Leader Array

Abstract

To provide not only improved delivery systems, but also means for producing such systems which find compatibility with molecules (payloads) having a range of sizes and molecular properties.SOLUTION: The present invention provides use of a Photorhabdus Virulence Cassettes (PVC) effector leader sequence, for packaging a payload into a PVC Needle Complex, and related methods for manufacturing a packaged PVC Needle Complex. The payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof, and the leader sequence and the payload form an effector fusion that is distinct from a wild-type PVC effector protein.SELECTED DRAWING: Figure 3B

Description

Technical Field

[0001] The present invention relates to leader sequences and the use of leader sequences for packaging molecules into protein complexes.

Background Art

[0002] Biological molecules (e.g., peptides, proteins, and nucleic acids) have great potential as widely applicable therapeutic agents. In fact, in recent years, there has been a tendency in the pharmaceutical industry to shift from "small molecule" drugs to more complex macromolecular therapeutic agents (also known as "biologics"). Such biologics include protein-based therapeutic agents (especially antibodies, hormones, growth factors, and cytokines) and nucleic acid-based therapeutic agents (e.g., short interfering RNAs, DNA / RNA vaccines, and gene therapies).

[0003] While the biologic market has been growing significantly in recent years, the low availability of effective delivery systems (and practicable methods for manufacturing such delivery systems) limits the diversity of molecular targets of such biotherapeutic agents, especially when the target is the cytoplasm. In fact, the majority of commercially approved peptide therapeutic agents act by targeting extracellular components of cells, such as membrane receptors or secreted molecules (e.g., present in the interstitial space). For example, Humira (the most successful therapeutic monoclonal antibody) targets the extracellularly secreted cytokine TNFα. Insulin acts by binding to its cognate receptor present on the cell membrane (the same applies to other hormonal peptide therapeutic agents).

[0004] A similar problem also exists in the agrochemical industry, and protein-based pesticidal agents are typically toxins that should target extracellular components of pest cells. As an example, Bacillus thuringiensis toxin is a commonly used natural pesticidal agent that should bind to membrane receptors and exert a toxic effect.

[0005] Methods for cytoplasmic delivery of biological molecules have been developed in laboratory studies and generally involve delivering molecules within a lipid vehicle that fuses with the cell membrane, and then releasing the payload into the cytoplasm. However, such methods have limited use in pharmaceuticals and veterinary medicines, for example, due to their non-specific nature in delivering molecules to cells.

[0006] Bacterial secretory systems are being explored as potential delivery systems due to their innate ability to secrete (or more specifically, "inject") molecules into target cells. The most studied such secretory system is the type III secretory system (T3SS) of "protein appendage" found in some Gram-negative bacteria. However, a significant drawback of these systems is that they always remain associated with the bacterial membrane and require the use of actual bacterial cells (including secretory systems) as delivery systems. Thus, it is difficult to have complete control over which molecules are transferred from the bacterium to the target cell (even if the target bioagent is overexpressed). This is because their secretory systems function by providing a link (or channel) between the bacterial cytoplasm and the target cell cytoplasm, allowing other components (potentially harmful to the host) to flow through it. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Therefore, not only are improved delivery systems needed, but also means for producing such systems that are compatible with molecules (payloads) of a wide range of sizes and molecular properties.

[0008] The present invention solves one or more of the above problems. [Means for solving the problem]

[0009] This invention is based on the surprising finding that the enterotoxigenic Photorhabdus virulence cassette (PVC) effector protein of Photorhabdus bacteria contains a previously unknown "leader sequence" (or "leader peptide") that functions to package (or "load") the PVC effector into a so-called PVC needle complex (e.g., a "nanosyringe"), which then delivers the PVC effector to target cells where it exerts its enterotoxigenic effect (the PVC effector represents the payload of such a nanosyringe). Furthermore, the inventors have found that such a leader sequence can be actually utilized to package a bound payload into the PVC needle complex (and associated / homologous complex) of the well-characterized molecular delivery system of Photorhabdus. Thus, the newly discovered leader sequence surprisingly functions to load a molecular payload (or "reaction site") into the PVC needle complex.

[0010] In addition to this finding, the inventors have developed the advantageous practical utility of such a leader sequence for packaging / loading "heterogeneous" payloads (e.g., non-Photorhabdus molecules) into PVC needle complexes, regardless of the size, molecular properties, or origin of the heterogeneous payloads. [Modes for carrying out the invention]

[0011] In a first aspect, the present invention relates to the use of a Photorhabdus villens cassette (PVC) effector reader array for packaging a payload in a PVC needle composite; The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); The leader sequence and payload form an effector fusion distinct from the wild-type PVC effector protein. Provides usage.

[0012] In one embodiment, an aspect of the present invention is the use of a PVC effector reader array for packaging a payload in a PVC needle composite; The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); The leader sequence and payload form a fusion distinct from the PVC effector protein (e.g., wild-type PVC effector protein). Provides usage.

[0013] In other words, the present invention provides, in one aspect, a method for packaging a payload in a PVC needle complex using a PVC effector leader sequence, comprising contacting an (effector) fusion with the PVC needle complex, wherein the payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); and the leader sequence and payload form an (effector) fusion distinct from a PVC effector protein (e.g., wild-type PVC effector protein).

[0014] The terms “fusion” and “effector fusion” are used interchangeably herein in the context of an (effector) fusion formed by a leader sequence and payload (and distinguished from wild-type PVC effector proteins).

[0015] This use (of the leader sequence) was demonstrated, as outlined in the examples, by expressing the effector fusion (tagged with the detection label) and the PVC needle complex in cells (e.g., host bacterial cells), therein packaging the effector fusion into the PVC needle complex (via the leader sequence), isolating the PVC needle complex, and then detecting the presence or absence of the payload within the PVC needle complex (e.g., its lysate) by Western blot detection of the detection label. The presence of the payload is detected only if it is fused to the leader sequence, but not if the payload lacks the leader sequence.

[0016] The term “PVC Effector Leader Sequence” refers to a leader region (polypeptide region) from a PVC effector polypeptide that can package a payload (e.g., an effector) within a PVC needle complex, preferably amino acids 1-50 of the PVC effector, or amino acids 2-50 if the initiating methionine is excluded. The inventors have demonstrated that leader sequences are encompassed within (or essentially comprised of) amino acids 1-50 of numerous identified PVC effector polypeptide sequences. However, it is intended that leader sequences of alternative length and located within the PVC effector be encompassed (e.g., provided that such leader sequences can package the payload within the PVC needle complex).

[0017] The remaining (non-leader sequence) portion of the PVC effector is referred to herein as the “effector portion” (e.g., payload). The effector portion preferably comprises, or essentially consists of, amino acids 51 to the C-terminus of the PVC effector protein.

[0018] Therefore, in one embodiment, the PVC effector leader sequence is contained within amino acids 1-50 or 2-50 (preferably 1-50) of the PVC effector polypeptide.

[0019] In embodiments, the PVC effector leader sequence comprises (or essentially consists of) amino acids 1 to 50 or 2 to 50 (preferably 1 to 50) of the PVC effector polypeptide.

[0020] The term “wild-type PVC effector protein” is used synonymously with the term “endogenous PVC effector protein” or simply “PVC effector protein” and refers to a PVC effector sequence (e.g., intact) that has an endogenous leader sequence (i.e., endogenous with respect to a given PVC effector, preferably amino acids 1-50 of the PVC effector) associated with an effector moiety (e.g., payload, preferably amino acids 51-C-terminus of the PVC effector protein). An example of a wild-type PVC effector may include (or essentially consist of) the amino acid sequences of one or more sequences selected from SEQ ID NOs: 1 to 46. Therefore, the fusions / effector fusions of the present invention described herein are distinguished from PVC effector proteins (e.g., wild-type PVC effector proteins) because the leader sequence is not fused to the effector moiety to which it can be fused in the case of a wild-type PVC effector protein. For example, a fusion / effector fusion may include the leader sequence of the "Pnf" PVC effector protein fused to the effector portion of the hvnA (gene Plu1649) PVC effector protein (e.g., amino acids 51-295 of SEQ ID NO: 46) (e.g., the leader of SEQ ID NO: 78), but is not intended to refer to the leader sequence of the "Pnf" PVC effector protein fused to the effector portion of the Pnf PVC effector protein (e.g., amino acids 51-340 of SEQ ID NO: 32) (e.g., the leader of SEQ ID NO: 78).

[0021] On the other hand, the fusion construct / effector fusion construct may include, for example, a non-effector portion, such as a non- Photorhabdus protein, such as the leader sequence (e.g., the leader of SEQ ID NO: 78) of the "Pnf" PVC effector protein fused to, for example, Cre recombinase. Thus, the leader sequence is useful in the packaging of a wide range of, for example, heterologous (non-wild type) agents into the PVC needle complex, opening up the possibility of using the PVC needle complex for the first time as a modular and diverse delivery system for delivering not only native effectors but also "non-native" payloads to cells. Thus, it is possible to produce a PVC needle complex having a selected payload.

[0022] Another aspect of the present invention is a method of producing a PVC needle complex comprising a payload (e.g., in other words, a method of producing a packaged PVC needle complex), comprising: a. contacting (e.g., within a host cell) the PVC needle complex with an effector fusion construct comprising a PVC effector leader sequence fused to the payload; b. the payload is one or more (preferably a polypeptide) selected from polypeptides, nucleic acids or combinations thereof; c. the effector fusion construct is distinguished from the wild-type PVC effector protein The method is provided.

[0023] One aspect of the present invention is a method of producing a PVC needle complex comprising a payload (e.g., in other words, a method of producing a packaged PVC needle complex), comprising: a. contacting (e.g., within a host cell) the PVC needle complex with a fusion construct, the fusion construct comprising a PVC effector leader sequence fused to the payload, the leader sequence and the payload forming a fusion construct that is distinguished from a PVC effector protein (e.g., a wild-type PVC effector protein); b. The payload is one or more (preferably, a polypeptide) selected from polypeptides, nucleic acids, or combinations thereof Provide a method.

[0024] In one embodiment, the contacting can be performed inside a cell (e.g., a bacterial host cell), in a cell lysate, or in a purified cell lysate (preferably, inside the cell). In one embodiment, the contacting can be performed in a cell-free expression system. Similarly, the uses described herein can include a contacting step (between the fusion / effector fusion and the PVC needle complex) performed inside a cell (e.g., a bacterial host cell), in a cell lysate, in a cell-free expression system, or in a purified cell lysate (preferably, in a cell, more preferably, inside a bacterial host cell).

[0025] A cassette (operon) encoding the PVC needle complex can be operably linked to a first promoter, and a gene encoding the fusion / effector fusion (payload) can be operably linked to a second (preferably, different) promoter. In one embodiment, the first and / or second promoter is an inducible promoter (e.g., an arabinose-inducible promoter, e.g., pBAD, and / or an IPTG-inducible promoter). Thus, the present invention encompasses an expression system in which an operon encoding PVC is present in a first vector / plasmid (optionally operably linked to a first promoter), and a sequence encoding an effector fusion (leader sequence fused to the payload) is present in a second (preferably, different) plasmid (optionally linked to a second promoter).

[0026] In one embodiment, the PVC needle complex and / or (preferably, and) the effector fusion can be expressed in one or more hosts selected from bacterial cells, yeast cells, insect cells, and / or mammalian cells. In a preferred embodiment, the PVC needle complex The combined and effector fusions can be expressed together in host cells selected from bacterial cells, yeast cells, insect cells, and mammalian cells (preferably bacterial cells). Suitable mammalian cells include HEK293 cells and / or CHO cells.

[0027] The PVC needle complex and / or (preferably, and) the effector fusion (payload) can be expressed in a heterologous bacterial expression system (preferably Escherichia coli (E. coli)). In one embodiment, the PVC needle complex and / or (preferably, and) the PVC effector can be expressed in Photorhabdus cells, and optionally, the PVC operon of the Photorhabdus cell is endogenous to the cell (and optionally, the PVC operon is operably bound to an inducible promoter that can be incorporated into the genome via genetic engineering and operably bound to the PVC operon). For example, the inducible promoter can be introduced into the genome of the Photorhabdus cell with PVC (operon) from 5', preferably by recombination as described in the example (e.g., Example 3).

[0028] The payload could be, for example, a therapeutic payload that makes the PVC needle complex useful in medical treatment.

[0029] In a further embodiment, the present invention relates to a (packaged) PVC needle composite for use in a method of treatment; a. An effector fusion containing (or essentially consisting of) a PVC effector leader array fused to the payload (e.g., packaged therein); b. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); c. Effector fusions are distinguished from wild-type PVC effector proteins. We provide PVC needle composites.

[0030] A further aspect of the present invention is a (packaged) PVC needle composite for use in a method of treatment; a. Holding a fusion that includes (or essentially consists of) a PVC effector leader array fused to the payload (e.g., packaged therein); b. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); c. The fusion is distinguished from the PVC effector protein (e.g., wild-type PVC effector protein). We provide PVC needle composites.

[0031] In one embodiment, the present invention is a method for treating a subject, comprising administering a (packaged) PVC needle complex to a subject (e.g., a patient); a. The PVC needle composite includes an effector fusion (e.g., packaged therewith) which includes (or essentially consists of) a PVC effector leader array fused to the payload; b. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); c. Effector fusions are distinguished from wild-type PVC effector proteins. Provide a method.

[0032] In other words, one aspect of the present invention is a method of treating a subject, comprising administering a (packaged) PVC needle complex to a subject (e.g., a patient); a. The PVC needle composite is fused to the payload, and the PVC effector leader is distributed It holds a fusion that includes (or essentially consists of) columns (for example, is packaged with them); b. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); c. The fusion is distinguished from the PVC effector protein (e.g., wild-type PVC effector protein). Provide a method.

[0033] In a preferred embodiment, the payload is a polypeptide.

[0034] The target may be mammals, preferably humans.

[0035] The terms "PVC needle composite holds effector fusion" and "PVC needle composite containing effector fusion" refer to a PVC needle composite having a packaged effector fusion, or in other words, a PVC needle composite packaged with an effector fusion.

[0036] The terms “packaged effector fusion,” “fusion,” and “effector fusion” (e.g., fusion / effector fusion is distinguished from wild-type PVC effector protein) encompass combinations of PVC effector leader sequences and payloads that remain in contact (e.g., fused) after packaging into the PVC needle complex (e.g., the leader sequence has not been cleaved from the payload), as well as combinations of PVC effector leader sequences and payloads that are no longer in direct contact (e.g., no longer fused after the leader sequence has been cleaved from the payload).

[0037] As used herein, the terms “to treat” or “to treat” include prophylactic treatment (e.g., to prevent the onset of a disease) and remedial treatment (treatment of a subject already suffering from a disease). Preferably, as used herein, the terms “to treat” or “to treat” mean remedial treatment. The terms “to treat” or “to treat” include treating both the disease and its symptoms. In some embodiments, “to treat” or “to treat” refers to the symptoms of the disease.

[0038] Therefore, the PVC needle complex can be administered to the target in a therapeutically effective or prophylactically effective dose.

[0039] "Therapeutic dose" is any amount of (packaged / loaded) PVC needle complex sufficient to produce such treatment of a disease or its symptoms when administered to a subject to treat the disease (or its symptoms) alone or in combination (e.g., with another therapeutic agent, administered concurrently or sequentially, acting additively or synergistically).

[0040] The “prophylactic effective dose” is any amount of the (packaged / loaded) PVC needle complex that, when administered to a subject alone or in combination (e.g., with another therapeutic agent, administered concurrently or sequentially, acting additively or synergistically), inhibits or delays the onset or recurrence of the disease (or its symptoms). In some embodiments, the prophylactic effective dose completely prevents the onset or recurrence of the disease. To “inhibit” the onset means to reduce the likelihood of the onset of the disease (or its symptoms) or to completely prevent its onset.

[0041] In a related embodiment, a packaged PVC needle composite comprising an effector fusion (e.g., holding / packaging it); a. The aforementioned effector fusion is a PVC effector leader fused to the payload. The array includes (or essentially consists of) (or, in other words, the effector fusion is formed by a PVC effector leader array and a payload); b. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof; c. Effector fusions are distinguished from wild-type PVC effector proteins. A packaged PVC needle composite is provided.

[0042] In other words, one aspect of the present invention is a packaged PVC needle composite that holds a fusion (for example, packaged with it); a. The fusion includes (or essentially consists of) a PVC effector leader array fused to the payload (or, in other words, the fusion is formed by the PVC effector leader array and the payload); b. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); c. The fusion is distinguished from the PVC effector protein (e.g., wild-type PVC effector protein). We provide (packaged) PVC needle composites.

[0043] In a preferred embodiment, the (packaged) PVC needle composite is an isolated (e.g., non-natural) PVC needle composite.

[0044] As described below, PVC needle complexes typically function to deliver toxicogenic PVC effectors to insect targets. By significantly expanding the number and types of payloads that can be packaged in PVC needle complexes, the present invention simultaneously expands the number and types of invertebrates (e.g., pests), such as amoebas, nematodes, helminths, and insects, that can be targeted and killed.

[0045] In a further embodiment of the present invention, a method for controlling pests: a. Including contact between a pest, or a target area containing a pest, and a PVC needle composite (e.g., one that holds / is packaged with it) containing an effector fusion; b. The effector fusion includes (or essentially consists of) a PVC effector leader array fused to the payload (or, in other words, the effector fusion is formed by the PVC effector leader array and the payload); c. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); d. Effector fusions are distinguished from wild-type PVC effector proteins. A method is provided.

[0046] One aspect of the present invention is a method for controlling harmful organisms: a. Including bringing a target area containing a pest or pest into contact with a PVC needle composite (e.g., packaged with it) that holds the fusion; b. The fusion includes (or essentially consists of) a PVC effector leader array fused to the payload (or, in other words, the fusion is formed by the PVC effector leader array and the payload); c. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); d. The fusion is distinguished from the PVC effector protein (e.g., wild-type PVC effector protein). Provide a method.

[0047] The terms "PVC needle composite holds effector fusion" and "PVC needle composite containing effector fusion" refer to a PVC needle composite having a packaged effector fusion.

[0048] The term "target area" refers to an area where pests are present and / or where pests may be present (e.g., where they are expected or suspected to be present).

[0049] Therefore, in one embodiment, the target area can be brought into contact with the pest before and / or while the pest is present. The target area may be near the pest (e.g., very close to it). Alternatively, the target area may be an area that the user wishes to protect from the pest. For example, the target area may include plants and / or plant products.

[0050] The term "suppressing pests" encompasses "pest control," "inhibiting the growth of pests," "inhibiting the proliferation of pests," and / or "death of pests."

