embodiment 12, the method according to Embodiment 11 is provided, wherein the intracellular pathogen is Listeria monocytogenes, Pseudomonas aeruginosa, or Salmonella enterica. 【0051】 According to Embodiment 13, the method according to Embodiment 11 is provided, wherein the intracellular pathogen is Listeria monocytogenes or Salmonella enterica. 【0052】 According to Embodiment 14, the intracellular pathogen is a viral pathogen, optionally a viral pathogen selected from the viral families of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Filoviridae, Orthomyxoviridae, or Retroviridae, and optionally the viral pathogen is a member of the Coronaviridae, and the method according to any one of Embodiments 1 to 9 is provided. 【0053】 According to Embodiment 15, the mammalian cell is treated in vivo by administering thereto a delivery vehicle that introduces a polynucleotide into the interior of the mammalian cell, the polynucleotide encodes a bacterial effector protein, and optionally the polynucleotide has a size similar to that of SEQ ID NO: 2 (e.g., at least 90% or 95% of the length of SEQ ID NO: 2) or the same size and encodes a polypeptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2, or encodes a continuous fragment of the polypeptide of SEQ ID NO: 2, the fragment being at least 10, 15, 20, 30, 50, 100, 300, 500, or 1,000 amino acids in length, and optionally the mammalian cell is a human cell, and the method according to any one of Embodiments 1 to 14 is provided. 【0054】 According to Embodiment 16, the mammalian cell is treated in vivo by administering thereto a composition that introduces a bacterial effector protein into the cell, and optionally the bacterial effector protein comprises a polypeptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2, and optionally the mammalian cell is a human cell, and the method according to any one of Embodiments 1 to 14 is provided. 【0055】 According to Embodiment 17, the method according to Embodiment 15 is provided, wherein the polynucleotide is introduced into the cell via a viral delivery vehicle. 【0056】 According to Embodiment 18, a polypeptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2 is introduced into the cell via a delivery vehicle that delivers the polypeptide to the cytoplasm of the cell, and optionally, the delivery vehicle is an extracellular vesicle, and the method according to Embodiment 16 is provided. 【0057】 According to Embodiment 19, there is provided a method according to any one of Embodiments 1 to 18, further comprising increasing the intracellular concentration of a second bacterial effector protein that is structurally different from the first bacterial effector protein. 【0058】 According to Embodiment 20, there is provided a method for inhibiting an intracellular pathogen present in a mammalian cell, the method comprising increasing the intracellular concentration of one or more bacterial effector proteins in the cell, and optionally, the effector protein comprises a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 (LegC4), or a continuous fragment thereof, the fragment being at least 10, 15, 20, 30, 50, 100, 300, 500, or 1,000 amino acids in length, provided that the intracellular bacterial pathogen is not Legionella pneumophilia, and optionally, the mammalian cell is a human cell. 【0059】 According to Embodiment 21, there is provided a method according to Embodiment 20, wherein increasing the intracellular concentration of the polypeptide comprises delivering an exogenous source of the polypeptide, or a polynucleotide encoding the polypeptide, into the interior of the mammalian cell. 【0060】 According to Embodiment 22, there is provided a method according to Embodiment 20 or 21, wherein the intracellular concentration of a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 is increased by transfecting the cell with a polynucleotide encoding the polypeptide. 【0061】 According to Embodiment 23, the method according to any one of Embodiments 20 to 22 is provided, wherein the cell is transfected with a polynucleotide encoding the polypeptide, the polynucleotide is operably linked to a regulatory element that enables expression of the polypeptide in mammalian cells, and optionally, the regulatory element is a non-native heterologous promoter that is not operably linked to the polypeptide encoding the native sequence. 【0062】 According to Embodiment 24, the method according to any one of Embodiments 20 to 23 is provided, wherein the cell is transfected with a polynucleotide having at least 95% sequence similarity to SEQ ID NO: 1. 【0063】 According to Embodiment 25, the method according to any one of Embodiments 20 to 24 is provided, wherein the polynucleotide is operably linked to an inducible promoter. 【0064】 According to Embodiment 26, the method according to any one of Embodiments 20 to 25 is provided, wherein the mammalian cell is a human cell. 【0065】 According to Embodiment 27, the method according to any one of Embodiments 20 to 26 is provided, wherein the intracellular pathogen is an intracellular bacterial pathogen, provided that the bacterial pathogen is not Legionella pneumophilia. 【0066】 According to Embodiment 28, the intracellular pathogen is selected from the group consisting of Yersinia pestis, Burkholderia pseudomallei, Burkholderia mallei, Listeria monocytogenes, Pseudomonas aeruginosa, Brucella abortus, Chlamydia trachomatis, Coxiella burnetii, Mycobacterium tuberculosis, Salmonella enterica, and Shigella flexneri species, and the method according to Embodiment 27 is provided. 【0067】 According to Embodiment 29, the intracellular pathogen is Listeria monocytogenes, Pseudomonas aeruginosa, or Salmonella enterica, and the method according to Embodiment 28 is provided. 【0068】 According to Embodiment 30, the intracellular pathogen is Listeria monocytogenes or Salmonella enterica, and the method according to Embodiment 28 is provided. 【0069】 According to Embodiment 31, the intracellular pathogen is a viral pathogen, optionally a viral pathogen selected from the viral families of Herpesviridae, Papillomaviridae, Coronaviridae, Flaviviridae, Filoviridae, Orthomyxoviridae, or Retroviridae, and optionally the viral pathogen is a member of the Coronaviridae, and the method according to any one of Embodiments 20 to 26 is provided. 【0070】 According to Embodiment 32, the mammalian cell is administered in vivo by administering a delivery vehicle that introduces into the interior of the mammalian cell a polynucleotide encoding a polypeptide having at least 80%, 85%, 95%, or 95% sequence identity to SEQ ID NO: 2, or a peptide fragment of SEQ ID NO: 2, and optionally the mammalian cell is a human cell, and the method according to any one of Embodiments 20 to 31 is provided. 【0071】 According to Embodiment 33, the mammalian cell is administered in vivo by administering a composition that introduces into the mammalian cell a polypeptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2, or a peptide fragment of SEQ ID NO: 2, wherein the fragment is at least 10, 15, 20, 30, 50, 100, 300, 550, or 1,000 amino acids in length, and optionally the mammalian cell is a human cell, and the method according to any one of Embodiments 20 to 31 is provided. 【0072】 According to Embodiment 34, a polypeptide having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2, or a peptide fragment of SEQ ID NO: 2, wherein the fragment is at least 10, 15, 20, 30, 50, 100, 300, 550, or 1,000 amino acids in length, is introduced into the cell by transfecting the cell with a polynucleotide encoding the polypeptide, the polypeptide is expressed in the cell, and optionally the mammalian cell is a human cell, and the method according to any one of Embodiments 20 to 33 is provided. 【0073】 According to Embodiment 35, there is provided the method of Embodiment 34, wherein the polynucleotide is introduced into the cell via a viral delivery vehicle. 【0074】 According to Embodiment 36, there is provided the method according to Embodiment 33, wherein the polypeptide is introduced into the cytoplasm of the cell via a delivery vehicle, and optionally, the delivery vehicle is an extracellular vesicle. 【0075】 According to Embodiment 37, the intracellular concentration of a polypeptide having at least 95% sequence to SEQ ID NO: 2 is increased by transfecting the cell with the polynucleotide encoding the polypeptide, and optionally, the polynucleotide has at least 95% sequence identity to SEQ ID NO: 1. There is provided the method according to any one of Embodiments 1 to 35. 【0076】 According to Embodiment 38, there is provided a pharmaceutical composition comprising a delivery vehicle and a pharmaceutically acceptable excipient, wherein the delivery vehicle is capable of delivering its contents into the interior of a mammalian cell, and comprising: i) a polypeptide having a size similar (e.g., at least 90% or 95% of the length of SEQ ID NO: 2) or identical to SEQ ID NO: 2, having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2 (LegC4), or a peptide fragment having at least 95% sequence identity to a contiguous fragment of SEQ ID NO: 2, wherein the fragment is at least 10, 15, 20, 50, 100, 300, 500, or 1,000 amino acids in length, or ii) A polynucleotide encoding a polypeptide having a size similar to that of SEQ ID NO: 2 (e.g., at least 90% or 95% of the length of SEQ ID NO: 2) or the same size, having at least 80%, 85%, 95%, or 99% sequence identity to SEQ ID NO: 2 (LegC4), or a peptide fragment having at least 95% sequence identity to a continuous fragment of SEQ ID NO: 2, wherein the fragment is at least 10, 15, 20, 50, 100, 300, 500, or 1,000 amino acids in length. 