[0051] Examples of such pests include one or more insects, mites, woodlice, pillbugs, centipedes, mollusks, millipedes, protists, fungi, helminths, and / or bloodborne parasites. Pests can be at any stage of development, for example, larval and / or adult pests (e.g., adults).

[0052] This invention can be used to target various agricultural, industrial, household, and horticultural pests.

[0053] In one embodiment, the pests are insects, mites, woodlice, pillbugs, centipedes, mollusks and / or millipedes. Preferably, the pests may be insects and / or mites (preferably insects).

[0054] Suitable insects include those of the orders Lepidoptera, Coleoptera, Diptera, Blattodea, Hymenoptera, Termites, Orthoptera, Silverfish, and / or Dermestidae. In one embodiment, the insects of the order Lepidoptera may be one or more moths and / or butterflies. Suitable moths include the tobacco hawk moth (Manduca Sexta) and / or the wax moth (Galleria mellonella).

[0055] In one embodiment, the insects of the order Coleoptera include European chafer larvae, northern masked chafer larvae, and Southern chafer larvae. It may be one or more of the following: southern masked chafer larva, Japanese beetle larva, scarab beetle larva, golden-lipped weevil, strawberry weevil, clay-colored weevil, Colorado leaf beetle, and / or click beetle. In another embodiment, the insects of the order Diptera may be one or more of the following: leather jackets (e.g., crane fly larvae), onion flies, cabbage flies, carrot rust flies, fungus gnats, and / or mosquitoes. In another embodiment, the insects of the order Blattodea may be one or more cockroaches selected from cockroaches, preferably the American cockroach and / or the German cockroach.

[0056] In one embodiment, the insect of the order Hymenoptera may be an ant. Preferably, the ant is a carpenter ant, a stinking house ant, a paving ant, an Argentine ant, a Pharaoh ant, a yellowish-brown crazy ant, a harvest ant, a red imported fire ant, a Southern fire ant, or a yellow squirrel ant. It may be one or more ants and / or imported fire ants. In another embodiment, an insect of the order Hymenoptera. This could be a yellow jacket.

[0057] In one embodiment, the insect of the order Termita may be a termite. Preferably, the termite may be one or more of the wetwood termites, drywood termites, and / or subterranean termites. In another embodiment, the insect of the order Orthoptera may be one or more of the crickets, grasshoppers, and / or locusts. In one embodiment, the insect of the order Silverfish may be a silverfish. In another embodiment, the insect of the order Dermestida may be an earwig.

[0058] Suitable examples of mollusks include slugs and / or snails.

[0059] In one embodiment, the harmful organism is a protist. In one embodiment, the protist is one or more selected from Chaos carolinense, Amoeba proteus, Naegleria fowleri, Dictyostelium discoideum, Entamoeba histolytica, Trichomonas vaginalis, Blastocystis hominis, Leishmania species, and Giardia lamblia. In one embodiment, the protist is one or more selected from Fonticula alba, Dictyostelium discoideum, Chlamydomonas reinhardtii, Crytomonas paramedium, Paulinella chromatophora, Nannochloropsis gaditana, and / or Tetrahymena Spp.

[0060] In one embodiment, the harmful organism is a fungus. In one embodiment, the fungus is Encephalitozoan cuniculi, Nasema apis. apis), Namema ceranae, Vittaforma carneae, Enterocytosoan bieneusi, Spraguea lophii, Vavra culiculis, Edharzardia aedes, Nematocida parisii, Razella Spp., Parasitella parasitica, Lichteimia ramose, Sporisorium scitamineum, Trametes versicolor, and / or Punctularia It is one or more fungi selected from strigosozonata.

[0061] In one embodiment, the fungus is a species of the genus Candida. This species of Candida includes C. albicans, C. ascalaphidarum, C. amphixiae, and C. antarctica. (C. Antarctica), C. argentea, C. atlantica, C. atmosphaerica, C. auris, C. bra C. blattae, C. bromeliacearum, C. carpophyta C. carpophila, C. carvajalis, C. cerambycidarum, C. chauliodes, C. corydalis, C. dosseyi, C. dubliniensis, C. ergatensis, C. fructus, C. glabrata, C. fermentati, C. guilliermondii, C. haemulonii, C. humilis, C. insectamens, C. insectum, C. interne C. intermedia, C. jeffresii, C. kefyr, C. keroseneae, C. krusei, C. lusitaniae, C. lyxosophila, C. maltose , C. marina, C. membranifaciens, C. C. mogii, C. oleophila, C. oregonensis, C. parapsilosis, C. quercitrusa, C. rugose, C. sake, C. shehatea, C. temnochilae, C. te C. tenuis, C. theae, C. tolerances, C. tropicalis, C. tsuchiyae, C. sinolaborantium, C. sojae, C. subhashii, C. viswanathii, C. u C. utilis, C. ubatubensis, and / or C. zemp It may be one or more selected from C. zemplinina. Preferably, the Candida spp. may be C. albicans.

[0062] In another embodiment, the pest is a helminth. The helminth may be one or more selected from the phyla Annelida, Platyhelminthes, Nematoda and / or Acanthocephala. In one embodiment, the helminth is , a parasitic flatworm. The parasitic flatworm may be one or more selected from the classes Cestoda, Trematoda and / or Monogenea. In one embodiment In this context, the worms are parasitic nematodes. These parasitic nematodes include roundworms (genus Ascaris), filarial worms, hookworms, pinworms (genus Enterobius), and / or whipworms. It may be one or more selected from (Trichuris trichiura).

[0063] In one embodiment, the harmful organism is a bloodborne parasite. The bloodborne parasite is a species of the genera Trypanosoma (e.g., Trypanosoma brucei and / or Trypanosoma cruzi), a species of Babesia (e.g., Babesia microti), a species of Leishmania, a species of Plasmodium (e.g., Plasmodium falciparum), and / or a species of Toxoplasma (e.g., It may be one or more of the following: .

[0064] PVC needle composites for pest control are preferably environmentally safe (e.g., environmentally safe pest-killing compositions).

[0065] Other advantageous uses include, for example, delivering payloads to cells during laboratory research. Such cells may be part of an in vitro cell line or may be cells from an animal (e.g., a research animal model). Furthermore, or alternatively, the cells may be contained within an ex vivo system, such as an organoid.

[0066] Another aspect of the present invention is an in vitro (and / or ex vivo) method for delivering a payload into a cell: a. The process involves bringing cells into contact with a packaged PVC needle complex containing an effector fusion (e.g., holding / packaging it); b. The effector fusion includes (or essentially consists of) a PVC effector leader array fused to the payload (or, in other words, the effector fusion is formed by the PVC effector leader array and the payload); c. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); d. Effector fusions are distinguished from wild-type PVC effector proteins. Provide a method.

[0067] One aspect of the present invention is an in vitro (and / or ex vivo) method for delivering a payload into a cell: a. This involves bringing the cells into contact with a PVC needle complex (packaged) that holds the fusion; b. The fusion includes (or essentially consists of) a PVC effector leader array fused to the payload (or, in other words, the fusion is formed by the PVC effector leader array and the payload); c. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); d. The fusion is distinguished from the PVC effector protein (e.g., wild-type PVC effector protein). Provide a method.

[0068] In one aspect, the present invention relates to an effector fusion (or, in other words, an effector fusion formed by a PVC effector leader array and a payload) comprising (or essentially consisting of) a PVC effector leader array fused to a payload; a. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof; b. Distinguished from wild-type PVC effector protein We provide effect pedal fusion products.

[0069] One aspect of the present invention is a fusion (or, in other words, a fusion formed by a PVC effector leader array and a payload) comprising (or essentially consisting of) a PVC effector leader array fused to a payload; a. The payload is one or more selected from polypeptides, nucleic acids, or combinations thereof (preferably polypeptides); b. Distinguished from PVC effector proteins (e.g., wild-type PVC effector protein) We provide a fusion product.

[0070] In one embodiment, the fusion / effector fusion is an isolated fusion / effector fusion (e.g., an isolated, naturally occurring fusion / effector fusion).

[0071] The present invention encompasses nucleic acids comprising nucleotide sequences encoding a fusion / effector fusion, and / or expression vectors comprising the nucleic acids. It also encompasses host cells comprising the nucleic acids and / or expression vectors.

[0072] As described above, the inventors of this invention have discovered and put into practical use the leader sequence described herein for the first time.

[0073] Therefore, another aspect of the present invention provides an isolated PVC effector reader array (for example, this isolated PVC effector reader array can package a payload within a PVC needle composite).

[0074] In a related embodiment, an isolated nucleic acid is provided that includes a nucleotide sequence encoding a PVC effector reader sequence.

[0075] The isolated PVC effector leader sequence may be recombinant, synthetic, and / or purified. The isolated nucleus encoding the PVC effector leader sequence may be recombinant, synthetic, and / or it may be a refined product.

[0076] Further details regarding the background of the present invention and the terms used herein are provided below.

[0077] The genus Photorhabdus belongs to the family Enterobacteriaceae. These are the three previously recognized species, namely P. luminescens, P. asymbiotica, and P. temperata. It is represented by the following. Important strains include P. asymbiotica subsp. australis and P. luminescens subsp. laumondii. Currently available genome sequences are available on GenBank. It is a Noh play (Photorhabdus asymbiotica ATCC43949) Complete genome - GenBank accession number FM162591.1; Photorhabdus laumondii subgenus Species *Photorhabdus laumondii* subsp. *laumondii*, strain TT01 chromosome, complete genome (GenBank accession number: CP024901.1).

[0078] References to "Photorhabdus luminescens subsp. laumondii" in this specification are interchangeable with "Photorhabdus luminescens subsp. laumondii TT01", "Photorhabdus laumondii subsp. laumondii strain TT01", and "P. luminescens TT01". It is possible.

[0079] The genome sequences of P. asymbiotica, specifically further strains of P. asymbiotica Kingscliff, are described in Wilkinson et al. (FEMS Microbiology Letters, Volume 309, Issue 2, August 2010, Pages 136-143), which are incorporated herein by reference. Further genome sequences are described in Thanwisai et al. (PLoS ONE 7(9):e43835), which are incorporated herein by reference.

[0080] Each of these species contains at least one operon known as a Photorhabdus virulence cassette (PVC) operon encoding a PVC needle complex which may be referred to herein as a “nanosyringe”. The genus Photorhabdus is typically found as an insecticidal bacterium after reflux from the (symbiotic) entomopathogenic species Heterorhabditis sp. nematodes (e.g., food and insects). Considering the need to avoid competition for resources from insects, it is understood that PVC needle complexes inherently function to suppress insects. In fact, it has been shown that insect larvae can be killed using isolated PVC needle complexes (which hold / package natural effector toxins, e.g., Pnf), see Example 2. Photorhabdus virulence cassettes represent one of at least four well-characterized toxin delivery systems of the genus Photorhabdus. Other major classes of Photorhabdus proteinicidal toxins include "Toxin Complex (Tc)", "Binary PirAB Toxin", and "Makes Caterpillars Floppy" (Mcf) toxin.

[0081] The term “Photorhabdus virulence cassette” (PVC) (as used herein synonymously with the term “PVC operon”) refers to a distinct operon of the Photorhabdus genome containing genes encoding polypeptide subunits that, when expressed, assemble to provide a macromolecule PVC needle complex. The molecular architecture of these cassettes is described, for example, in The Molecular Biology of Photorhabdus Bacteria (Springer International Published), which is incorporated herein by reference. The PVC (operon) is well characterized and described in ng AG 2017, ISBN:978-3-319-52714-7, Chapter 10, pages 159-177). A PVC typically comprises about 16 genes (pvc1-pvc16) encoding a structural protein that assembles to provide a “PVC needle complex,” and one or more genes at the 3' end that typically encode a PVC effector gene having subsequent toxic activity (and typically homologous to a typical T3SS-like effector). The Photorhabdus genome typically comprises multiple such cassettes (e.g., at least four) that often associate with different effector payloads, or even more than one effector payload.

[0082] Three classes of PVC structural operons (classes I, II, and III) have been observed in the genomes of the genus Photorhabdus and other genera. Within each class, PVCs are similar in the number and type of genes encoding the structural proteins they contain (see Figure 1(B)). More specifically, class I PVCs (which may be referred to herein as “prototype PVCs”) contain 16 conserved genes (pvc1-16). Class II lacks the pvc13 host cell binding fiber, and (though not theoretically constrained) pvc3, as conceived by the inventors, may be a minor specialized sheath subunit that attaches the pvc13 fiber protein onto the PVC needle complex (nanosyringe). Therefore, this class may be “non-specific” and is thought to inject its payload into multiple (potentially any) cell types. Class III is similar to Class I but has an additional Pvc0 gene at the initiation of the operon (of unknown function) and two additional genes encoded between pvc13 and pvc14, which are similar to the "invasion" type protein genes. This class is typically found in human clinical isolates of the genus Photorhabdus, and we have shown that when these isolates (containing the PVC operon encoding the PVC class III operon) are grown at 37°C and exposed to human serum, optimal transcription of PVC class III can occur, suggesting that this class may be a mammalian-compatible form of the PVC needle complex.

[0083] An example cassette (PVC) is shown in Figure 1(D), which is Photorhabdus asymbiotica ATCC43949 (available from ATCC, accession number) The map shows the model "Class I" PVC operon of part number ATCC43949, and the operon is It associates with the downstream effector gene "PAU_03332" (encoding a Pnf protein effector, e.g., SEQ ID NO: 32). This model operon is called PaATCC43949PVCpnf. This operon contains 16 structural genes (pvc1~16) and two effector-encoding genes (3' end) (in this case, pvc17 / Rhs-like, encoding an Rhs-like effector, and pvc21, encoding a Pnf effector). The aforementioned genes pvc1~16 correspond to the genes PAU_03353 to PAU_03338 in the sequence of GenBank accession number FM162591.1, and SEQ ID NO: 9 It is represented by an array of 3s.

[0084] One example of a PVC operon (e.g., one that codes for a structural gene but not a PVC effector) is provided in SEQ ID NO: 93 (coding the operon schematically shown in Figure 1(D)), while other examples are SEQ ID NOs: 94 and 95. These sequences begin at the ATG start codon of the first structural gene (pvc1) of the PVC cassette / operon and end at the TAA stop codon of the last structural gene (pvc16).

[0085] PVC needle complexes from any one of classes I to III can be used for a variety of applications. However, certain classes of PVC needle complexes are particularly suitable for delivery to specific cell types. For example, a PVC needle complex for delivering payloads to mammalian cells may preferably be a member of class III. A PVC needle complex for delivering payloads to insect cells (e.g., insects) may preferably be a member of class I. In the chromosome (for example, P. asymbiotica PVCpnf, encoded by sequence number 93, which is expressed in Escherichia coli from a cosmid clone) could be.

[0086] Therefore, as will be understood by those skilled in the art, the term “PVC needle complex” (as used herein synonymously with the terms “PVC needle complex delivery system” and “nanosyringe”) means a macromolecular protein complex containing polypeptide subunits encoded by the PVC (operon) of bacteria of the genus Photorhabdus. The PVC needle complex is assembled into a nanosyringe structure having a physical structure (surface) similar to that of antimicrobial R-type pyosin (see Example 3). Functional and molecular tests have shown that the PVC needle complex can be packaged (loaded) with PVC effector protein (i.e., the PVC effector protein is packaged in or on it), the packaged PVC needle complex can be released from the bacteria, and then the PVC effector can be injected into target cells, resulting in the PVC effector protein exhibiting toxicity.

[0087] The term "PVC needle complex" preferably encompasses PVC needle complex-like structures / complexes encoded by an operon containing genes homologous to those of the Photorhabdus PVC operon. The PVC-like elements are not limited to the Photorhabdus genus, and well-characterized homologous operons (relative to the PVC operon) exist on the pADAP plasmid of the entomopathogenic bacterium Serratia entomophila. Furthermore, similar and (at least partially) homologous PVC-like "injectosome" needle complex systems are used by the bacterium Pseudoalteromonas luteoviolacea (e.g., the marine worm Kasanekan). (Used to control the metamorphosis of *Hydroides elegans*). PVC operon and phase Structures encoded by operons of the same sex are present in other Enterobacteriaceae (e.g., Yersinia Spp.), and the leader described herein They can be used in conjunction with rows. Each of these (PVC-like) structures is encompassed by the term "PVC needle composite" as used herein.

[0088] Therefore, the PVC needle complex is a "nanosyringe" complex with a polypeptide that represents the "payload" or "reaction site" of the PVC needle complex, encoded by an effector gene packaged (loaded) within the PVC needle complex or at its end (tip). The inventors have demonstrated that the PVC needle complex itself (still loaded with the payload) is freely released (e.g., secreted) from Photorhabdus cells and then interacts with the membrane of target cells to inject the payload into the cytoplasm of those cells. In fact, the inventors successfully expressed and loaded the PVC needle complex in heterologous expression systems, then isolated / purified the PVC needle complex and used them to suppress (e.g., kill) insect larvae (see Example 2). Thus, the PVC needle complex acts as a long-range protein delivery system.

[0089] In one embodiment, the PVC needle composite is encoded by a sequence having at least 75% sequence identity (preferably at least 85% sequence identity; more preferably at least 95% sequence identity) with a sequence selected from sequence numbers 93, 94, and 95 (e.g., sequence number 93).

[0090] In one embodiment, the PVC needle composite is coded by a sequence selected from sequence numbers 93, 94, and 95 (for example, sequence number 93).

[0091] The leader / signal sequence is typically a peptide and is located at the N-terminus of the vast majority of (newly) expressed proteins that will be directed toward the secretory pathway (e.g., the aforementioned protein Many proteins have a signal sequence of 10-30 amino acids in length (to direct them to protein conduction channels on the cell membrane). Many proteins require a signal sequence for entry into the Golgi or endoplasmic reticulum.

[0092] In this specification, the term “leader sequence” as used in the context of “PVC effector leader sequence” (and used interchangeably with the terms “leader peptide,” “signal sequence,” “targeting signal,” “localization signal,” “localization sequence,” and “transport peptide”) means a polypeptide sequence that functions to direct a PVC effector into the interior or end (tip) of a PVC needle complex; therefore, the leader sequence functions to package the PVC effector within the PVC needle complex. The PVC needle complex can then deliver (e.g., inject) the PVC effector into a target cell. The PVC needle complex may be an assembled PVC needle complex. The term “PVC needle complex” may refer to a fragment of the PVC needle complex (e.g., the leader sequence is in contact with the fragment, and optionally, the PVC needle complex is assembled around the leader sequence-payload “effector fusion”).