【0077】 According to Embodiment 39, there is provided the composition according to item 38, wherein the delivery vehicle comprises a polynucleotide encoding a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 2. 【0078】 According to Embodiment 40, there is provided the composition according to item 38, wherein the delivery vehicle comprises a polypeptide having at least 95% sequence identity to the polypeptide of SEQ ID NO: 2. 【0079】 According to Embodiment 41, there is provided the composition according to item 38 or 39, wherein the delivery is a viral delivery vehicle. 【0080】 According to Embodiment 42, there is provided the composition according to item 38 or 40, wherein the delivery is extracellular vesicles. 【0081】 According to Embodiment 43, there is provided the composition according to any one of items 38 to 42, further comprising an antimicrobial agent, optionally an antibiotic. 【0082】 According to Embodiment 44, a viral vector is provided, the vector can deliver its contents into the interior of mammalian cells, and further includes a polynucleotide encoding a polypeptide having at least 95% sequence identity to SEQ ID NO: 2 or a continuous peptide fragment of SEQ ID NO: 2, the fragment being at least 10, 15, 20, 30, 50, 100, 300, 500, or 1,000 amino acids in length, and further, the polynucleotide is operably linked to a heterologous promoter capable of expressing the polypeptide in mammalian cells. 【0083】 According to Embodiment 45, there is provided a viral vector according to Embodiment 44, wherein the viral vector is derived from a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes simplex virus, and optionally, the viral vector is an adenovirus or an adeno-associated virus-derived vector. 【0084】 According to Embodiment 46, there is provided a viral vector according to Embodiment 44 or 45, wherein the promoter is an inducible promoter. 【0085】 According to Embodiment 47, a recombinant polynucleotide is provided, the polynucleotide i) has a size similar to (e.g., at least 90% or 95% of the length of SEQ ID NO: 2) or the same size as SEQ ID NO: 2 and has at least 95% sequence identity to a polypeptide of SEQ ID NO: 2, or ii) is a polypeptide fragment of SEQ ID NO: 2, the fragment having at least 95% sequence identity to a continuous 10, 15, 20, 50, 100, 300, 500, or 1,000 amino acid fragment of SEQ ID NO: 2 encoding the same, the polynucleotide is operably linked to a promoter capable of expressing the polypeptide in mammalian cells, and optionally, the promoter is an inducible promoter. 【0086】 According to Embodiment 48, a method or composition according to any of Embodiments 1 to 47 is provided, wherein a cytomegalovirus promoter is operably linked to a polynucleotide encoding a bacterial effector protein, and optionally, the encoded protein comprises a polypeptide having at least 95% sequence identity with SEQ ID NO: 2 or a fragment thereof. 【Example】 【0087】 Example 1: Antibacterial Activity of LegC4 in Reducing Intracellular Bacterial Populations 【0088】 To evaluate the antibacterial effect of LegC4 expressed in host cells against intracellular pathogens, LegC4 was delivered to host cells, and the host cells were challenged by contact with an intracellular bacterial population. 【0089】 Method 【0090】 HeLa (ATCC CCL2) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C and 5% CO2. Cells were seeded into 24-well cell culture plates 24 to 48 hours before the start of transfection (until reaching 70 - 80% confluence per well). LegC4 expression plasmids (legC4_pCMV6-AN-GFP; legC4_pCMV6) with or without a green fluorescent protein (GFP) tag were purchased from Blue Heron Bio and purified from Escherichia coli stab cultures in the laboratory by Midi-Prep (Qiagen Midi-Prep kit, Qiagen). 【0091】 Cells were transfected with Lipofectamine 3000 at a concentration of 500 ng DNA per well according to the manufacturer's instructions (Thermo Fisher Scientific). 