[0093] PVC leader sequences are typically located at the N-terminus of a PVC effector or its homologue (characterized by or encompassed by the first 50 amino acids). However, the present invention encompasses leader sequences of PVC effectors and PVC effector homologues that can be found in regions other than the N-terminal region of such PVC effectors / homologies (e.g., in the C-terminal region).

[0094] In one embodiment, the leader sequence includes (or essentially consists of) amino acid residues 1-50 of a PVC effector (e.g., a PVC effector protein). The reference to “amino acid residues 1-50” includes “amino acid residues 2-50” that have been cleaved, e.g., with the N-terminal methionine excluded. The leader sequence may be a fragment of the N-terminal 50 amino acids of the PVC effector (e.g., a fragment containing or essentially consisting of ≤45, ≤35, ≤25, or ≤15 amino acids), provided that the fragment can package the payload into the PVC needle complex.

[0095] In one embodiment, the leader sequence of the present invention (e.g., isolated leader sequence) comprises (or essentially comprises) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with one or more sequences selected from SEQ ID NOs. 47 to 92 (preferably SEQ ID NOs. 50, 68, 71, 76, 78, or 92), provided, for example, that the leader sequence can package the payload into the PVC needle complex. In a preferred embodiment, the leader sequence comprises (or essentially comprises) an amino acid sequence having at least 60% sequence identity with one or more sequences selected from SEQ ID NOs. 47 to 92 (preferably SEQ ID NOs. 50, 68, 71, 76, 78, or 92), provided, for example, that the leader sequence can package the payload into the PVC needle complex. In a more preferred embodiment, the leader sequence includes (or essentially consists of) one or more amino acid sequences selected from SEQ ID NOs: 47 to 92 (preferably SEQ ID NOs: 50, 68, 71, 76, 78, or 92). In one embodiment, the leader sequence includes (or essentially consists of) an amino acid sequence selected from SEQ ID NOs: 47 to 92 (preferably SEQ ID NOs: 50, 68, 71, 76, 78, or 92).

[0096] In one embodiment, the leader sequence includes (or essentially consists of) an amino acid sequence selected from SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 78, and SEQ ID NO: 92.

[0097] In one embodiment, the leader sequence includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 50. In one embodiment, the leader sequence includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 68. In one embodiment, the leader sequence includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 71. In one embodiment, the leader sequence includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 76. In one embodiment, the leader sequence includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 78. In one embodiment, the leader sequence includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 92.

[0098] While not constrained by theory, the leader sequence is thought to share a “chemical composition consensus” based on amino acid properties. More specifically, the leader sequence contains a similar charge pattern, which includes two negatively charged regions followed by positively charged regions (e.g., [-ve][+ve][-ve][+ve]), see Figure 9. This matches the leader sequence of type 2 secretion system toxins, which contains a charge / property pattern of [+ve][hydrophobic][+ve][C]. A further theory assumes that the leader sequence shares a typical “helix-turn-helix” structure. Another theory is that the leader sequence forms a structure recognized by ATPase enzymes located within the PVC needle complex or at its terminal (e.g., the tip) (e.g., encoded by the gene PAU_03339(pvc15) in the model operon in Figure 1(D)).

[0099] The term “PVC effector” (used synonymously with the terms “PVC operon-coded effector” and “PVC effector protein”) refers to an effector polypeptide encoded by a Photorhabdus PVC operon, more specifically (and typically) located immediately downstream (3') of the structural gene of the operon (preferably immediately downstream of pvc16, and typically within 5kb). The term “PVC effector” preferably includes its homologs. Thus, the leader sequence may also be from a polypeptide encoded by a gene that is a homolog of the gene encoding the PVC effector; see Table 1 for such homologs. In fact, the identification of PVC effectors is aided by detecting homology of downstream genes of pvc16 to known toxin polypeptides (e.g., the gene encoding the toxin polypeptide). As will be understood by those skilled in the art, the term “homologous” preferably means a gene that originates from the same ancestor and shares similar functions, such a gene (or polypeptide encoded by it) is homologous to the gene encoding the PVC effector. The homologous gene may be from the genome of a species of the genus Photorhabdus, or from a species other than Photorhabdus. Examples of suitable homologous genera are outlined in Table 1.

[0100] The inventors identified three of the most common (best characterized) strains of the genus Photorhabdus, as well as the P. asymbiotica Thai strain PB68.1. We elucidated and characterized in detail the genes encoding the PVC effectors of these PVC needle complexes. This was done based on the proximity of gene linkage to the 3' end of the PVC structural genes of the operon, and the predicted function of the protein sequences of the effectors (e.g., homologs of known effector / toxin proteins). More specifically, the PVC effectors (e.g., genes encoding PVC effectors) were typically homologous to genes encoding known toxin polypeptides (e.g., homologs outlined in Table 1) and typically located within a distance of 1 to 5 kilobases (kb) downstream of the last structural gene of the PVC operon (e.g., pvc16) (typically with several intervening genes or no intervening genes) in an open reading frame (O It was identified as an RF. Typically, there is no “non-toxin-like” ORF between the operon terminal (encoding the PVC needle complex) and the PVC effector gene. Other small predictor genes (e.g., one or two) may be present in those regions, but these other genes are not assigned to the PVC effector (due to a lack of homology with the known effector / toxin genes mentioned above).

[0101] To assign a putative PVC effector gene (e.g., an ORF within 5kb downstream of the last structural gene of the PVC operon, e.g., within 1kb) as the PVC effector gene, the inventors used a combination of BlastP and HHPRED (https: / / toolkit.tuebingen.mpg.de / # / tools / hhpred). The putative PVC effector gene was identified as a known toxin co Based on direct homology to the do gene, similarity to the toxin protein family, proximity to the PVC operon (e.g., within 1-5 kb downstream of the last structural gene of the PVC operon, pvc16), and / or domain similarity of the predicted secondary structure to known toxins, it was assigned as a PVC effector gene.

[0102] Therefore, PVC effectors (genes) can be identified by (i) identifying pvc16 (e.g., via sequence homology with known pvc16), (ii) identifying ORF3' for pvc16 (preferably downstream of pvc16 ≤ 5kb), and (iii) confirming that the ORF encodes a PVC effector by identifying sequence homology with known genes encoding toxin polypeptides (e.g., toxin proteins listed in the column of Table 1 labeled "Homogene") (within the Photorhabdus genome).

[0103] For example, the PVC effector gene PAU_03337 (referred to herein as "sepC" due to its homology with the virrent sep gene) is located 325 base pairs (bp) downstream of pvc16 (PAU_03338) of the PVC operon referred to herein as PVCpnf (e.g., the one with sequence number 93) found in P. asymbiotica ATCC43949. That is, the start codon of PAU_03337 begins 325 bp downstream of the end of the stop codon of PAU_03338.

[0104] This is P. asymptomatic, accessible via GenBank accession number FM162591.1. This can be explained by reference to the complete genome of P. asymbiotica ATCC43949 (see, for example, Wilkinson et al, BMC Genomics volume 10, article number: 302 (2009), which is incorporated herein by reference), and the effector gene PAU_03337 is as follows: PAU_03338 is annotated when located in the genome as follows: complementary strand side (3913237..3914247) - i.e., at nucleotide positions 3913237..3914247; and PAU_03338 is annotated when located in the genome as follows: complementary strand side (3914573..3915454). No other ORFs (encoding effectors or non-effectors) are found between these two genes.

[0105] Further PVC effector genes that associate with the PVC operon referred to herein as PVCpnf (for example, SEQ ID NO: 93), namely PAU_03332 (referred to herein as "pnf"), are located 3535 bp downstream of pvc16 (PAU_03338).

[0106] The PVC effector gene PAU_02095 (referred herein to as the "Rhs-like toxin effector" due to its homology with the virulent Rhs toxin gene) is located 3961 bp downstream of pvc16 (PAU_02099) in the PVC operon referred herein as PVClopT (e.g., sequence number 94) found in P. asymbiotica ATCC43949. That is, the start codon of PAU_02095 is It begins 3961bp downstream from the end of the stop codon of PAU_02099.

[0107] In a further example, the PVC effector of gene PAU_02009 (referred herein to as "cif" due to its predictive function as a cell cycle inhibitor / ATP / GTP binding protein) is located 157 bp downstream of pvc16 (PAU_02008) of the associated PVC operon referred herein to as PVCcif, found in P. asymbiotica ATCC43949.

[0108] In a further example, with respect to the PVC operon of P. luminescens TT01, referred to herein as the PVCunit4 operon, PVC F The effector gene "pvc17" (e.g., "plu1651") is located 104 bp downstream of pvc16 (gene "plu1655"); with respect to the PVC operon of Photorhabdus temperata subsp. temperata Meg1, referred herein as the PVCcif operon, the PVC effector gene "CIF toxin effector" (e.g., MEG1DRAFT_03529) is located 4216 bp downstream of the related pvc16 gene.

[0109] These examples illustrate that genes encoding PVC effectors are typically located within ≤5kb downstream of the last gene of the PVC operon (e.g., pvc16), and more typically within ≤1kb downstream of the last gene of the PVC operon.

[0110] In summary, there are 46 PVC effectors identified in these four strains (based on currently available sequence data) (see Table 1). The first 50 amino acids of each of these PVC effectors represent (or encompass) their endogenous leader sequence, and we have demonstrated that the leader sequence can be cloned and fused to various payloads to be packaged in the PVC needle complex (see Examples 3 and 4). Thus, a (as-translated) PVC effector comprises at least two major domains: the leader sequence (amino acids 1 to 50) and the actual effector polypeptide (amino acids 51 to the C-terminal amino acid), the latter of which may be referred to herein as the “effector” (e.g., the “effector portion”) or the “payload”.

[0111] Although the genome sequences of the genus Photorhabdus are constantly being modified, this integrated list of PVC effector genes represents a comprehensive description of such effectors and, based on currently available sequence data of the most common (best-characterized) Photorhabdus strains, provides those skilled in the art with an understanding of the term “PVC effector” and the sequences of those PVC effectors (and, for example, how to search / mine further PVC effectors in alternative (genome) sequences). As described above, the inventors have found that PVC effector proteins contain the necessary (and sufficient) leader sequences to direct the PVC effector protein (e.g., payload) into the PVC needle complex.

[0112] [Table 1]

[0113] [Table 2]

[0114] [Table 3]

[0115] The accession numbers provided in Table 1 are provided for illustrative purposes and provide illustrative amino acid sequences of (or those having a high degree of similarity to) the PVC effectors described herein. The sequences of the accession numbers can be accessed via GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0116] Local tags (starting with "PAU" or "Plu") are available via GenBank as described above. Corresponds to locus tags assigned to effectors in the genome sequence. "PAT" (referring to strain P. asymbiotica Thai strain PB68.1) and "PAK" (strain Locus tags beginning with P. asymbiotica (referring to Kingscliff) are: The PVC effector gene in the genome of the aforementioned strain was assigned by the inventors (in a manner consistent with the locus tag of a publicly available sequence) during its identification.

[0117] This locus tag can be used herein to refer to the corresponding PVC effector polypeptide.

[0118] In one embodiment, the PVC effector is PAK_1985 (SEQ ID NO: 1), PA K_1987 (SEQ ID NO: 2), PAK_1988 (SEQ ID NO: 3), PAK_2075 (SEQ ID NO: 4), PAK_2077 (SEQ ID NO: 5), PAK_2892 (SEQ ID NO: 6), PAK_2893 (SEQ ID NO: 7), PAK_2894 (SEQ ID NO: 8), PAK_3525 (SEQ ID NO: 9), PAT_00148 (SEQ ID NO: 10), PAT_00149 (SEQ ID NO: 11), PAT_00150 (SEQ ID NO: 12), PAT_00152 (SEQ ID NO: 13), PAT_02308 (SEQ ID NO: 3), PAT_00150 (SEQ ID NO: 12), PAT_00152 (SEQ ID NO: 13), PAT_02308 (SEQ ID NO: 3), PAT_00148 (SEQ ID NO: 3), PAT_00150 (SEQ ID NO: 4), PAT_00152 (SEQ ID NO: 13), PAT_02308 (SEQ ID NO: 3), PAT_00150 (SEQ ID NO: 12), PAT_00152 (SEQ ID NO: 13), PAT_02308 (SEQ ID NO: 3), PAT_0014 Column number 14), PAT_02309 (sequence number 15), PAT_02310 (sequence number 16), PAT_02956 (sequence number 17), PAT_02957 (sequence number 18), PAT_03171 (sequence number 19), PAT_03172 (sequence number 20), PAT_03177 (sequence number 21), PAU_02009 (sequence number 22), PAU_02010 (sequence number 23), PAU_02095 (sequence number 24), PAU_02096 (sequence number 25), PAU_ 02097 (SEQ ID NO: 26), PAU_02098 (SEQ ID NO: 27), PAU_02230 (SEQ ID NO: 28), PAU_02805 (SEQ ID NO: 29), PAU_02806 (SEQ ID NO: 30), PAU_02807 (SEQ ID NO: 31), PAU_03332 (SEQ ID NO: 32), PAU_03337 (SEQ ID NO: 33), Plu1651 (SEQ ID NO: 34), Plu1671 (SEQ ID NO: 35), Plu1672 (SEQ ID NO: 36), Plu1690 (SEQ ID NO: 37), Plu16 Encoded by one or more genes selected from 91 (sequence number 38), Plu1712 (sequence number 39), Plu1713 (sequence number 40), Plu1714 (sequence number 41), Plu2400 (sequence number 42), Plu2401 (sequence number 43), Plu2514 (sequence number 44), Plu2515 (sequence number 45), Plu1649 (sequence number 46), or combinations thereof (having the sequence number of the encoded PVC effector protein in parentheses).

[0119] In one embodiment, the PVC effector is PAU_02009 (sequence number 22), PAU_02010 (sequence number 23), PAU_02095 (sequence number 24), PAU_02096 (sequence number 25), PAU_02097 (sequence number 26), PAU_02098 (sequence number 27), PAU_02230 (sequence number 28), PAU_02805 (sequence number 29), PAU_02806 (sequence number 30), PAU_02807 (sequence number 31), PAU_03332 (sequence number 32), PAU_03337 (sequence number 33), Plu1651 (sequence number 34), Plu16 Encoded by one or more genes selected from 71 (sequence number 35), Plu1672 (sequence number 36), Plu1690 (sequence number 37), Plu1691 (sequence number 38), Plu1712 (sequence number 39), Plu1713 (sequence number 40), Plu1714 (sequence number 41), Plu2400 (sequence number 42), Plu2401 (sequence number 43), Plu2514 (sequence number 44), Plu2515 (sequence number 45), Plu1649 (sequence number 46), or combinations thereof (having the sequence numbers of the encoded PVC effector proteins in parentheses). These gene names correspond to the "locus tags" of PVC effector genes in the Photorhabdus genome sequence accessible via GenBank as described above. PA The T and PAK locus tags were generated by the inventors so that the terms match the PAU and Plu locus tags of publicly available genome sequences.

[0120] Therefore, the PVC effector can be encoded by one or more of the genes listed above.

[0121] In one embodiment, the PVC effector is encoded by one or more genes selected from PAK_02075 (sequence number 4), PAU_02009 (sequence number 22), PAU_02096 (sequence number 25), PAU_02806 (sequence number 30), PAU_03332 (sequence number 32), Plu1651 (sequence number 34), Plu1649 (sequence number 46), or a combination thereof (having the sequence number of the encoded PVC effector in parentheses).

[0122] In a preferred embodiment, the PVC effector is encoded by one or more genes selected from PAU_02806 (sequence number 30), PAU_03332 (sequence number 32), Plu1651 (sequence number 34), Plu1649 (sequence number 46), or a combination thereof (having the sequence number of the encoded PVC effector in parentheses).

[0123] The PVC effector may have a sequence that has at least 80% sequence identity (preferably at least 90% sequence identity; more preferably 100% sequence identity) with an amino acid sequence selected from SEQ ID NOs: 1 to 46. For example, the PVC effector may have a sequence that has at least 80% sequence identity (preferably at least 90% sequence identity; more preferably 100% sequence identity) with an amino acid sequence selected from SEQ ID NOs: 22 to 46.

[0124] The inventors have identified that the leader sequences of gogB1(PAU_02806) and Pnf(PAU_03332) PVC effectors are particularly efficient in packaging (fusion) payloads into PVC needle complexes. In one embodiment, the PVC effector is encoded by PAU_02806 (e.g., having the amino acid sequence of SEQ ID NO: 30). In one embodiment, the PVC effector is encoded by PAU_03332 (e.g., having the amino acid sequence of SEQ ID NO: 32).

[0125] In one embodiment, the PVC effector includes (or essentially consists of) one or more amino acid sequences selected from SEQ ID NOs: 1 to 46 (e.g., SEQ ID NOs: 22 to 46), or combinations thereof. For example, the PVC effector may include (or essentially consist of) sequences selected from SEQ ID NOs: 4, 22, 25, 30, 32, and 46.

[0126] In one embodiment, the PVC effector includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 4. In one embodiment, the PVC effector includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 22. In one embodiment, the PVC effector includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 25. In one embodiment, the PVC effector includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 30. In one embodiment, the PVC effector includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 32. In one embodiment, the PVC effector includes (or essentially consists of) the amino acid sequence of SEQ ID NO: 46.

[0127] The term “package” (used synonymously with the terms “transpackage” and “load”) refers to the direction of a payload by the leader sequence of the present invention (to which the payload is bound / fused) into the interior or end (tip) of an assembled PVC needle complex, such that the PVC needle complex is subsequently configured for delivery (e.g., injection) of the payload into target cells. Thus, the payload can be packaged within the PVC needle complex or at the end (or tip) of the PVC needle complex (e.g., at least a portion of the payload may be outside the PVC needle complex).

[0128] The term “payload” (as used herein synonymously with the term “reaction site”) refers to a molecule packaged within the assembled PVC needle complex or at its tip, and subsequently delivered (e.g., injected) into the (target) cell. In wild-type Photorhabdus, the payload is a PVC effector (more specifically, the effector portion of the PVC effector) encoded (as described above) by a gene downstream (3') of the structural gene of the PVC operon. For example, the effector gene PAU_03337 (PVCpn) encoding the adenylate cyclase effector. See the model PVC operon in Figure 1(D), which has PAU_03332 (listed as PVCpnf21) (e.g., sequence number 32) that codes for a Pnf effector (listed as f17).

[0129] The leader sequences and payloads of the present invention form an "effector fusion" (or simply "fusion") that is "distinguishable from (e.g., wild-type) PVC effectors (e.g., polypeptides encoded by one of the genes outlined in Table 1)." For example, the effector fusion may be a chimera formed from a leader sequence from a first PVC effector fused to a second (different) PVC effector (the effector portion thereof) (preferably amino acids 51 to the C-terminal amino acids of the second PVC effector), where the first and second PVC effectors are different. The effector fusion may be a chimera comprising (or essentially consisting of) the leader sequence described herein fused to a non-PVC effector polypeptide. The effector fusion may be a chimera comprising (or essentially consisting of) the leader sequence described herein fused to a non-Photorhabdus polypeptide. The effector fusion may be a leader sequence-nucleic acid fusion (preferably a conjugate) comprising the leader sequence described herein fused to a nucleic acid.

[0130] Effector fusions are not limited to fusion complexes containing a leader sequence fused to a toxic payload (for example, a leader could be fused to a therapeutic payload). Therefore, as used in the context of “effector fusion,” the term “effector” means the payload packaged within the PVC needle complex (which could provide various effects, e.g., toxicogenicity and / or therapeutic effects). Thus, the term “effector fusion” can be used interchangeably with the term “fusion” herein.

[0131] The term "effector fusion" can be used synonymously with the terms "leader sequence-payload fusion" and / or "leader sequence-payload complex."

[0132] Alternatively, the payload can be distinguished from the PVC effector protein (for example, from amino acid 51 to the C-terminal amino acid of the PVC effector). For example, the payload may be a polypeptide or nucleic acid not found in wild-type Photorhabdus bacteria.

[0133] Analysis of the size (e.g., polypeptide length) and structure of various natural PVC effector payloads encoded by the genus Photorhabdus demonstrates the existence of a wide range of different PVC effector lengths and structures, demonstrating that the applicability of the PVC needle composite delivery system of the present invention is not limited by the size or characteristics of the target payload. In summary, it is supported that PVC needle composites can be used as versatile and multifunctional delivery vehicles without requiring specific secondary structures, biophysical properties, or payload lengths.

[0134] The payload may be one or more selected from polypeptides (e.g., polypeptide payloads), nucleic acids (e.g., nucleic acid payloads), or combinations thereof. In a preferred embodiment, the payload is a polypeptide.

[0135] Examples of polypeptide payloads include antibodies (e.g., anti-MDM antibodies), nanobodies, peptide vaccines (e.g., tyrosinase-related protein 2 (TRP2) peptide vaccines), nuclear factor-κB inhibitors, T3SS payloads (e.g., T3SS payloads that inhibit the NF-κB and / or MAPK pathway), anti-apoptotic peptides (e.g., BH4), and nicotinamide adenine dinucleotide quinone internal oxidoreductase (Ndi1). Examples include PHOX complex subunits, myotubularin, nucleic acid (preferably DNA) modifying enzymes, or combinations thereof. Suitable examples of nucleic acid modifying enzymes include recombinases (e.g., Cre recombinase), transposases, Cas enzymes (e.g., Cas9), and / or Mad7 (preferably Mad7, more preferably Cre recombinase). The payload may be, for example, tBid (SEQ ID NO: 109) and / or BaxBH3 peptides (aa59-73) (SEQ ID NO: 111).

[0136] Any polypeptide possessing enzymatic activity can be a payload.

[0137] Nucleic acid payloads can be conjugated / crosslinked to the leader sequence of the present invention. For example, nucleic acids can be crosslinked to the leader sequence using copper-free click chemistry (e.g., strain-enhanced alkyne azide cycloaddition (SPAAC)). Examples of nucleic acid payloads include primers, mRNA, nucleic acid analogs, aptamers, small interfering RNA (siRNA), microRNA therapeutic inhibitors (anti-miRs), microRNA therapeutic mimes (pro-miRs), long non-coding RNA modulators, single guide RNAs (sgRNAs), or combinations thereof.

[0138] The leader sequence can be fused directly or indirectly (e.g., by a spacer) to the payload. The leader sequence can be fused to the payload covalently or non-covalently. In a preferred embodiment, the leader sequence is covalently fused to the payload. For example, the fusion / effector fusion may be a (recombinant) fusion protein containing (or essentially consisting of) a PVC effector leader sequence fused to the (polypeptide) payload.

[0139] Another aspect of the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the PVC effector reader sequence of the present invention. Another aspect of the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the effector fusion (e.g., fusion) of the present invention, and optionally a nucleotide sequence encoding the PVC needle complex.

[0140] Another aspect of the present invention provides an expression vector comprising a nucleic acid (preferably isolated nucleic acid) comprising a nucleotide sequence encoding the PVC effector reader sequence of the present invention. Another aspect of the present invention provides an expression vector comprising a nucleic acid (preferably isolated nucleic acid) comprising a nucleotide sequence encoding the effector fusion (e.g., fusion) of the present invention, and optionally a nucleotide sequence encoding the PVC needle complex.

[0141] Another aspect of the present invention provides a host cell comprising an isolated nucleic acid, wherein the isolated nucleic acid comprises a nucleotide sequence encoding the PVC effector reader sequence of the present invention. Another aspect of the present invention provides a host cell comprising an isolated nucleic acid, wherein the isolated nucleic acid comprises a nucleotide sequence encoding the effector fusion (e.g., fusion) of the present invention, and optionally, a nucleotide sequence encoding the PVC needle complex.

[0142] The term "nucleic acid" can be used synonymously with the term "polynucleotide."

[0143] Another aspect of the present invention provides a host cell comprising an expression vector, wherein the expression vector comprises a nucleotide sequence encoding the PVC effector reader sequence of the present invention. Another aspect of the present invention provides a host cell comprising an expression vector, wherein the expression vector comprises a nucleotide sequence encoding the effector fusion (e.g., fusion) of the present invention, and optionally, a nucleotide sequence encoding the PVC needle complex.

[0144] The host cell may be a mammalian cell, an insect cell, a yeast cell, a bacterial cell (e.g., Escherichia coli), or a plant cell. In a preferred embodiment, the host cell is a bacterial cell (preferably Escherichia coli).

[0145] In one embodiment, the host cell is a Photorhabdus cell, which optionally contains a PVC operon operably bound to an inducible promoter (see, e.g., Example 3). The PVC operon may be endogenous to the Photorhabdus cell (e.g., the PVC operon may be PVCu4). Preferably, the Photorhabdus cell may be available from ATCC under accession number ATCC29999.

[0146] The sequences of the present invention (e.g., leader sequences and / or nucleic acid sequences) include sequences isolated from naturally occurring environments, recombinants or cloned (e.g., DNA) isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0147] The leader sequences and / or polynucleotides of the present invention can be prepared by any means known in the art. For example, large quantities of leader sequences and / or polynucleotides can be produced by replication and / or expression in suitable host cells. Natural or synthetic DNA fragments encoding a desired fragment are typically incorporated into recombinant nucleic acid constructs, typically DNA constructs, that can be introduced into and replicated within prokaryotic or eukaryotic cells. DNA constructs are usually suitable for self-replication in single-cell hosts, such as yeast or bacteria, but may also be intended for introduction and integration into the genomes of cultured bacteria, insects, mammals, plants, or other eukaryotic cell systems.

[0148] The leader sequences and / or polynucleotides of the present invention can also be produced by chemical synthesis, for example, by the phosphoramidite method or triester method, and can be carried out on a commercially available automated oligonucleotide synthesizer. Double-stranded (e.g., DNA) fragments can be obtained from single-stranded products by chemical synthesis by synthesizing a complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

[0149] When applied to a leader sequence or nucleic acid sequence, the term “isolated” in the context of the present invention means that a leader sequence and / or polynucleotide sequence has been removed from its native genetic environment and is therefore free from other extraneous or undesirable coding sequences (but may include naturally occurring 5' and 3' untranslated regions, e.g., promoters and termination factors) and is in a form suitable for use in a genetically engineered protein production system. Such an isolated molecule is one that has been separated from its native environment.

[0150] sequence homology The identity percentage can be determined using any of the various sequence alignment methods, including, but not limited to, global, local, and hybrid methods, such as the segment approach. The protocols for determining the identity percentage are standard procedures within the scope of the skills of those skilled in the art. The global method determines the best alignment by aligning the sequence from the beginning to the end of the molecule, adding the scores of individual residual pairs, and imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Quence Alignments by Iterative Refinement as Assessed by Reference to Structural See Alignments, 264(4) J.MoI. Biol. 823-838 (1996). The local method aligns sequences by identifying one or more conserved motifs shared by all input sequences. Non-restrictive methods include, for example, the following: Match-box, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, e.g., CELawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, e.g., Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0151] Therefore, the sequence identity percentage is determined by conventional methods. For example, Altschul See et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, the two amino acid sequences are aligned as follows: As shown (amino acids are indicated by standard single-letter codes), a gap opening penalty of 10, a gap elongation penalty of 1, and Henikoff and Henikoff (the above part The alignment score is optimized using the "blosum62" score matrix from [source].

[0152] The "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore, % identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. The calculation of % sequence identity may also take into account the number of gaps that need to be introduced to optimize the alignment of the two or more sequences, and the length of each gap. Sequence comparison and determination of the percentage of identity between two or more sequences can be performed using a predefined mathematical algorithm, such as BLAST, which is familiar to those skilled in the art. [ka]

[0153] Next, the identity percentage was,

number

[0154] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, including conservative amino acid substitutions (see below) and other substitutions that do not significantly affect polypeptide folding or activity; small deletions, typically of 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, e.g., amino-terminal methionine residues, small linker peptides up to about 20-25 residues, or affinity tags.

[0155] Conservative amino acid substitutions Basic: Arginine, lysine, histidine Acidic: Glutamic acid, aspartic acid Polarity: Glutamine, Asparagine Hydrophobic: Leucine, Isoleucine, Valine Aromatic compounds: Phenylalanine, tryptophan, tyrosine Small molecules: Glycine, Alanine, Serine, Threonine, Methionine

[0156] In addition to 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can be substituted for amino acid residues in the polypeptide of the present invention. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can be substituted for polypeptide amino acid residues. The polypeptide of the present invention may also include amino acid residues that do not exist in nature.

[0157] Amino acids that do not exist in nature include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating amino acid residues that do not exist in nature into proteins are known in the art. For example, an in vitro system can be used to suppress nonsense mutations using chemically aminoacylated suppressor tRNA. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing Escherichia coli (E. coli) S30 extract and commercially available enzymes and other reagents. The proteins are purified by chromatography. For example, Robertson et al. See also al., J.Am.Chem.Soc.113:2722,1991; Ellman et al., Methods Enzymol.202:301,1991; Chung et al., Science 259:806-9,1993; and Chung et al., Proc.Natl.Acad.Sci.USA 90:10145-9,1993). In the second method, translation occurs in Xenopus oocytes. This is carried out by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, *E. coli* cells are cultured in the absence of the native amino acid to be replaced (e.g., phenylalanine) and in the presence of a desired non-native amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-native amino acid is incorporated into the polypeptide in place of its native equivalent. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted into non-natural species through in vitro chemical modification. Chemical modification, when combined with site-directed mutagenesis, can further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0158] A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, amino acids not found in nature, and unnatural amino acids can be substituted for amino acid residues in the polypeptide of the present invention.

[0159] The essential amino acids in the polypeptide of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). The site of biological interaction can also be determined by physical analysis of the structure, as can be determined by techniques such as nuclear magnetic resonance, crystallographic analysis, electron diffraction, or photoaffinity labeling, along with mutations of the putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J.Mol.Biol.224:899-904, 1992; Wlodaver et al., FEBS Lett.309:59-64, 1992. Identity can also be inferred from homology analysis with related components of the polypeptide of the present invention (e.g., translocation or protease components).

[0160] Multiple amino acid substitutions can be prepared and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutagenerated polypeptide to determine the spectrum of acceptable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, International Publication No. 92 / 06204) and region-specific mutagenesis (Derbyshire). Examples include et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0161] Multiple amino acid substitutions can be prepared and tested using known mutagenesis and screening methods, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide, selecting a functional polypeptide, and then sequencing the mutagenerated polypeptide to determine the spectrum of acceptable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent No. 5,223,409; Huse, International Publication No. 92 / 06204) and region-specific mutagenesis (Derbyshire). Examples include et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0162] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this disclosure pertains. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, N ew York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provides to those skilled in the art much of the general knowledge of the terms used in this disclosure. To provide.

[0163] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein may be used in the implementation and testing of embodiments of this disclosure. Numerical ranges include the numbers that define the range. Unless otherwise specified, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation, respectively.

[0164] The headings provided herein are not limitations on any particular aspect or embodiment of the present disclosure.

[0165] In this specification, amino acids are referred to by their full names, three-letter abbreviations, or one-letter abbreviations. The term "protein" as used herein includes proteins, polypeptides, and peptides. The term "amino acid sequence" as used herein is synonymous with the terms "polypeptide" and / or "protein." In some examples, the term "amino acid sequence" is synonymous with the term "peptide." In some examples, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. Conventional one-letter and three-letter codes for amino acid residues may be used in this disclosure and claims, as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). A three-letter code for amino acids. It is also understood that polypeptides can be coded by two or more nucleotide sequences due to the degeneracy of the gene code.

[0166] Other definitions of terms may appear throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that this disclosure is not limited to and is therefore subject to change in the specific embodiments described. It should also be understood that, since the scope of this disclosure is defined solely by the appended claims, the terms used herein are for the purpose of describing specific embodiments only and are not intended to limit them.

[0167] Where a range of values ​​is provided, it is understood that the intermediate values ​​between the upper and lower limits of that range are also disclosed specifically to one-tenth of the lower limit unit unless the context specifically indicates otherwise. Any smaller range between any descriptive value or intermediate value in a descriptive range and any other descriptive value or intermediate value in that descriptive range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from that range, and if a smaller range includes one or both limits, each range is also included in this disclosure according to the limits specifically excluded from the descriptive range. If a descriptive range includes one or both limits, the range excluding either or both of the limits that they include is also included in this disclosure.

[0168] As used herein and in the appended claims, the singular "a" "an" and "the" can refer to multiple objects unless the context explicitly indicates otherwise. It must be noted that, for example, a reference to “effector” includes multiple such effectors, and a reference to one or more effectors and their equivalents known to those skilled in the art includes references to one or more effectors and their equivalents.

[0169] The publications discussed herein are provided only for disclosures prior to the filing date of this application. This specification should not be construed as permission for such publications to constitute prior art with respect to the claims to which they are attached.

[0170] Embodiments of the present invention are described below, by reference only to the following drawings and examples. [Brief explanation of the drawing]