48 hours after transfection, the cells were washed twice with Dulbecco's Phosphate Buffered Saline (DPBS) and the medium was replaced with DMEM containing 10% FBS and no antibiotics. In each 24-well plate, three wells were left untransfected, and three wells were transfected with LegC4-GFP or LegC4 alone. GFP expression was imaged using a Zeiss Axiovert Microscope. 【0092】 Shigella flexneri strain Castellani and Chalmers (ATCC 700930) and Salmonella enterica serovar typhimurium (ATCC 14208) were cultured overnight at 37 °C on trypticase soy agar (TSA) plates. Both strains were cultured overnight at 37 °C in tryptone soy broth (TSB). On the day of infection (48 hours after cell transfection), the bacteria were subcultured in TSB and grown in a shaking flask at 37 °C until the absorbance (A600) reached approximately 1.00 (exact values: S. flexneri, 1.301; S. enterica, 1.054). 【0093】 The bacterial suspension was prepared such that the multiplicity of infection (MOI) was 10 when 35 mL of the suspension was added to each well of a 24-well plate with DMEM (10% FBS, no antibiotics). Before adding the bacterial suspension, the medium cultured from the cells was removed. After inoculation, the cells were cultured at 37 °C and 5% CO2 for 1 hour. After 1 hour, the supernatant was removed from each well, and the cells were washed twice with phosphate buffered saline (PBS) to remove non-infected bacteria. Extracellular bacteria were killed by adding 50 μg / mL gentamicin (in the cell culture medium) at 37 °C and 5% CO2 for 30 minutes. Then, the cells were washed twice and fresh medium containing 5 μg / mL gentamicin was added. 【0094】 The supernatant was collected at each time point (1 hour, 3 hours, 24 hours, or 48 hours post-infection), the cells were washed twice, and lysed with 0.35 mL of 1% Triton X-100 for 15 minutes at room temperature. The lysate was recovered by scraping the wells with a pipette tip and pipetting. The cell lysate was kept on wet ice during the procedure. 【0095】 The lysate was serially diluted 10-fold with PBS, and 0.1 mL aliquots were plated in triplicate on TSA plates and incubated overnight at 37°C. The bacterial inoculum suspension was also serially diluted 10-fold and plated on TSA for quantification of the inoculum dose. 【0096】 Bacteria were quantified by counting and recording the colony-forming units (CFU) within the countable range of 30 - 300 colonies per plate. The log 10 decrease in intracellular bacteria in each sample was measured by subtracting the mean value of log 10 CFU recovered from transfected wells from the log 10 CFU recovered from non-transfected samples. Statistical analysis was performed using GraphPad Prism. All supernatant and lysed samples were stored at -80°C for future analysis. 【0097】 Results 【0098】 Transfection and expression of LegC4 in HeLa cells 【0099】 The transfection efficiency was evaluated by fluorescence microscopy. The expression of LegC4-AN-GFP was detected by the signal in the FITC channel due to GFP expression. GFP expression was not detected above background levels in transfected cells with LegC4 alone (without the GFP reporter tag). Images were taken on the day of infection (48 hours post-transfection). 【0100】 Expression of LegC4 controls intracellular Salmonella infection 【0101】 Shigella did not survive after 3 hours in either non-transfected HeLa cells or transfected HeLa cells (in this experiment), so no further analysis of Shigella was performed. Salmonella survived in all three cell conditions until 48 hours post-infection. However, expression of LegC4-GFP or LegC4 alone resulted in a nearly significant decrease in intracellular S. enterica compared to non-transfected cells (LegC4-GFP, p-value = 0.053; LegC4 alone, p-value = 0.053) (Figure 1A). 【0102】 When the data were analyzed as the logarithmic decrease of S. enterica relative to non-transfected cells (Figure 1B), a clear trend of increasing antibacterial effect (logarithmic decrease) over time was observed. Notably, a significant increase (p = 0.0076) in the logarithmic decrease of S. enterica was observed at 48 hours in cells expressing LegC4 alone compared to LegC4-GFP, suggesting that the presence of the GFP reporter tag may have an inhibitory effect on the activity of the LegC4 protein. 【0103】 Finally, in cells transfected with LegC4 alone, a significant increase in logarithmic decrease was observed between 24 and 48 hours post-transfection (p = 0.0107), suggesting that the activity of LegC4 as an immune effector may have the greatest effect on reducing or preventing the survival of intracellular bacteria rather than preventing bacteria from invading cells at the initial stage of infection. 