[0171] Brief explanation of the drawing [Figure 1A] This shows a schematic representation of one PVC operon layout (a gene cluster located within a variable region of the origin genome) encoding the PVC needle complex. [Figure 1B] Schematic representation of Class I, II, and III PVC operon layouts. Homologous subunit types between these classes are indicated by similar shading (grayscale). [Figure 1C] Description of the assembled PVC needle complex. Correlate the gene clusters in (A) with the location of the encoded protein in the structure in (C) using the numbering shown (for example, the cap cluster "16" in A is indicated as "16" in the leftmost cap region of (B)). [Figure 1D] A map of the model class I PaATCC43949PVCpnf operon (e.g., encoded by SEQ ID NO: 93) showing two effector genes (Rhs-like adenylate cyclase and PAU_03332) in the payload region. [Figure 2] This outlines the cloning procedure for preparing PVC needle complex expression plasmids based on overlapping PCR. The PCR fragment (containing the overlapping region) is provided from a template gDNA of P. asymbiotica ATCC43949 (available from ATCC under accession number ATCC43949) along with the relevant primers that target the PVC operon. [Figure 3A] The image shows a transmission electron microscope image of an (in vitro) sample of a PVC needle complex (prepared, for example, from cells containing the expression vector described above). The PVC needle complex assembles into a distinct "nanosyringe" structure, consistent with its role as a contractile structure. [Figure 3B]This shows a 3D rendering model of a PVC needle composite derived from a high-resolution single-particle cryoEM tomography structure. [Figure 4A] Transmission electron micrographs of PVC needle complexes containing the Pnf payload after immunogold staining with anti-Pnf (Immunogold) antibody are shown, confirming the association of the Pnf-payload toxin with the PVC needle complex (referred to as PVCpnf). The PVCpnf needle complex was prepared from the supernatant of an Escherichia coli (E. coli) cosmid clone encoding the PVCpnf operon. The payload toxin protein was localized using an antipeptide antibody against the Pnf (TGQKPGNNEWKTGR, SEQ ID NO: 96) epitope. The Pnf toxin could only be detected at the terminals of the disrupted or contracted needle complex, providing evidence that the toxin is contained within the complex (arrow). [Figure 4B] Western blot analysis confirms that, if the PVC needle complex is chemically or physically disrupted, the Pnf protein (toxin) can only be detected using an anti-peptide antibody. These preparations were taken from the PaATCC43949 supernatant. The inability to detect Pnf in the clarified supernatant confirms that all proteins are associated with the PVC needle complex concentrated preparation. Lanes 1+5: sonicated samples, 2+6: treated with 1M NaCl, 3+7: treated with 1% SDS, 4+8: treated with 1M urea. Note that the PVC needle complex appears to be stable in 1M NaCl. [Figure 5A] This image shows cryo-SEM images of ex vivo hematopoietic cells (insect macrophage / neutrophil equivalents) from 5th instar tobacco moths (Manduca sexta) injected with a natural concentrate preparation of the PaATCC43949PVCpnf needle complex (nanosyringe), heterologously produced by an E. coli cosmid clone. Note the abundant linear structures corresponding to the PVC needle complex (nanosyringe) (small arrow) and membrane ruffling effect (large arrow), which are consistent with the mechanism of action of the Pnf payload toxin absent from the control treatment. Scale bar = 50 μm. 25 kV; magnification 40 K. [Figure 5B]This image shows cryo-SEM images of ex vivo hematopoietic cells (insect macrophages / neutrophil equivalents) from fifth-instar tobacco moths (Manduca sexta) injected with a heat-inactivated and concentrated preparation of PaATCC43949PVCpnf needle complex (nanosyringe) heterologously produced by an E. coli cosmid clone. Scale bar = 50 μm. 25 kV; magnification 50 K. [Figure 6A] We present experimental results demonstrating that the (toxic) cellular phenotype following contact with the PVC needle complex is due to intracellular toxin delivery. When the Pnf-loaded PVC needle complex was injected into insects (Galleria mellonella insect larvae), it showed potent activity within 15 minutes for a given dose (described in the examples). Note that the mortality / morbidity was typically associated with a "melanization" immune response in those dead / dying insects. [Figure 6B] Experimental results demonstrate that the (toxic) cellular phenotype after contact with the PVC needle complex is due to intracellular toxin delivery. Controls and denatured (boiling-mediated) Pnf-loaded PVC needle complexes injected into animals showed no activity. [Figure 6C] Experimental results demonstrating that the (toxic) cellular phenotype after contact with the PVC needle complex is due to intracellular toxin delivery are shown. Purified Pnf (payload) lacking the PVC needle complex (i.e., Pnf not packaged in the complex) showed no activity in either animals (left) or HeLa cell lines (right). [Figure 6D] We present experimental results demonstrating that the (toxic) cellular phenotype after contact with the PVC needle complex is due to intracellular toxin delivery. Pnf (payload) delivered into the cytoplasm of HeLa cells via a protein-containing "BioPorter" liposome preparation exhibited potent activity / toxicity, as evidenced by multinucleation within the cells. [Figure 6E]We present experimental results demonstrating that the (toxic) cellular phenotype following contact with the PVC needle complex is due to intracellular toxin delivery. Pnf (payload), delivered into the cytoplasm of HeLa cells via intracellular expression after translocation with an appropriate plasmid, exhibited potent activity / toxicity, as evidenced by multinucleation within the cells. [Figure 6F] This document presents experimental results demonstrating that the (toxic) cellular phenotype after contact with the PVC needle complex is due to intracellular toxin delivery. The effect of PVCpnf+Pnf on the respiration rate of THP1-derived human macrophages, as measured by a resazurin plate reader assay, is shown. Note that heat-denatured and empty PVCpnf nanosyringes did not show potent adverse effects. These same samples were examined by injection into Galleria larvae. PVCpnf+Pnf samples showed a mortality rate of over 50% within minutes (darkened larvae in the two lower panels), while all insects injected with heat-denatured and empty PVCpnf remained healthy (non-darkened larvae in the two upper panels). [Figure 7A] This study presents the (in silico) predicted secondary structures of a series of endogenous payloads (toxins) that associate with various PVC operons, demonstrating a wide variety of structural types. [Figure 7B] Amino acid lengths of various payloads (toxins) plotted against predicted isoelectric points. [Figure 8A] This document provides evidence that the leader sequence of the present invention (e.g., 50 amino acids) is necessary and sufficient for the (trans)packaging of payload proteins / peptides into PVC needle complexes (nanosyringes) expressed in the genus Photorhabdus. 1-6: Schematic map of chimeric effector protein expression constructs (trans-expressed in arabinose-inducible pBAD30 vectors) including those expressing Pnf and non-natural cre recombinase and Myc tags. The C-terminal Myc tag epitope is indicated as a black arrow. [Figure 8B]This invention provides evidence that the leader sequence (e.g., 50 amino acids) is necessary and sufficient for the (trans)packaging of payload proteins / peptides into PVC needle complexes (nanosyringes) expressed in the genus Photorhabdus. Western blot using anti-Myc mouse antibody. Samples are from purified PVC(u4) needle complexes (nanosyringes) overexpressed from chromosomally modified P. luminescens TT01 possessing transpackaging expression constructs 1-6 shown in (A). A blank pBAD30 plasmid was used as a negative control and showed no signal. Arrows indicate the exact band size for the predicted product. [Figure 9] The alignment of the leader sequences is shown, demonstrating the existence of a chemical compositional consensus between the leader sequences based on amino acid properties. More specifically, the leader sequences contain similar charge patterns of two negatively charged regions followed by positively charged regions [-ve][+ve][-ve][+ve], one after the other. [Figure 10A]Western blot analysis of PVC needle complexes and payloads from particulate preparations is shown (cesium chloride gradient and monolithic FPLC preparations as described in Materials and Methods). [1] (pBADPVCpnf, PVC16 in the nanosyringe is FLAG-tagged, providing PVC16::FLAG detectable with anti-FLAG Ab) shows a signal from the tagged cap protein of "PVCPnf" (PVC needle complex with Pnf payload), confirming the presence of the PVC needle complex in the purified fraction. [2] (pBADPVCpnf+Cre::Myc, detectable with anti-Myc Ab, Cre has the N-terminal fusion of the Pnf reader, e.g., SEQ ID NO: 78) shows a signal from the large amount of packaged Myc-tagged payload protein in the same sample as in (1), confirming the presence of the Cre payload in the purified PVC needle complex (nanosyringe). [3] (PVCU4+Cre::Myc, detectable with anti-Myc Ab, Cre has a Pnf reader, e.g., the N-terminal fusion of SEQ ID NO: 78) different PVC needle composite chassis ("PVCU4") purified products were probed for Myc-tagged Cre to reveal the corresponding band for packaged (packaged Myc-tagged Cre). This is highlighted in the blot for clarity. [Figure 10B] Transmission electron micrographs of the PVC needle complexes show that both wild-type (with Pnf payload) and atypical (non-natural) recombinase (Cre) payload PVC needle complexes do not affect the morphology of the PVC needle complexes in any of the chassis examined, ensuring that they are not assembled abnormally. [Figure 10C](A) provides additional / supplementary data. More specifically, (C) provides further evidence via Western blot analysis of the (trans)package of Cre recombinase into purified PVCpnf expressed in Escherichia coli (E. coli). The Western blot demonstrates the detection of a considerably large Cre payload (using anti-Myc tagged antibody) for a given amount of anti-FLAG antibody Western signal (a specific probe for nanosyringes due to PVC16::FLAG incorporation). Numbers indicate 2-fold dilutions. Note that upon dilution, the anti-FLAG signal from the nanosyringe is lost, while the payload remains strong in most lanes. CsCl indicates purification by cesium chloride density gradient centrifugation. "Mon" indicates further anion exchange of the sample via a "monolithic" column. "Post-elution," "Interphase," and "Sub-Interph." indicate the liquid fraction from which signals are detected from the purification process. [Figure 10D] Western blot analysis of Cre transpackaged in PVCpnf in Escherichia coli (E. coli). The payload was probed for its incorporated "Myc" tag (C-terminal fusion) after purification of the nanosyringe-payload complex. Western blot analysis of the particle preparations confirms that all four leaders were able to efficiently transpackage the exogenous Cre enzyme. [Figure 10E] A phylogenetic tree demonstrating that the exemplified leader sequences are well distributed throughout, and therefore the diversity is maximally continuous or close to it (see Example 4.2). [Figure 11]Western blot analysis of PVC needle complexes expressed with and without (1) co-expression of (Myc-tagged) Pnf from separate plasmids, simultaneously probed with anti-FLAG and anti-Myc antibodies, is shown. In lane 1, the PVC needle complex (nanosyringe) was expressed and purified in E. coli (E. coli) without the presence of a "payload plasmid" (an expression plasmid encoding a payload protein bound to the leader sequence). This yields a band corresponding only to the FLAG tag present on the syringe (PVC needle complex) itself. For lane 2, the same approach was taken, but a culture containing a (separate) plasmid carrying the tagged payload (Myc-Pnf) was also used. Bands corresponding to the FLAG and Myc tags can be seen, confirming the presence of the Pnf payload (the four lanes in 1 and 2 are simply different purified fractions from a cesium chloride gradient). [Figure 12] This shows a Western blot analysis of a transpack experiment in a P. luminescens TT01 PVCu4 overexpressing strain. The results demonstrate the transpackaging of myc-tagged Pvc17 (Plu1651whole::Myc). [Figure 13A]Further Western blot analysis of transpackaging experiments (described in the examples) in the P. luminescens TT01 PVCunit4 overexpressing strain is shown. The results demonstrate the need for transpackaging of Myc-tagged Pvc17 (Plu1651::Myc) and Myc-tagged Pvc17 alone, using a Pnf reader (PAU_03332 reader), as well as the reader itself. (A) Lane 1 shows the package of the leader (PAU_03332::Myc) fused to the Myc tag; Lane 3 shows the missing package when the leader sequence is absent (Myc alone is not packaged); Lane 4 shows the missing package of HvnA (natural effector) when the leader sequence is absent; Lane 6 shows the package of Myc-tagged PAU_03332::Plu1649, i.e., a chimeric package of the leader from PAU_03332 (i.e., amino acids 1-50 of PAU_03332) and the effector from Plu1649 (i.e., amino acids 51-C terminus). The high-intensity bands in lanes 1 and 6 demonstrate that the Pnf(PAU_03332) leader is particularly effective for packaging the payload. [Figure 13B] Further Western blot analysis of transpackaging experiments (described in the examples) in the P. luminescens TT01 PVCunit4 overexpressing strain is shown. The results demonstrate the need for transpackaging of Myc-tagged Pvc17 (Plu1651::Myc) and Myc-tagged Pvc17 alone, as well as its leader, using a Pnf reader (PAU_03332 reader). (B) Lane 1 shows the packaging of Plu1651 with a C-terminal Myc tag using an anti-Myc antibody Western blot. [Figure 14]Further Western blot analysis is shown demonstrating extremely high levels of transpackaging of Myc-tagged Pnf (PAU_03332::Myc) using the PAU_02806 (GogB) reader (second lane, excluding ladder lane). The first lane demonstrates the use of the Plu1649 reader (Myc-tagged Plu1649::PAU_03332) for packaging the PAU_03332 effector. The band is considered weak due to the relative intensity of the band in the second lane. The experiment involved filter-sterilized 50 mL of culture containing 8 M final concentration urea added to degrade PVC. Samples were collected from 10 mL of supernatant. [Figure 15] Further Western blot analysis demonstrates the transpackaging of Plu1651 (pvc17) with the C-terminal Myc tag shown in Figure 13 into PVCunit4 expressed from the genus Photorhabdus. Raw represents particulate preparation from supernatant, while Be, B2, and IP represent different "cuts" from cesium chloride gradient purification. [Figure 16A] This provides a schematic explanation of the mechanism of action of Cre in the mouse organoid experiment (Example 6) and how the positive control (TAM) promotes Cre activation. White arrows indicate the localization of cells expressing the tdTom fluorescent reporter gene. [Figure 16B] Demonstration of delivery of active transpackaged Cre recombinase into mouse bile duct organoids by purified PVCpnf expressed from Escherichia coli (E. coli). White circles indicate the localization of groups of cells expressing the fluorescent reporter gene. The upper image shows a direct grayscale conversion of the image obtained via light microscopy observation. The lower image shows a corresponding image of positive cells with enhanced false color, which is provided simply to aid in identifying the difference between affected cells and the surrounding unaffected cells within the former grayscale conversion. [Figure 17]Dot blot analysis of nanosyringe expression with and without the payload (Cas9-like protein MAD7) is shown. Some leaky expression of IPTG-inducible MAD7 is observed pre-induction (T1), as is common in this expression system. As predicted, no Myc signal is present from PVC-only samples at any point, and the MAD7 signal grows throughout the expression cycle of approximately 24 hours. The robust Myc signal is maintained after purification by ultracentrifugation, as described elsewhere, indicating that the protein is incorporated into the nanosyringe chassis system. The FLAG signal is robust in the MAD7 sample, occurring post-induction as predicted and persisting after purification, because this promoter system reduced leaky expression. It can be concluded that nanosyringes and MAD7 are compatible with each other in terms of expression, and that the largest protein MAD7 examined to date can be packaged in the nanosyringe system. [Figure 18] Western dot blot analysis (7 and 8) confirms the transpackaging of the pro-apoptotic tBid protein domain and BaxBH3 peptide (both having the leader sequence of SEQ ID NO: 78 fused to the N-terminus) into purified PVCpnf expressed from Escherichia coli (E. coli). Nanosyringes containing the cognitive toxin "Pnf" are shown as purified by two different methods (5 and 6) as positive controls. The blots in the lower row of the panel represent the same examples as 7 and 8 in the panel above. These blots were prepared from another purification of the same construct to demonstrate the reproducibility of the purification. This experiment demonstrated that the "tBid protein domain and BaxBH3 peptide" pack sample (nanosyringe) used in the apoptosis delivery system of Example 9 can be well prepared. [Figure 19A]The image shows TUNEL staining microscopy analysis of cells exposed to packaged nanosyringes for only 20 minutes. The first bar (left) = DNase I-treated cells (+ control); second bar = no DNase I treatment or nanosyringe treatment (- control); third bar = cells exposed to nanosyringes packaged with tBid (via the leader sequence of SEQ ID NO: 78 fused to the N-terminus); fourth bar (right) = cells exposed to nanosyringes packaged with the Bax_BH3 domain (via the leader sequence of SEQ ID NO: 78 fused to the N-terminus). [Figure 19B] Representative micrographs from Example 9 showing TUNEL staining of PBMCs after treatment with nanosyringes and controls. PBMCs were treated with tBID, Bax-loaded nanosyringes, and positive (DNase I-treated cells) and negative (untreated DNase I) controls at room temperature for 20 minutes, followed by TUNEL staining to determine the apoptotic response. In the original (non-grayscale) micrographs: Cells negative for the apoptotic response show blue or light brown staining. Blue staining (methyl green) or light brown staining indicates healthy cells where apoptotic signals are absent. Dark brown staining indicates cells undergoing apoptosis. [Examples]

[0172] Examples Materials and methods cloning Plasmids encoding the PVC needle complex were prepared using standard molecular techniques known in the art. In short, P. asymbiotica ) Genomic DNA from ATCC43949 (from ATCC, accession number ATCC43949) Multiple (e.g., four) overlapping regions of the PVC operon were amplified using PCR (with appropriate primers) (available). The entire operon was prepared using overlap / extension PCR and fused into an appropriate expression vector as detailed in Figure 1 (overlapping PCR was also used here) (SEQ ID NOs: 101-101). (Use primer 06).

[0173] In short, four overlapping PVC fragments were prepared to cover a PVC operon (e.g., the one in SEQ ID NO: 93) using primers for SEQ ID NO: 101(F1) and 105(R1); SEQ ID NO: 102(F2) and 106(R2); SEQ ID NO: 103(F3) and 107(R3); and SEQ ID NO: 104(F4) and 108(R4), respectively. These were then cleaved at the required insertion sites in the target cloning vector. These five DNA fragments were then assembled by overlapping PCR (using primers for SEQ ID NO: 101 and 108), and the resulting fragments were ligated into the cloning vector. The product was transformed into laboratory Escherichia coli (E. coli) and recovered by vector marker selection (e.g., due to ampicillin resistance).

[0174] The operon is typically operably conjugated to an inducible promoter (e.g., arabinose-inducible and / or IPTG-inducible) as is known in the art. This is commonly done with pBAD family plasmids (arabinose-inducible) (Invitrogen, catalog number: V43001) and pVTRa (IPTG-inducible) (Biomedal, SL This is achieved by cloning into a vector (however, any combination of compatible expression vector systems should be sufficient).

[0175] The PVC needle complex can be expressed independently of the payload (toxin), and vice versa. Separate expression vectors (e.g., those with different inducible promoters) may contain the PVC needle complex and the payload, respectively.

[0176] Expression of PVC needle complex in Escherichia coli (e.g., laboratory-scale expression) / purification A typical process for purifying PVC needle complexes from a 1L culture of an E. coli (transformed with an appropriate expression vector / cosmid) is as follows: 1. Prepare an overnight culture of bacteria (transformed with the PVC needle complex expression vector) by picking colonies from the plate and inoculating them into 100 ml of LB medium. Grow the culture at 37°C with shaking. a. Typically, supplementing the culture medium with 0.2% d-glucose can help suppress gene constructs for optimal cell health. b. Supplement the culture medium with the appropriate antibiotics for maintaining the expression (PVC needle complex) vector. If a payload vector is also being used, supplement the medium with the appropriate antibiotics for that vector as well. 2. The following day, inoculate a 1L flask with a 1:100 ratio dilution from the overnight culture. The culture medium for the 1L flask is the same as the overnight medium, but typically does not contain glucose. 3. The culture is grown to approximately the mid- or late-stage exponential stage (OD600nm with an OD of approximately 0.8), and the plasmid is induced at that point. a. For PVC needle complex (nanosyringe) plasmids, expression is typically induced by adding 0.2% arabinose. For payload plasmids (payloads, e.g., plasmids encoding Pnf), the IPTG concentration can typically be optimized based on protein, with a typical number of starts of 0.1 mM being preferred. 4. After induction, return the culture to the incubator and incubate at 18°C ​​until the next day. 5. Harvest the culture by centrifugation in a suitable centrifuge / bottle / rotor at 5000 × g for 30 minutes. 6. Next, the cell pellet is dissolved to release the PVC needle complex (nanosyringe). a. The following dissolution methods can be used: (i) Overnight lysozyme incubation. (ii) Ultrasonic treatment with a needle sonicator (with or without initial lysozyme treatment). (iii) Cell disruption device / homogenizer. 7. Optionally, DNases and protease inhibitors may be added to the lysate. 8. Remove the cell debris by centrifugation at 50,000 × g at 4°C for 20 minutes in a high-speed centrifuge. The lysate is concentrated and its volume reduced by passing it through a 9-100,000 kDa MWCO centrifugation column, and small proteins are removed. Once the volume is reduced to a manageable level, it is centrifuged several times, and the retention solution is replaced with an appropriate sample buffer, e.g., TM (20 mM Tris-HCl, 8 mM MgCl2, pH 7.4) and dialyzed.

[0177] The subsequent purification process via a cesium chloride density gradient is as follows: 1. Prepare a CsCl density solution as follows: (a) 1.7 g / mL CsCl in H2O; (B) 1.5 g / mL CsCl in H2O; (C) 1.45 g / mL CsCl in H2O 2. Next, set the gradient (from the bottom to the top of the tube) in the ultracentrifuge tube, for example: (1) (bottom of tube) - 2 mL density, 1.7 CsCl; (2) - 3 mL density, 1.5 CsCl; (3) - 3 mL density, 1.45 CsCl; (4) (top of tube) - sample in TM buffer. Preferably, each density is carefully applied to the side of the tube so that the boundary does not blend with the pre-density layer. 3. Next, the equilibration tube is subjected to ultracentrifugation at 35,000 RPM for 2 hours at 4°C in an SW40Ti swing bucket rotor equivalent to 155,000 × g. 4. The precise gradient fraction is the area directly above the appearing "bluish-white" halo. Extract the fraction by puncturing the tube with a syringe and needle. 5. A PVC needle composite of good purity can thus be obtained and stored in a buffer at 4°C. Preferably, it is dialyzed again in TM buffer to remove CsCl.

[0178] Following or instead of CsCl gradient purification, PVC can be extracted via monolithic anion exchange chromatography as follows (note that all steps can be performed manually using a peristaltic pump or syringe, or via F / HPLC): 1. Unless already done, dialyze the sample extract into a bound mobile phase (typically TM buffer) containing a low concentration of salt (20 mM NaCl). 2. Equilibrate the column according to the manufacturer's guidelines, in short: a. At least 5 column volumes (CV) of dH2O; b. A binding buffer (TM, with a low concentration of salt) of at least 5 CV; c. At least 5 CV of elution buffer (high-concentration salt, TM with >= 1 M NaCl); d. Add at least 10 CV of binding buffer again. 3. Apply the sample to the column at a low flow rate (1-2 mL / min). 4. Wash the column with TM buffer containing up to 200 mM NaCl. Elute with 5.1M NaCl-containing TM buffer (or, if using an FPLC instrument, use gradient elution). 6. The PVC needle complex is present in the elution fraction. When using a fraction collector, subsequent SDS-PAGE or an equivalent may be required to accurately identify the fraction.

[0179] The column (for example, the one in step 2) is a CIMmultus® quaternary amine anion exchanger. It was a column (BIA Separations doo). For example, CIMmultus® QA-1, a monolithic column with a channel size of 1.3 μm and a column volume of 1 mL.

[0180] Alternatively, a DEAE (weak anion exchanger) column can be used.

[0181] Alternatively, for use with Photorhabdus expression systems, the PVC needle complex can be purified from the cell pellet and / or the supernatant, with the following additions / modifications: 1. After cell harvesting according to the standard protocol above, transfer the supernatant to a Pyrex bottle and proceed as desired. Optionally, the sample can be enriched via a 100,000 MWCO column if necessary. a. DNase (0.25 U / mL) and protease inhibitors may be optionally added. 2. Add NaCl to a final concentration of 0.5M, and also add 80g / L of PEG6000. Mix the solution overnight at 4°C. 3. The solution is centrifuged at 8000 × g at 4°C for 30 minutes to pelletize the PEG6000. 4. Resuspend the pellet in a small volume (approximately 5 mL) of TM buffer (or a similar solution) and incubate at room temperature for 2 hours while shaking. 5. The material is pelletized by centrifugation at 5.13,000 × g for 10 minutes, and the supernatant is collected in a new tube. Continue with the selected purification method.

[0182] Other methods for purifying PVC needle complexes are described elsewhere, for example, by reference to Yang et al. (J Bacteriol. 2006 Mar;188(6):2254-2261), which are incorporated herein by reference. .

[0183] P. luminescens TT01 PVCunit4 (gene plu16) Construction of arabinose-inducible overexpression strains for the chassis encoded by 67~plu1652 Using chromosome recombination, a selective PVC (operon) (for example, an operon encoding the PVCunit4 needle complex was used here) was placed under the control of an arabinose-inducible transcription promoter to prepare a Photorhabdus strain that overexpressed the PVC needle complex. The recombinant strain was then genetically transformed with an effector expression plasmid (e.g., based on the arabinose-inducible expression vector pBAD30) to promote PVC needle complex overexpression, PVC effector expression, PVC effector transpackage secretion, and complete complex secretion solely through the addition of arabinose sugars.

[0184] Recombinant Photorhabdus PVC overexpression strain construction P. luminescens strain DJC (also known as strain TT0) as a template 1) The promoter region of PVCunit4 was amplified using primers PVCpromF(5'-TATCATATGTCTACAACTCCAGAACAAATTGCTG-3', SEQ ID NO: 97) and PVCpromR(5'-ATCTCTAGAACAGATATTCCAGCCAGC-3', SEQ ID NO: 98) with genomic DNA from 1). A suitable P. luminescens strain is available from ATCC under accession number ATCC29999. The PCR product was digested with NdeI and XbaI, and suicide vector pC was generated using Escherichia coli (E. coli) DH5α λ-pir (Biomedal SL) as a carrier strain. The plasmid was introduced by ligation into EP (ThermoFisher, catalog number: V04450). The resulting plasmid was transferred to the *E. coli* donor strain S17.1λ-pir (Biomedal SL) for conjugation into the genus *Photorhabdus*. Briefly described below. Belt, P. luminescens DJC donor strain and rifampicin-resistant (RifR) Overnight cultures of the isolated strain were diluted in LB supplemented with 10 mM MgSO4 and grown to the mid-phase exponential stage (OD600 approximately 0.5). Then, 3 ml of each culture was harvested, washed twice, and diluted in 100 μl LB supplemented with 10 mM MgSO4. Resuspended. 80 μl of P. luminescens DJC RifR was added. μl of donor bacteria were mixed (resulting in a 4:1 recipient-donor ratio) and placed in the center of an LB agar plate supplemented with 0.1% pyruvate and 10 mM MgSO4. The plate was incubated overnight at 30°C, and the resulting growth was harvested in 1.5 ml of LB agar. Aliquots were placed on plates containing rifampicin (50 μg / ml) and chloramphenicol (25 μg / ml) to select transconjugans, and the plates were incubated at 30°C for 3 days. Potential transconjugans were re-streaked and confirmed by PCR using primers ParaINF (5'-GGCGTCACACTTTGCTATG-3', SEQ ID NO: 99) and tPVCpR (5'-TCGGTGGCAGTAAATTGTCC-3', SEQ ID NO: 100).

[0185] Overexpression and purification of PVC needle complex from the genus Photorhabdus. P. luminescens DJC PVCunit4::pCEP overnight cultivation The culture was diluted in 2 × 250 ml LB containers supplemented with chloramphenicol (25 μg / ml) and incubated at 180 rpm and 28°C. After 2-3 hours, arabinose (0.2%) was added, and the culture was returned to the incubator for a further 26 hours. The cells were pelletized by centrifugation (7000 g, 30 minutes), and the supernatant was collected. Any extracellular DNA was degraded by adding DNase I at a concentration of 0.25 U / ml to the supernatant. After incubation at room temperature for 30 minutes, polyethylene glycol 8000 (8%) and NaCl (0.5 M) were added to precipitate the proteins. The supernatant was incubated overnight at 4°C with stirring. The precipitated proteins were then collected by centrifugation at 8000 g at 4°C for 30 minutes. The pellet was resuspended in 8 ml of TM buffer (20 mM Tris-HCl, 20 mM MgCl2, pH 7.4) and incubated at room temperature for 2 hours with gentle shaking. Any residue was removed by centrifugation at 13000 g for 10 minutes, and the supernatant containing the PVC needle complex was applied to a CsCl density gradient and centrifuged at 35000 rpm in a Beckman Coulter Optima L-90K or XPN-80K ultracentrifuge. The mixture was centrifuged for 2 hours. A CsCl density gradient was created by layering TM buffer containing CsCl from the bottom of the tube at ρ = 1.7 (2 ml), 1.5 (3 ml), and 1.45 (3 ml), respectively. The fraction containing the PVC needle complex was collected, CsCl was removed using an Ultracel-100K instrument (Amicon), and the buffer was subjected to TMS (20 The solution was replaced with mM Tris-HCl, 8 mM MgSO4, pH 7.4. CIMmultus (trademark) The PVC needle complex was further purified using a quaternary amine 2 μm pore anion exchange column (BIA separations). The column was washed with TMS buffer containing 200 mM NaCl, and the PVC needle complex was eluted in TMS containing 1 M NaCl. NaCl was removed by buffer exchange using an Ultracel-100K instrument, and then tested for final purification. The sample was applied to a CIMmultus™ DEAE 2μm pore column (BIA separations). The column was washed in TMS containing 200 mM NaCl, and the sample was eluted in TMS containing 500 mM NaCl.

[0186] This can be performed with or without cell lysis (to release the PVC needle complex) (for example, since the PVC needle complex is thought to be secreted from viable cells, it can be recovered in the supernatant).

[0187] Transmission electron microscope observation Pioloform coating 3 coated with a fine carbon layer for transmission electron microscopy (TEM) observation. A 00 mesh copper grid was used as the substrate for protein fractionation. The preferred aqueous negative staining agent was 3% methylamine tungstate. The coated grid was exposed to UV light for 16 hours immediately before use to ensure proper wetting of the substrate. A 10 μl droplet was applied to the TEM grid, and the protein was allowed to settle for 5 minutes. The liquid was absorbed from the edge of the grid with filter paper and immediately replaced with 10 μl of filtration-negative staining agent. The droplet was partially removed with filter paper, and the grid was etched. The samples were completely air-dried and then observed using a JEOL1200EX transmission electron microscope (JEOL, Tokyo, Japan) at 80kV.

[0188] BioPORTER assay and actin stress fiber analysis For the BioPORTER assay (Genlantis), use 80 μl of purified wild-type and mutant Pnf protein (500 μg ml-1), or PBS as a negative control, in one BioPORTER assay. The sample was added to a tube (Genlantis) and resuspended in 920 μl of DMEM. The sample was then divided into 6 layers. The sample was added to HeLa cells grown in a refrigerated plate and incubated for 4 hours. BioPORTER / The protein or PBS mix was replaced with fresh complete medium, and the cells were incubated for 20 - 48 hours. To visualize cell morphology and the actin cytoskeleton, the cells were fixed in 4% PBS-formaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100, and stained with tetramethylrhodamine B isothiocyanate (TRITC) phalloidin (Sigma) and D API dihydrochloride (Sigma). Images were acquired with an LSM510 confocal microscope (Leica).

[0189] Example 1 Cloning and expression of the PVC needle complex The inventors successfully excised (cloned) the required expression genes from the host bacterium, Photorhabdus (e.g., those contained within SEQ ID NO: 93, SEQ ID NO: 94, and / or SEQ ID NO: 95), and devised a reliable and scalable expression system in laboratory Escherichia coli (E. coli) as described above. Trans-expression on separate plasmids was demonstrated to enable the payload (e.g., Pnf) to be incorporated into the syringe, creating a multi-plasmid (modular) platform.

[0190] After purification from E. coli, electron microscopy analysis demonstrated that the purified PVC needle complex retained the exact "nanosyringe" structure (see Figure 3). Furthermore, the PVC needle complex remained precisely associated with the payload (e.g., Pnf) after purification (see Figure 4), demonstrating that the inventors had successfully prepared a PVC needle complex (nanosyringe) with the exact structure for payload delivery to cells.

[0191] Furthermore, electron microscopy analysis demonstrated that the purified complex was appropriately localized on the cell surface of the cells, and that the PVC needle complex with the Pnf payload (PVCpnf) induced a phenotype (ruffling) consistent with the hypothesized mechanism of the effector (PVC), see Figure 5.

[0192] Example 2 2.1 Demonstration that the PVC needle complex exerts its effects through intracellular delivery of the effector. Polypeptide Pnf was identified as a PVC effector as follows. It was identified within the complete genome of Photorhabdus asymbiotica ATCC43949 - GenBank accession number: FM162591.1.

[0193] The last gene of the PVC operon (P. asymbiotica ATCC43949PVCpnf operon, having the sequence of SEQ ID NO: 93), namely pvc16 (e.g., PAU_03338), was identified. The location of the pvc16 gene in the PVC locus is illustrated in Figures 1(A), (B), and (D). An ORF immediately 3' of pvc16 (e.g., within approximately 5kb downstream of pvc16) was identified, and one such ORF (PAU_03332) is 3535bp downstream of pvc16. The predictive function of the polypeptide encoded by this putative effector ORF (having the sequence of SEQ ID NO: 32) was obtained using a combination of BlastP and HHPRED (https: / / toolkit.tuebingen.mpg.de / # / tools / hhpred). This ORF was then... Based on direct homology with known bacterial toxins (e.g., those of the CNF1 family from Escherichia coli), it was possible to assign it as a PVC effector.

[0194] Next, a Pnf-loaded PVC needle composite was prepared according to Example 1.

[0195] The inventors have demonstrated that these packaged (e.g., loaded) PVC needle complexes exert cellular effects consistent with the origin of the cargo they carry. For example, cells and complete insect animals exposed to PVC needle complexes loaded with the cytoskeletal toxin Pnf undergo cell death in a manner consistent with cytoskeletal toxicity.

[0196] Injection experiments (injection into insect larvae) were performed by injecting 10 μl of supernatant, using a culture of Escherichia coli (E. coli) containing a PVC needle complex with Pnf (PVCPnf), for example, a cosmid clone encoding PVC as specified by sequence number 93 packaged in sequence number 32 PVC effector, after centrifugation (pelletization) of an overnight culture (typically 1 L).

[0197] We demonstrated that the PVC needle complex is responsible for the phenotype due to the intracellular delivery (e.g., injection) of the Pnf payload. Therefore, the toxic effect could only be reconstituted by providing the same protein (Pnf) through a different pathway to access the cell's cytoplasm (transfer and expression of an expression plasmid, or conduction via a liposome preparation containing the protein), see Figure 6. Conversely, denatured (boiling-mediated) PVC needle complex preparations, toxin proteins layered on tissue culture cells, or toxin proteins injected into complete animals did not show activity.

[0198] 2.2 Evidence of delivery of toxic effector enzyme Pnf into cultured human macrophages To supplement the data outlined above, the inventors conducted additional experiments to provide further evidence of the delivery of the toxic effector enzyme Pnf into cultured human macrophages.

[0199] Concept: The inventors examined purified PVCpnf expressed from Escherichia coli (E. coli) (trans)packaged with natural Pnf toxin against cultured human THP1-derived macrophages. Unlike the lethal effect of Pnf in insect models, prior liposome-mediated Pnf protein translocation experiments showed a more elusive phenotype in human HeLa cells. In those experiments, the cells showed actin stress fiber formation at 24 hours and multinucleation at 48 hours. Therefore, the inventors examined the effect of purified PVCpnf (nanosyringe) containing / packaged with Pnf PVC effectors on macrophage respiration rate using a resazurin colorimetric assay.

[0200] method: Background of the Resazurin Assay. The blue compound resazurin was investigated for use in the assay to determine the activity of PVCs against macrophages (M0). Resazurin is metabolically reduced in cellular mitochondria, producing the pink and highly fluorescent compound resolphin. The effect of PVCs on macrophage metabolism can be determined by introducing resazurin into the culture medium. The number of macrophages affected by PVCs can be estimated by comparing the measured fluorescence with that of a cell density optimization curve (see Czekanska, Methods in Molecular Biology, 2011, 740, 27-32, incorporated herein by reference).

[0201] Optimization of resazurin use for THP1-derived macrophages. The optimal cell density for use in this assay using PVC was determined by evaluating macrophage metabolism over 18 hours at different seeding densities. 30 mL cultures of THP-1 cells were pelleted at 1000 rpm for 4 minutes and then resuspended in 2 mL of RPMI medium (also containing 10% FBS(v / v) and 2 mM L-glutamine). Cells were counted using a hemocytometer. The cells were then diluted in culture medium to a density of 2 × 10⁶ cells per mL⁻¹. Next, the THP-1 cells were activated with phorbol 12-myristate-13-acetate (PMA) immediately before plating. 200 μL of cells were plated in sets of four in a 96-well plate, and 2-fold serial dilutions were performed until a final cell density of 1.5625 × 10³ cells per mL⁻¹ was reached. 125 μL of the starting cell dilution was also plated in sets of four on the same plate for 5-fold serial dilutions until a cell density of 0.32 × 10³ cells per mL⁻¹ was reached. Four blank wells containing RPMI and PMA were also prepared. The plates were incubated at 37°C in 5% CO₂ for 48 hours. The medium was aspirated from the wells and replaced with fresh RPMI, and the macrophages were incubated for a further 24 hours. Resazurin tablets (VWR) were lysed in RPMI (12.5 mg / mL), and 10 μL was added to each well. The solution was successively added to the wells (well concentration of 1.25 mg / mL). The resulting fluorescence was measured every 30 minutes for 18 hours on a plate reader (excitation: 530-570 nm, fluorescence: 580-620 nm, maintained at 37°C and 5% CO2). Subsequently, the optimal cell density over time for use with PVC was determined.

[0202] Assay for PVC testing. THP-1 cells diluted to 1.25 × 10⁵ cells in mL⁻¹ were activated and seeded in a 96-well plate, with each well containing 100 μL of cells at a final well density of 1.25 × 10⁴ cells in mL⁻¹. Four blank wells containing cells without PVC samples, as well as four wells containing only medium and PMA, were also prepared. The plates were incubated at 37°C in 5% CO₂ for 48 hours. The medium was then replaced with fresh RPMI, and 10 μL of each PVC sample was added. The plates were incubated for a further 24 hours, after which 10 μL of resazurin (12.5 mg / mL) was added to each well, and fluorescence was measured every 30 minutes for 18 hours (excitation: 530–570 nm, fluorescence: 580–620 nm, maintained at 37°C and 5% CO₂).

[0203] Results: Figure 6F shows that the challenge with PVCpnf+Pnf did indeed reduce macrophage respiration, while heat-denatured or empty PVCpnf nanosyringes did not have a potent adverse effect. Nevertheless, control cells without the sample still showed the best respiration rate. The effect on macrophages was correlated with an insect injection toxicity assay. In this case, the two PVCpnf+Pnf preparations showed mortality rates of more than half of the insect cohort, while insects injected with heat-denatured and empty PVCpnf remained healthy.

[0204] Example 3 Demonstration that the leader sequence is responsible for packaging the payload into the PVC needle complex. Surprisingly, the inventors have found that, preferably, assuming a “leader” peptide sequence at the N-terminus of the payload (toxin) protein can direct the payload to the PVC complex and enable (e.g., trigger) the packaging of the payload into the PVC needle complex. The inventors have demonstrated that amino acid residues 1-50 of the PVC effector protein are / contain the leader sequence.

[0205] To demonstrate this, an expression construct (overexpression in chromosomally engineered P. luminescens TT01) was prepared, and Plu1649 (shown as "hvn" in the figure) was expressed in it. The payload expressed by (Myc-tagged) Plu1649 ("hvnA"), which has the sequence of sequence number 46, lacked the leader sequence (N-terminal amino acid residues 1-50) for detection purposes (see Figure 8A - Construct 1). After expression (both payload and PVC needle complex) and isolation (and running of its components including any packaged payload on gel), (Myc-tagged) Plu1649 ("hvnA") was undetectable within the PVC needle complex via Western blot analysis, indicating that the payload (lacking the leader sequence) was not packaged within the complex (see Figure 8B, ray (See Lane 1), thus demonstrating that it does not associate with the isolated complex. However, good packaging was observed for hvnA retaining the leader sequence, see Lane 2 (note that the band is considered weak due to the relative intensity of the band in Lane 3).