【0104】 【0105】 Example 2: Antibacterial and antiviral activities of LegC4-expressing mammalian cells 【0106】 Methods 【0107】 Overview of the intracellular bacterial survival assay 【0108】 HeLa cells were either left untransfected or transfected with LegC4 expression plasmids (legC4_pCMV6-AN-GFP; legC4_pCMV6) with or without a green fluorescent protein (GFP) tag. GFP expression was imaged. Subsequently, the cells were infected with Listeria monocytogenes and Pseudomonas aeruginosa. At each time point, the supernatant and lysate were collected. The lysate was serially diluted, plated, and incubated overnight as described in detail in Example 1. 【0109】 Overview of virus survival assay 【0110】 MRC-5 cells were either left untransfected or transfected with LegC4 expression plasmids (legC4_pCMV6-AN-GFP; legC4_pCMV6) with or without a GFP tag. Subsequently, the cells were infected with HCoV-OC43, a surrogate for human coronavirus. At each time point, the supernatant was collected and stored. Cytopathic effect (CPE) was read after a 4-day culture period. After the last time point, a virus TCID-50 assay was performed to quantify the virus titer. 【0111】 Results 【0112】 Transfection and expression of LegC4 in HeLa cells 【0113】 The transfection efficiency was evaluated by fluorescence microscopy. The expression of LegC4-AN-GFP was detected by the signal resulting from the expression of GFP. GFP expression was not detected above the background level in cells transfected with LegC4 alone. 【0114】 Pseudomonas aeruginosa 【0115】 A significant decrease in P. aeruginosa was observed between non-transfected cells and Leg-GFP expressing cells at 24 hours post-infection (see Figure 2). 【0116】 Listeria monocytogenes 【0117】 A nearly significant decrease in L. monocytogenes (p-value = 0.0791) was observed between non-transfected cells and LegC4 stable expressing cells at the 24-hour time point (see Figure 3). 【0118】 【0119】 Expression of LegC4 controls HCoV-OC43 viral infection 【0120】 A significant decrease in HCoV-OC43 was observed in cells expressing LegC4 at both 48 and 72 hours post-infection (Figure 4). Notably, a 1000-fold decrease in viral titer was confirmed at 48 hours. CPE observations indicate that non-transfected cells began to show morphological changes at 48 hours post-infection. No CPE was observed up to the 96-hour time point under the LegC4 condition, suggesting that the presence of LegC4 may delay the onset of infection (Figure 5).

Claims

[Claim 1] A pharmaceutical composition for treating mammalian cells in order to suppress the number of intracellular pathogens present in mammalian cells that have come into contact with intracellular pathogens compared to untreated mammalian cells that have come into contact with intracellular pathogens, a) A polypeptide having at least 95% sequence identity with SEQ ID NO: 2 (LegC4) or a polypeptide having at least 95% sequence identity with a peptide fragment of SEQ ID NO: 2, wherein the fragment is at least 20 amino acids long; or b) A nucleic acid sequence encoding a polypeptide having at least 95% sequence identity with SEQ ID NO: 2 (LegC4) or a polypeptide having at least 95% sequence identity with a peptide fragment of SEQ ID NO: 2, wherein the fragment is at least 20 amino acids long; However, the intracellular pathogen is not Legionella pneumophila; this is a pharmaceutical composition. [Claim 2] The pharmaceutical composition according to claim 1, comprising a polynucleotide encoding a polypeptide having at least 95% sequence identity with SEQ ID NO:

2. [Claim 3] The pharmaceutical composition according to claim 2, comprising a polynucleotide having at least 95% sequence identity with respect to SEQ ID NO:

1. [Claim 4] The pharmaceutical composition according to claim 3, wherein the mammalian cells are human cells. [Claim 5] The pharmaceutical composition according to claim 4, wherein the intracellular pathogen is an intracellular bacterial pathogen. [Claim 6] The intracellular pathogens include Brucella abortus, Listeria monocytogenes, Chlamydia trachomatis, Coxiella burnetii, Mycobacterium tuberculosis, Salmonella enterica, Pseudomonas aeruginosa, Yersinia pestis, and Burkholderia pseudomalai. The pharmaceutical composition according to claim 5, selected from the group consisting of *pseudomallei* and *Burkholderia mallei*. [Claim 7] The pharmaceutical composition according to claim 6, wherein the intracellular pathogen is Listeria monocytogenes, Pseudomonas aeruginosa, or Salmonella enterica. [Claim 8] The pharmaceutical composition according to claim 4, wherein