[0206] Surprisingly, as demonstrated by Western blot detection of Myc-tagged hvnA, hvnA with leader sequences from different (non-hvnA) PVC effectors (i.e., corresponding to N-terminal amino acid residues 1-50 from the PAU_03332 effector) (see Figure 8A, construct 3) were precisely packaged within the complex and remained associated with the PVC needle complex upon isolation / purification (see Figure 8B, lane 3). Thus, we demonstrate the remarkable ability of the "PAU_03332" leader sequence (associating with different payload Pnfs) to package hvnA payloads (i.e., payloads different from those of PAU_03332). This demonstrates the ability to swap leader sequences of PVC effectors and allows for the use of the optimal leader sequence (with optimal packaging activity) for packaging.

[0207] Example 4 4.1 Demonstration that the leader sequence directs the packaging of atypical / exogenous payloads (into the PVC needle complex) Among the unforeseen technical effects of the present invention, the inventors have found that the fusion of the leader sequences described herein to exogenous (non-Photorhabdus) polypeptides (preferably at the N-terminus) enables the packaging of said exogenous polypeptides into PVC needle complexes and that the exogenous polypeptides remain associated with the PVC needle complexes upon isolation / purification. As an example, FIG. 8B (lane 4) demonstrates that a non-Photorhabdus "Myc" polypeptide (<10 kDa), when fused to the leader sequence, is packaged into the PVC needle complex, and lane 6 shows that an even larger non-Photorhabdus "Cre recombinase" polypeptide (>32 kDa) can likewise be properly packaged into the PVC needle complex when fused to the leader polypeptide of the present invention.

[0208] The inventors have conducted a detailed analysis of the sizes (e.g., polypeptide lengths) and structures of various native PVC effector payloads encoded by Photorhabdus (see FIG. 7), which show a wide range of different lengths and structures, demonstrating that the applicability of the PVC needle complex (nanosyringe) delivery system of the present invention is not limited by the size or properties of the target payload protein. In summary, neither a specific secondary structure, nor biophysical properties, nor payload length are required, supporting the use of the PVC needle complex (nanosyringe) chassis as a versatile and multifunctional delivery vehicle.

[0209] Furthermore, this packaging of the exogenous polypeptide is independent of the selected PVC needle complex chassis and has been achieved using both, for example, the "PVCpnf" chassis (SEQ ID NO: 93) and the "PVCU4" (e.g., PVCunit4) chassis (endogenous for Photorhabdus overexpression strains) (see Figure 10A). Importantly, the inventors ensured that the packaging of the exogenous payload in either chassis does not affect the morphology of the PVC needle complexes and that they are not assembled abnormally (see Figure 10B).

[0210] In the data presented herein, the payload protein is supplied “trans” to a separate gene construct. Surprisingly, the leader sequence is sufficient to target those separately synthesized proteins for packaging into the PVC needle complex vehicle (see Figure 11). This is true in *E. coli* when the chassis (PVC) gene itself is also present on the plasmid, and in the host organism *Photorhabdus* genus. Chassis genes are integrated into the chromosome, as in the case of Photorhabdus.

[0211] Figure 10(C) provides further examples of transpackaging of high levels of Cre site-specific recombinase into PVCpnf nanosyringes expressed in Escherichia coli (E. coli). More specifically, we constructed a laboratory Escherichia coli (E. coli) expression strain possessing (i) an arabinose-inducible expression plasmid of the P. asymbiotica (ATCC43949) PVCpnf operon (having a C-terminal FLAG tag on Pvc16, for example, at the immediate 3' of SEQ ID NO: 93) and (ii) a second IPTG-inducible expression plasmid containing a Cre recombinase having an N-terminal fusion of a natural Pnf effector 50 amino acid leader sequence (e.g., the leader of SEQ ID NO: 78) and a C-terminal Myc-tagged epitope. The PVC operon and effector (Cre+ leader sequence) were co-induced for 24 hours, and the chimeric nanosyringes were purified. Western blot analysis was used to confirm the presence of purified FLAG-tagged Pvc16 cap protein (and therefore nanosyringe chassis) and transpackaged Myc-tagged Cre recombinase.

[0212] 4.2 Transpackage using additional readers to demonstrate functionality for larger and more diverse array spaces To supplement the data outlined in Example 3, Figure 10D demonstrates the (trans)packaging of Cre into PVCpnf (in E. coli) using the following four additional leader sequences (thus demonstrating the functionality of a larger sequence space): - Lane 1: Leader of PAU_02096 (leader sequence = sequence number 71), experiment referred to as "nanosyringe + lopt50::cre::Myc" in Figure 10D; - Lane 2: PAK_02075 leader (leader sequence = sequence number 50), experiment referred to as "nanosyringe + cnf50::cre::Myc" in Figure 10D; - Lane 3: Leader of PAU_02009 (leader sequence = sequence number 68), experiment referred to as "nanosyringe + cif50::cre::Myc" in Figure 10D; and - Lane 4: PAU_02806 leader (leader sequence = sequence number 76), experiment referred to as "nanosyringe + gog50::cre::Myc" in Figure 10D.

[0213] These results also demonstrate the usefulness of leader sequences that show greater sequence diversity for the (trans)packaging of the payload. In fact, to provide further validation, the inventors performed CLUSTALW sequence comparisons of a panel of leader sequences to determine diversity. PVC effectors are identified as proteins encoding a recognizable toxin-like domain immediately downstream of the pvc16 structural gene. Each PVC operon may encode only a single effector or several different effector genes in a tandem array. The phylogenetic tree is shown in Figure 10E, and the identity of the leader sequences exemplified herein for packaging payload proteins in nanosyringe complexes is either or both of the P. asymbiotica ATCC43949 PVCpnf operon (solid arrow) or the P. luminescens TT01 PVCunit4 operon (dotted arrow). It is produced by [method].

[0214] As can be seen from the tree in Figure 10E, the example leader sequences are well distributed throughout, and therefore the diversity is at least maximally continuous.

[0215] Example 5 Tail fiber / binding domain modification It is known that PVC needle complexes include tail fibers (3D rendered PVC structure, see asterisk on the left of the rightmost image) which may enable cell type-specific targeting of the PVC complex. The inventors have developed a tail fiber for incorporating non-natural amino acids. We successfully demonstrated that modifications to the fiber region (e.g., substitution of amino acids in the wild-type sequence with alternative amino acids for 20 standard amino acids) do not affect tail fiber expression.

[0216] Example 6 Demonstration of delivery of active (exogenous) enzyme / payload into ex vivo mouse organoids using a PVC needle complex with a packaged leader sequence. Concept: To obtain data on the delivery of exogenous functional enzymes to mammalian tissues. The inventors demonstrated the delivery of a transpackaged bacteriophage-derived recombinase protein known as "Cre" into ex vivo mouse bile duct organoids. The organoids are derived from a mouse line in which the expression of a chromosome-encoded red fluorescent protein (RFP) reporter is normally disrupted by a termination signal flanked by the loxP recognition site for Cre-recombinase. In the presence of recombinase, the termination signal is recombinated, and the cell subsequently expresses the reporter protein. The general principle of this experimental demonstration is summarized in Figure 16A.

[0217] Methods: Bile duct organoid preparation: Primary mouse bile ducts were isolated and expanded for 12 passages as organoids in Matrigel using "BD Expansion Medium" according to the Huch et al (Regen Med. 2013 Jul;8(4):385-7. PMID:23826690;DOI:10.2217 / rme.13.39) protocol. Cells were then plated in 2D and cultured in BD Expansion Medium. Mouse genotype: LSL-Tom reporter + Axyn 2CreRT (inducible upon 4OHT treatment) in Rosa26 locus. Cells were cultured in uncoated polystyrene plates at a seeding density of 10,000 cells / well. Nanosyringes were prepared as syringe preparations of 30% volume in PBS + 70% culture medium. Total volume was 100 μl per well. The positive control represented 500 nM 4OHT (in ethanol) at a 1:1000 (v / v) ratio as a positive control for recombinant DNA. The negative control represented only the 1:1000 (v / v) ethanol dilution. Cells were seeded, grown for 48 hours, added via nanosyringe, then cultured for a further 24 hours, fixed (4% PFA fixation for 15 minutes at room temperature), and stained for microscopic examination. Staining: Primary antibody anti-RFP from Rockland (1:1000). Secondary anti-rabbit 568 (used at 1:500 v / v). Samples were visualized on a laser confocal microscope.

[0218] Results: Figure 16B includes representative micrographs from these experiments, demonstrating that a signal for the RFP protein could be detected in a large number of cells when treated with Cre-loaded PVCpnf nanosyringes. Since these are ex vivo organoids rather than simple cell monolayers, some randomness in the number of cells administered is expected, which is also observed in the positive control, which is a small molecule inducer (not a large protein complex). Because these are organoids, it is expected that there is some level of cellular differentiation present that may alter the binding characteristics of the nanosyringes. Further interesting observations from this preliminary run are that, while information on the total amount of nanosyringes applied to the system is still unavailable, we demonstrate that TAM small molecule inducers do not appear to have significantly greater tissue penetration than nanosyringes, suggesting that their distribution ability is not so hindered by their size.

[0219] Additional interpretation: In summary, the inventors have demonstrated the ability to deliver (e.g., administer) exogenous enzymes to cellular targets. Furthermore, this “nanosyringe + Cre” experiment provides promising evidence for the concept of a biotechnology tool / aid by demonstrating its ability to provide DNA alterations resulting in transformed cells. Thus, this experiment specifically demonstrates the use of exogenous payloads (non-bacterial viral proteins) and nucleic acid-modifying enzymes. It is clear that Cre enzymes are functionally delivered, can traverse the cell interior to the nucleus, and influence its DNA modification.

[0220] Example 7 Transpackaging of MAD7 site-specific recombinase (exogenous payload) expressed in Escherichia coli (E. coli) into PVCpnf nanosyringes. Concept: With regard to the Cre data (of Example 6) and other examples of packaged payloads provided herein, the inventors demonstrated the packaging of Cas-like enzyme MAD7 into a nanosyringe via a leader sequence. This is an example of the largest exogenous component in the payload described herein (MAD7 = 147.9 kDa).

[0221] Methods: Briefly, the chassis gene and the MAD7 gene (the latter tagged with the C-terminal Myc tag for detection as described herein and a leader sequence for nanosyringe incorporation) were co-expressed in Escherichia coli (E. coli) during induction. During harvesting and purification of the nanosyringe complexes, the payload packages were probed via dot blot analysis (e.g., for detection of the Myc tag). Using the purification method described herein (using ultracentrifugation), (e.g.) protein complexes / biological substances with (e.g.) very high molecular weights can be selected, allowing for the recovery of the nanosyringes and any loads (payloads) they carry. "Loose" / unpackaged payloads remain in solution and are not subjected to sufficient centrifugal force and are therefore lost during purification unless contained within a considerably larger nanosyringe "shell" (i.e., if well packaged). Good packaging of MAD7 is demonstrated by Figure 17.

[0222] Example 8 Transpackaging of an apoptosis-inducing payload into PVCpnf expressed in Escherichia coli (E. coli). Using the Escherichia coli (E. coli) PVCpnf reader::payload::Myc transpackaging system (PVCpnf reader = SEQ ID NO: 78) described in Figure 10C, the inventors demonstrated the ability to transpackage at least two pro-apoptotic human-derived protein sequences or peptides (e.g., sequences of SEQ ID NO: 109 and SEQ ID NO: 111). A Pnf effector protein reader sequence (e.g., SEQ ID NO: 78) was fused to the N-terminus, and a Myc epitope tag was fused to the C-terminus. Western dot blot analysis (similar to that of Example 7) confirmed the presence of these human-derived proteins in purified nanosyringes (Figure 18).

[0223] Example 9 Demonstration of induction of apoptosis in cultured ex vivo human cells by nanosyringe delivery of (trans)packaged pro-apoptotic human polypeptides. Preliminary testing involved delivering transpackaged human protein sequences (e.g., packaged according to Example 8) using PVCpnf nanosyringes produced in Escherichia coli (E. coli) to confirm their ability to induce apoptosis in ex vivo circulating PBMC cells from human donors. The assay was TUNEL staining microscopy analysis of cells exposed to the packaged nanosyringes for only 20 minutes. The results are shown in Figure 19A, demonstrating the delivery of the tBid p15 fragment and BaxBH3 domain (via favorable induction of apoptosis). The tBid p15 fragment (SEQ ID NO: 109) is part of the normal human apoptosis regulatory pathway. Cellular effect: Pro-apoptosis member of the Bcl-2 family. The C-terminal portion of Bid (tBid) translocates to mitochondria, where it induces the release of cytochrome c. Bid is normally cleaved from its latent cytoplasmic full-length proBid form by caspase 8. BaxBH3(aa59~73)(SEQ ID NO: 111) is a minimal BH3 domain synthetic peptide containing 15 key residues of the standard Bax BH3 domain. Cellular effect: These 15 residues bind to Bcl-xL and functionally antagonistize it. It also contains sufficient information to specifically induce Bax / Bak. It is thought to terminate the Bak / Bcl-2 interaction and release apoptosis-promoting factors.

[0224] This report describes a more detailed examination of the delivery of pro-apoptotic human peptides into ex vivo peripheral blood mononuclear cells (PBMCs). The objective of this study was to investigate whether PVC nanosyringes loaded with pro-apoptotic peptides could induce apoptosis in ex vivo human peripheral blood mononuclear cells. The nanosyringes were first evaluated for any immediate cytotoxicity using a trypan blue dye exclusion assay, and then for the apoptotic response using a TUNEL assay.

[0225] Trypan Blue Exclusion Test for Cell Viability: Trypan blue is a diazo dye commonly used to selectively stain dead tissue or cells; therefore, dead cells appear as a distinguishable blue under a microscope, while viable cells or tissues with intact cell membranes remain unstained. Since viable cells are excluded from staining, this staining method is also described as a dye exclusion method. Trypan blue is commonly used to assess tissue or cell viability. A suitable number of cells (2 × 10⁵) were exposed in nanosyringes and empty nanosyringes for 20 minutes. A suitable volume of cells (30 μL) was added to an equal volume of 0.4% trypan blue, and the number of viable (unstained) and dead (stained) cells was counted using a hemocytometer. Each compound was tested at three concentrations. Hematologic cells from two independent human donors were tested for each compound at each concentration, and each sample was tested in pairs.

[0226] Cell processing and preparation for microscopic observation: The viability of peripheral blood mononuclear cells (PBMCs) from two independent healthy human donors was determined after 20 minutes of processing in two chimeric nanosyringes (e.g., loaded with exogenous pro-apoptotic peptides) at three test concentrations in two independent tests. PBMCs were harvested by centrifugation and resuspended in culture medium at 1 × 10⁶ cells / ml. Cells were fixed in 2.5% formalin and incubated at room temperature for 20 minutes. Poly-L-lysine coated slides were prepared by spraying with 70% ethanol and allowing to stand and air dry. Cells were centrifuged for 30 seconds. The supernatant was removed and cells were resuspended in 200 μl of dH₂O. 5 μl of cell suspension was added to each slide / fixture. Two fixations were performed per slide to allow for double staining. Cell suspensions were allowed to stand and air dry.

[0227] Results of PBMC cell viability assay: The trypan blue viability assay confirmed that the PVC preparation was not immediately toxic to PBMCs collected from healthy human donors (Table 2). Nanosyringe treatment showed a viability of >60% and low toxicity at the maximum dose concentration (Table 2). The inventors then subsequently tested the ability of chimeric nanosyringes to induce apoptosis.

[0228] [Table 4]

[0229] Examination of chimeric nanosyringe-induced apoptosis using the TUNEL assay: Apoptotic nuclei were then identified in single-cell suspensions fixed on slides using the TUNEL assay. In this assay, terminal deoxynucleotidyltransferase (TdT) binds to the exposed 3'-OH end of DNA fragments generated in response to apoptotic signaling factors. This subsequently catalyzes the addition of biotin-labeled deoxynucleotides, which can be detected using a streptavidin-horseradish peroxide (HRP) conjugate. Diaminobenzidine (DAB) reacts with the HRP-labeled sample to produce an insoluble brown substrate at the DNA fragmentation sites. Methyl green counterstaining allows visualization of normal and apoptotic cells.

[0230] We determined the induction of apoptosis after exposure of human PBMCs to nanosyringes. The TUNEL assay kit (Abcam) was used to detect apoptotic cells. The assay was performed by the manufacturer. The procedure was carried out according to the instructions. Briefly, the slides were coated with 100 μL of proteinase K solution for 5 minutes, and then rinsed with 1× TRIS-buffered saline (TBS). The nanosyringe or DNase I positive kit control was treated at room temperature for 20 minutes. The slides were rinsed with TBS. The slides were then incubated with TdT equilibrium buffer for 30 minutes, and then the TdT labeling reaction mix was added. The slides were incubated at 37°C for 19 minutes. The slides were then washed with TBS, stopped with stop buffer, and incubated at room temperature for 5 minutes. The slides were washed again with TBS, and then blocked with block buffer at room temperature for 10 minutes. Detection was performed by applying the conjugate to the sample for 30 minutes. The slides were rinsed with TBS and then treated with DAB solution for 15 minutes. The slides were rinsed with dH2O and then counterstained with methyl green. The slides were dehydrated in 100% ethanol, then in xylene, and mounted on glass coverslips. All staining was performed in pairs. Apoptosis endpoints showing positive staining in the apoptosis detection assay are represented by a dark brown (DAB) signal. Lighter brown shading and / or blue / green to green / brown shading indicate unresponsive, negative cells for apoptosis.

[0231] Analysis was performed by selecting five random portions of cells on a slide, counting positively stained cells (dark brown) and negatively stained cells (blue or light brown), and determining the proportion of cells exhibiting apoptosis.

[0232] To generate a positive control, slides were treated with 1 μg / μl DNase I (kit positive control) at room temperature for 20 minutes after the proteinase K treatment step detailed below. DNase I treatment fragments DNA in normal cells, generating free 3'OH groups identical to those produced during apoptosis. A negative control was generated by substituting DNase I in the reaction mix with dH2O during the treatment stage.

[0233] Results of PBMC Apoptosis Assay: TUNEL staining was performed using PBMCs after treatment with nanosyringes loaded with intact tBID and Bax, using appropriate positive and negative kit controls. Treatment was performed for 20 minutes to determine whether the nanosyringe induced an apoptotic signal. Positive controls (DNase I treatment) and negative controls (no DNase I treatment) were included. The results showed that both nanosyringes containing either tBID or Bax showed a strong apoptotic signal to PBMCs (89% and 78% positive, respectively). The positive controls showed a strong apoptotic signal (79%), while the negative controls showed no apoptotic signal (100% negative). A significant loss of adherent cell count in the nanosyringe-treated samples was also observed, which was thought to indicate a rapid and comprehensive apoptotic response and unsuccessful retention after washing. Note that this effect was considerably more pronounced than in the kit-positive controls, suggesting a more rapid response. Representative micrographs are shown in Figure 19B.

[0234] Conclusion: Nanosyringes loaded with tBID and Bax can rapidly induce widespread apoptosis in human peripheral blood mononuclear cells. Furthermore, trypan blue elimination assays confirmed that these chimeric nanosyringes did not cause rapid lethal lysis or widespread cell membrane damage.

[0235] Example 10 Demonstration of the practical utility of leader sequences and PVC needle complexes - intracellular delivery of atypical (non-Photorhabdus) payloads (1) An anti-MDM (p53 inhibitor) antibody is conjugated to the leader sequence described herein and expressed together with a PVC needle complex for packaging. The isolated PVC needle complex (containing the antibody payload) is brought into contact with a tumor for intracellular delivery of the antibody (the tumor cells are characterized by having high p53 activity and MDM suppression for MDM inhibition). The tumor is suppressed by the activity of the anti-MDM antibody. (2) An antitumor peptide vaccine is delivered (intracellularly) using a PVC needle complex to activate the MHC-1-dependent cytotoxic T-cell lymphocyte (CTL) response. The tyrosinase-related protein 2 (TRP2) peptide vaccine is delivered to CTLs to enhance cross-presentation, resulting in an antitumor effect against TRP2-expressing tumors. Tumors are suppressed by the activity of the peptide vaccine. (3) A nuclear factor-κB inhibitor (used to control inflammatory disorders, e.g., rheumatoid arthritis) is delivered (intracellularly) to cells using a PVC needle complex. Subsequently, the cells demonstrate a reduction in the expression of pro-inflammatory cytokines. (4) The T3SS payload (which inhibits the NF-κB and MAPK pathways) is delivered (intracellularly) using the PVC needle complex. This is completed with the isolated (purified) PVC needle complex, and it is not necessary for the PVC needle complex to remain associated with the bacterial cell from which it originates. (5) Using the PVC needle complex, the cells are subjected to peptide inhibition of anti-apoptotic peptides, such as BH4, Bcl-xL-protein, and / or c-Jun N-terminal kinase. It delivers (intracellularly) harmful agents (which can protect the heart and brain from ischemic injury (restriction of blood supply to tissues, causing a lack of oxygen and glucose necessary for cellular metabolism)). For example, Jun kinase inhibition via the 20 amino acid linkage motif of Jun kinase is sufficient. For example, the release of cytochrome c in cells is inhibited. (6) Using a PVC needle complex, nicotinamide adenine dinucleotide quinone internal oxidoreductase (Ndi1), a single subunit yeast analog of complex I (which provides significant cardioprotective effects), is delivered (intracellularly) to complex I-deficient mutant cells. The Ndi1 protein is precisely targeted to the matrix side of the inner mitochondrial membrane, restoring NADH oxidase activity in complex I-deficient cells. (7) Using a PVC needle complex, one of the two essential subunits of the PHOX complex (used in enzyme substitution therapy to restore ROS production in chronic granulomatous disease) is delivered to chronic granulomatous disease cells. Restoration of ROS production is observed. (8) Myotubularin (used to improve local and distal muscle performance in patients with X-linked myotubular myopathy) is delivered (intracellularly) (e.g., intramuscularly) using a PVC needle complex. Myotubularin dephosphorylation of phosphatidylinositol triphosphate and phosphatidylinositol (3,5) diphosphate is observed. (9) Using a PVC needle complex, the recombinase "Cre" (capable of cleaving a specific gene cassette) is delivered (intracellularly) into a mouse cell line having a loxP recombination site in which the genome flanks the termination signal upstream of the mCherry gene. The Cre payload cleaves the recombination site, removes the termination signal, and enables the expression of the mCherry gene in the cell. (10) A PVC needle complex is used to deliver approximately 15 kDa nanobodies (antibody fragments) that have affinity for intracellular components (intracellularly). The nanobodies-intracellular complex is detected. (11) Atypical (non-Photorhabdus) polypeptide toxins for insect crop pests and animal parasites are delivered intracellularly (e.g., into insect cells) using a PVC needle complex. Suppression of pests is observed. (12) Using a PVC needle complex, a nuclease (e.g., Cas9 and / or Mad7) is delivered (intracellularly) into a target cell containing guide RNA. The nuclease performs site-directed gene inactivation.

[0236] All publications cited in the above specification are incorporated herein by reference. Various modifications and variations of the methods and systems described in the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described with specific preferred embodiments, it should be understood that the claimed invention should not be excessively limited to such specific embodiments. In fact, various modifications of the methods described for carrying out the present invention, which will be apparent to those skilled in the art in biochemistry and biotechnology or related fields, are intended to fall within the scope of the following claims.

[0237] array The first Met amino acid residue or the corresponding first codon may be optional if it is represented by one of the following sequence numbers.

[0238] Sequence ID 1 (PAK_1985) [ka]

[0239] Sequence ID 2 (PAK_1987) [ka]

[0240] Sequence ID 3 (PAK_1988) [ka]

[0241] Sequence ID 4 (PAK_2075) [ka]

[0242] Sequence ID 5 (PAK_2077) [ka]

[0243] Sequence ID 6 (PAK_2892) [ka]

[0244] Sequence ID 7 (PAK_2893) [ka]

[0245] Sequence ID 8 (PAK_2894) [ka]

[0246] Sequence ID 9 (PAK_3525) [ka]

[0247] Sequence ID 10 (PAT_00148) [ka]

[0248] Sequence ID 11 (PAT_00149) MIFKMLNLAVFYLLGNIFHYLICQKFICYFCSVLKSVTMFLTKVAVQIALYLNILPTMAGIAGLHAEVQALNNLFISGDRGTEKRENWKYIRNMLESTIFTQRLTAGQAGKDFAACHNCSGILSSPVNVITGKVESAGGNFFINIISI

[0249] Sequence ID 12 (PAT_00150) [ka]

[0250] Sequence ID 13 (PAT_00152) [ka]

[0251] Sequence ID 14 (PAT_02308) [ka]

[0252] Sequence ID 15 (PAT_02309) [ka]

[0253] Sequence ID 16 (PAT_02310) [ka]

[0254] Sequence ID 17 (PAT_02956) [ka]

[0255] Sequence ID 18 (PAT_02957) [ka]

[0256] Sequence ID 19 (PAT_03171) [ka]

[0257] Sequence ID 20 (PAT_03172) [ka]

[0258] Sequence ID 21 (PAT_03177) [ka]

[0259] Sequence ID 22 (PAU_02009) [ka]

[0260] Sequence ID 23 (PAU_02010) [ka]

[0261] Sequence ID 24 (PAU_02095) [ka]

[0262] Sequence ID 25 (PAU_02096) [ka]

[0263] Sequence ID 26 (PAU_02097) [ka]

[0264] Sequence ID 27 (PAU_02098) [ka]

[0265] Sequence ID 28 (PAU_02230) [ka]

[0266] Sequence ID 29 (PAU_02805) [ka]

[0267] Sequence ID 30 (PAU_02806) [ka]

[0268] Sequence ID 31 (PAU_02807) [ka]

[0269] Sequence ID 32 (PAU_03332) [ka]

[0270] Sequence ID 33 (PAU_03337) [ka]

[0271] Sequence ID 34 (Plu1651) [ka]

[0272] Sequence ID 35 (Plu1671) [ka]

[0273] Sequence ID 36 (Plu1672) [ka]

[0274] Sequence ID 37 (Plu1690) [ka]

[0275] Sequence ID 38 (Plu1691) [ka]

[0276] Sequence ID 39 (Plu1712) [ka]

[0277] Sequence ID 40 (Plu1713) [ka]

[0278] Sequence ID 41 (Plu1714) MKKTDEKYGQYEYKDEDITSYPIAWTNPDNGKIYIGINSPEYSHLNNKGESELNLAKIISTIIHESLHASSHQHKGLQSQTDTGADNLNYDEYVTDYFAREVYKQILPDKDYVANCFTKGLGGENKIWGGNIVEFMIQ

[0279] Sequence ID 42 (Plu2400) [ka]

[0280] Sequence ID 43 (Plu2401) [ka]

[0281] Sequence ID 44 (Plu2514) [ka]

[0282] Sequence ID 45 (Plu2515) [ka]

[0283] Sequence ID 46 (Plu1649) [ka]

[0284] [Table 5]

[0285] Sequence ID 93 (Photorhabdus asymbiotica strain ATCC43949PVCPnf operon, pvc1~pvc16; for example, GenBank accession number) (The sequence of gene PAU_03353 to PAU_03338 corresponds to sequence FM162591.1) [ka] [ka] [ka] [ka] [ka]

[0286] Sequence ID 94 (Photorhabdus asymbiotica strain ATCC43949 PVClopT operon, pvc1~pvc16; for example, corresponding to gene sequences PAU_02112 to PAU_02099 of GenBank accession number FM162591.1) [ka] [ka] [ka] [ka]

[0287] Sequence ID 95 (Photorhabdus asymbiotica strain ATCC43949PVCPaTox operon, pvc1~pvc16) [ka] [ka] [ka] [ka] [ka]

[0288] Sequence ID 96 (Pnf epitope) TGQKPGNNEWKTGR

[0289] Sequence ID 97 (PVCpromF) TATCATATGTCTACAACTCCAGAACAAATTGCTG

[0290] Sequence ID 98 (PVCpromR) ATCTCTAGAACAGATATTCCAGCCAGC

[0291] Sequence ID 99 (ParaINF) GGCGTCACACTTTGCTATG

[0292] Sequence ID 100 (ParaINF) TCGGTGGCAGTAAATTGTCC

[0293] Sequence ID 101 (F1 primer) ATGTCTACAAGTACATCTCAAATTGCG

[0294] Sequence ID 102 (F2 primer) GACTCCCTTGAGGGTACGG

[0295] Sequence ID 103 (F3 primer) TTCTGATGAGAGTGATGGTAC

[0296] Sequence ID 104 (F4 primer) TGAATAAAGAATTCAGTCAATATC

[0297] Sequence ID 105 (R1 primer) TAGTGGCTGATGAAAGTCTG

[0298] Sequence ID 106 (R2 primer) GGAAGCCAAAGATAATGAAGTG

[0299] Sequence ID 107 (R3 primer) CATTTCTTCCCTATGGTTG

[0300] Sequence ID 108 (R4 primer) TTAAATTCCTACAAGATTATCTTT

[0301] Sequence ID 109 (tBid amino acid sequence) RSSHSRLGRIEADSESQEDIIRNIARHLAQVGDSMDRSIPPGLVNGLALQLRNTSRSEEDRNRDLATALEQLLQAYPRDMEKEKTMLVLALLLAKKVASHTPSLLRDVFHTTVNFINQNLRTYVRSLARNGMD

[0302] Sequence ID 110 (E. coli sequence-optimized tBid base) [ka]

[0303] Sequence ID 111 (BaxBH3 peptide (aa59~73)) LSESLKRIGDELDSN

[0304] Sequence ID 112 (E. coli sequence optimized BaxBH3 base) CTGTCGGAGAGTTTGAAGCGTATAGGTGACGAGCTGGACAGCAAT

Claims

1. The use of a leader peptide of Photorhabdus virulence cassette (PVC) effector protein for packaging a payload within a PVC needle complex, The leader peptide has an amino acid sequence that packages the polypeptide payload into the PVC needle complex. The polypeptide payload is a therapeutic polypeptide, an enterogenic polypeptide, and / or a nucleic acid-modifying enzyme. The leader peptide and the payload form an effector fusion distinct from the wild-type PVC effector protein. The aforementioned use includes bringing the PVC needle composite into contact with the effector fusion, use.

2. The use according to claim 1, wherein the PVC needle complex and the effector fusion are expressed in a heterologous bacterial expression system, yeast cells, insect cells, and / or mammalian cells.

3. The use according to claim 1 or 2, wherein the leader peptide comprises amino acid residues 1 to 50 or 2 to 50 of the PVC effector protein.

4. The use according to any one of claims 1 to 3, wherein the leader peptide comprises an amino acid sequence having at least 60% sequence identity with one or more sequences selected from SEQ ID NOs: 47 to 92.

5. The use according to any one of claims 1 to 4, wherein the PVC effector protein comprises an amino acid sequence of one or more sequences selected from SEQ ID NOs: 1 to 46.

6. The use according to any one of claims 1 to 5, wherein the PVC effector protein comprises an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO:

46.

7. The use according to any one of claims 1 to 6, wherein the leader peptide is covalently fused to the payload, preferably at the N-terminus of the payload.

8. A method for manufacturing a PVC needle composite containing a payload, comprising: a. The method involves contacting a PVC needle complex with an effector fusion product containing a leader peptide of a PVC effector protein fused to a payload, wherein the leader peptide has an amino acid sequence that packages the polypeptide payload into the PVC needle complex; b. The polypeptide payload is a therapeutic polypeptide, an enterogenic polypeptide, and / or a nucleic acid modifying enzyme; c. The effector fusion is distinguishable from the wild-type PVC effector protein. method.

9. The method according to claim 8, wherein the contact is carried out in cells of an in vitro cell system, in a cell lysate, or in a purified cell lysate.

10. An in vitro and / or ex vivo method for delivering a payload into cells, comprising: a. The method involves bringing cells into contact with a PVC needle complex containing an effector fusion; b. The effector fusion comprises a leader peptide of a PVC effector protein fused to the payload, wherein the leader peptide has an amino acid sequence that packages the polypeptide payload into the PVC needle complex; c. The polypeptide payload is a therapeutic polypeptide, an enterogenic polypeptide, and / or a nucleic acid modifying enzyme; d. The effector fusion is distinguishable from the wild-type PVC effector protein. method.

11. An in vitro method for controlling harmful organisms: a. Including contact between a pest or a target area containing a pest and a PVC needle composite containing an effector fusion; b. The effector fusion comprises a leader peptide of a PVC effector protein fused to the payload, wherein the leader peptide has an amino acid sequence that packages the polypeptide payload into the PVC needle complex; c. The polypeptide payload is a therapeutic polypeptide, an enterogenic polypeptide, and / or a nucleic acid modifying enzyme; d. The effector fusion is distinguishable from the wild-type PVC effector protein. method.

12. A PVC needle composite for use in therapeutic methods; a. The PVC needle complex comprises an effector fusion product containing a leader peptide of a PVC effector protein fused to the payload, wherein the leader peptide has an amino acid sequence that packages the polypeptide payload into the PVC needle complex; b. The polypeptide payload is a therapeutic polypeptide, an enterogenic polypeptide, and / or a nucleic acid modifying enzyme; c. The effector fusion is distinguishable from the wild-type PVC effector protein. PVC needle composite.

13. A PVC needle composite containing an effector fusion; a. The effector fusion comprises a leader peptide of a PVC effector protein fused to the payload, wherein the leader peptide has an amino acid sequence that packages the polypeptide payload into the PVC needle complex; b. The polypeptide payload is a therapeutic polypeptide, an enterogenic polypeptide, and / or a nucleic acid modifying enzyme; c. The effector fusion is distinguishable from the wild-type PVC effector protein. PVC needle composite.

14. An effector fusion comprising a leader peptide of a PVC effector protein fused to the payload; a. The leader peptide has an amino acid sequence that packages the polypeptide payload into the PVC needle complex; b. The polypeptide payload is a therapeutic polypeptide, an enterogenic polypeptide, and / or a nucleic acid modifying enzyme; c. The effector fusion is distinguishable from the wild-type PVC effector protein. A fusion of effects pedals.

15. The method according to any one of claims 8 to 11, wherein the leader peptide comprises amino acid residues 1 to 50 or 2 to 50 of the PVC effector protein.

16. The PVC needle complex according to claim 12 or 13, wherein the leader peptide comprises amino acid residues 1 to 50 or 2 to 50 of the PVC effector protein.

17. The effector fusion according to claim 14, wherein the leader peptide comprises amino acid residues 1 to 50 or 2 to 50 of the PVC effector protein.

18. The method according to any one of claims 8 to 11, wherein the leader peptide comprises an amino acid sequence having approximately 60% sequence identity with one or more sequences selected from SEQ ID NOs: 47 to 92.

19. The PVC needle complex according to claim 12 or 13, wherein the leader peptide comprises an amino acid sequence having approximately 60% sequence identity with one or more sequences selected from SEQ ID NOs: 47 to 92.

20. The effector fusion according to claim 14, wherein the leader peptide comprises an amino acid sequence having approximately 60% sequence identity with one or more sequences selected from SEQ ID NOs: 47 to 92.

21. The method according to any one of claims 8 to 11, wherein the PVC effector protein comprises an amino acid sequence of one or more sequences selected from SEQ ID NOs: 1 to 46.

22. The PVC needle complex according to claim 12 or 13, wherein the PVC effector protein comprises an amino acid sequence of one or more sequences selected from SEQ ID NOs: 1 to 46.

23. The effector fusion according to claim 14, wherein the PVC effector protein comprises one or more amino acid sequences selected from SEQ ID NOs: 1 to 46.

24. The method according to any one of claims 8 to 11, wherein the PVC effector protein comprises a sequence selected from SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO:

46.

25. The PVC needle complex according to claim 12 or 13, wherein the PVC effector protein comprises a sequence selected from SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO:

46.

26. The effector fusion according to claim 14, wherein the PVC effector protein comprises a sequence selected from SEQ ID NO: 4, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO:

46.

27. The method according to any one of claims 8 to 11, wherein the leader peptide is covalently fused to the payload.

28. The PVC needle complex according to claim 12 or 13, wherein the leader peptide is covalently fused to the payload.

29. The effector fusion product according to claim 14, wherein the leader peptide is covalently fused to the payload.

30. An isolated nucleic acid comprising a nucleotide sequence encoding an effector fusion according to claim 14, 17, 20, 23, 26, or 29.

31. An expression vector comprising the isolated nucleic acid molecule described in claim 30.

32. A host cell comprising an isolated nucleic acid molecule according to claim 30, or an expression vector according to claim 31.

33. The host cell according to claim 32, which is one or more selected from mammalian cells, insect cells, yeast cells, bacterial cells, and / or plant cells.

34. The host cell according to claim 33, wherein the bacterial cell is an E. coli cell.

35. The host cell according to claim 32, which is a cell of the genus Photorhabdus.

36. The host cell according to claim 35, wherein the Photorhabdus cell comprises a Photorhabdus PVC operon operably bound to an inducible promoter.

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