the intracellular pathogen is a viral pathogen. [Claim 9] The pharmaceutical composition according to claim 1, further comprising a delivery vehicle for delivering the polypeptide and / or the polynucleotide into the cytoplasm of a target cell. [Claim 10] The pharmaceutical composition according to claim 9, wherein the delivery vehicle is an extracellular vesicle. [Claim 11] The pharmaceutical composition according to claim 9, wherein the delivery vehicle is a viral vector. [Claim 12] A pharmaceutical composition that inhibits infection of mammalian cells by intracellular pathogens, a) A polypeptide having at least 95% sequence identity with SEQ ID NO: 2 (LegC4) or a polypeptide having at least 95% sequence identity with a peptide fragment of SEQ ID NO: 2, wherein the fragment is at least 20 amino acids long; or b) A nucleic acid sequence encoding a polypeptide having at least 95% sequence identity with SEQ ID NO: 2 (LegC4) or a polypeptide having at least 95% sequence identity with a peptide fragment of SEQ ID NO: 2, wherein the fragment is at least 20 amino acids long; However, the intracellular pathogen is not Legionella pneumophila, In some cases, mammalian cells are human cells; this is a pharmaceutical composition. [Claim 13] The intracellular pathogens include Brucella abortus, Listeria monocytogenes, Chlamydia trachomatis, Coxiella burnetii, Mycobacterium tuberculosis, Salmonella enterica, Pseudomonas aeruginosa, Yersinia pestis, and Burkholderia pseudomalai. The pharmaceutical composition according to claim 12, wherein the bacterial intracellular pathogen is selected from the group consisting of *Pseudomallei* and *Burkholderia mallei*. [Claim 14] The pharmaceutical composition according to claim 13, wherein the intracellular pathogen is Listeria monocytogenes, Pseudomonas aeruginosa, or Salmonella enterica. [Claim 15] The pharmaceutical composition according to claim 12, wherein the intracellular pathogen is a viral pathogen. [Claim 16] The pharmaceutical composition according to claim 12, comprising a polynucleotide encoding a polypeptide having at least 95% sequence identity with respect to Sequence ID No.

2. [Claim 17] The pharmaceutical composition according to claim 12, comprising a polynucleotide having at least 95% sequence identity with respect to SEQ ID NO:

1. [Claim 18] It is a delivery vehicle, i) A polypeptide having at least 95% sequence identity with SEQ ID NO: 2, or a peptide fragment having at least 95% sequence identity with a continuous fragment of SEQ ID NO: 2, wherein the fragment is at least 10, 15, 20, 30, 50, 100, 300, 500, or 1,000 amino acid lengths, ii) A polynucleotide encoding a polypeptide having at least 95% sequence identity with SEQ ID NO: 2, or a peptide fragment having at least 95% sequence identity with a consecutive fragment of SEQ ID NO: 2, wherein the fragment has a length of at least 10, 15, 20, 30, 50, 100, 300, 500, or 1,000 amino acids. A delivery vehicle including a delivery vehicle. [Claim 19] The delivery vehicle according to claim 18, comprising a polynucleotide encoding a polypeptide having at least 95% sequence identity with the polypeptide of Sequence ID No.

2. [Claim 20] A delivery vehicle according to claim 18 or 19, which is a virus delivery vehicle. [Claim 21] The delivery vehicle according to claim 18 or 19, which is an extracellular vesicle. [Claim 22] A delivery vehicle according to claim 18, i) A polypeptide having at least 95% sequence identity with respect to Sequence ID No. 2, or ii) A polypeptide fragment of Sequence ID No. 2, wherein the fragment has at least 95% sequence identity with the consecutive 10, 15, 20, 50, 100, 300, 500, or 1,000 amino acid fragments of Sequence ID No. 2, It contains polynucleotides that code, A delivery vehicle comprising a polynucleotide operably linked to a promoter capable of expressing the polypeptide in mammalian cells, wherein the promoter is optionally an inducible promoter. [Claim 23] Recombinant polynucleotides, i) The polypeptide of SEQ ID NO: 2, or a polypeptide having at least 95% sequence identity with respect to SEQ ID NO: 2, ii) A polypeptide fragment of Sequence ID No. 2, wherein the fragment has at least 95% sequence identity with the consecutive 20, 50, 100, 300, 500, or 1,000 amino acid fragments of Sequence ID No. 2, The polynucleotide is operably linked to a promoter capable of expressing the polypeptide in mammalian cells, and optionally, iii) The promoter is an inducible promoter, or iv) The promoter is a cytomegalovirus promoter, Recombinant polynucleotides.

Images