Leader sequence

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 to package molecules into protein complexes. [Background technology]

[0002] Biological molecules (e.g., peptides, proteins, and nucleic acids) have great potential as broadly applicable therapeutic agents. Indeed, in recent years, there has been a trend in the pharmaceutical industry to move away from "small molecule" drugs toward more complex macromolecular therapeutic agents (also known as "biological agents"). Such biological agents include protein-based therapeutic agents (especially antibodies, hormones, growth factors, and cytokines) and nucleic acid-based therapeutic agents (e.g., short interfering RNA, DNA / RNA vaccines, and gene therapy).

[0003] While the biologics market has grown significantly in recent years, the low availability of effective delivery systems (and feasible methods for manufacturing such delivery systems) has limited the diversity of molecular targets for such biotherapeutics, especially when the target is the cytoplasm. Indeed, the majority of commercially available approved peptide therapeutics act by targeting extracellular components, 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 is true for other hormone peptide therapeutics).

[0004] Similar challenges exist in the agrochemical industry, where protein-based pesticides are typically toxins that target extracellular components of pest cells. For example, Bacillus thuringiensis toxins are commonly used natural pesticides that bind to membrane receptors to exert their toxic effects.

[0005] Methods for cytoplasmic delivery of biological molecules have been developed in laboratory studies, which generally involve delivering the molecule in a lipid vehicle that fuses with the cell membrane and then releases its payload into the cytoplasm. However, such methods have limited use in, for example, pharmaceutical and veterinary medicine due to the nonspecific nature of how they deliver molecules into cells.

[0006] Bacterial secretion systems have been explored as potential delivery systems, given their natural ability to secrete (or more specifically, "inject") molecules into target cells. The most studied such secretion system is the "protein appendage" type III secretion system (T3SS) found in some Gram-negative bacteria. However, a notable drawback of these systems is that they always remain associated with the bacterial membrane, requiring the use of the actual bacterial cell (including the secretion system) as a delivery system. Therefore, it is difficult to fully control which molecules are transferred from the bacterium to the target cell (even when the target biological agent is overexpressed). This is because these secretion systems function by providing a connection (or channel) between the bacterial cytoplasm and the target cell cytoplasm, through which other components (potentially harmful to the host) can flow. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there is a need not only for improved delivery systems, but also for means to produce such systems that are compatible with molecules (payloads) having 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] The present invention is premised on the surprising finding that the toxigenic Photorhabdus Virulence Cassette (PVC) effector protein of the bacterium Photorhabdus contains a previously unknown "leader sequence" (or "leader peptide") that functions to package (or "load") the PVC effector into so-called PVC-needle complexes (e.g., "nanosyringes"), which subsequently deliver the PVC effector to target cells where it exerts its toxigenic effect (the PVC effector represents the payload of such nanosyringes). Furthermore, the inventors have found that such leader sequences can indeed be utilized to direct the packaging of payloads attached thereto into the PVC-needle complexes (and related / homologous complexes) of the well-characterized molecular delivery system of Photorhabdus. Thus, the newly discovered leader sequence surprisingly functions to load the PVC-needle complexes with molecular payloads (or "reactive sites").

[0010] Building on this finding, the inventors have developed advantageous practical utility for such leader sequences to package / load "heterologous" payloads (e.g., non-Photorhabdus molecules) into PVC needle composites, regardless of the size, molecular properties, or origin of the heterologous payload. DETAILED DESCRIPTION OF THE INVENTION

[0011] In a first aspect, the present invention provides the use of a Photorhabdus virulence cassette (PVC) effector leader sequence for packaging a payload into a PVC needle complex; the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably a polypeptide); The leader sequence and payload form an effector fusion that is distinct from the wild-type PVC effector protein. Provide use.

[0012] In one aspect, the present invention relates to the use of a PVC effector leader sequence to package a payload into a PVC needle complex; the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably a polypeptide); The leader sequence and payload form a distinct fusion with the PVC effector protein (e.g., wild-type PVC effector protein). Provide use.

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

[0015] This use (of the leader sequence) was demonstrated by expressing an effector fusion (tagged with a detection label) and a PVC-needle complex in a cell (e.g., a host bacterial cell), where the effector fusion is packaged 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 crushed form) via Western blot detection of the detection label, as outlined in the Examples. 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 is capable of packaging a payload (e.g., an effector) into a PVC needle complex, and is 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 can be contained within (or consist essentially of) amino acids 1-50 of a number of identified PVC effector polypeptide sequences. However, leader sequences of alternative lengths and located within PVC effectors are intended to be encompassed (e.g., provided that the leader sequence is capable of packaging a payload into a 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 consists essentially of amino acids 51 to the C-terminus of the PVC effector protein.

[0018] Thus, in one embodiment, the PVC effector leader sequence is comprised 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 consists essentially of) amino acids 1-50 or 2-50 (preferably 1-50) of a PVC effector polypeptide.

[0020] The term "wild-type PVC effector protein" is used interchangeably with the term "endogenous PVC effector protein" or simply "PVC effector protein" and refers to a (e.g., intact) PVC effector sequence having an endogenous leader sequence (i.e., endogenous to a given PVC effector, preferably amino acids 1-50 of the PVC effector) associated with an effector moiety (e.g., a payload, preferably amino acids 51 to the C-terminus of the PVC effector protein). Exemplary wild-type PVC effectors can comprise (or consist essentially of) the amino acid sequence of one or more sequences selected from SEQ ID NO:1-SEQ ID NO:46. Thus, the fusions / effector fusions of the 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 would be fused in the case of a wild-type PVC effector protein. By way of example, a fusion / effector fusion may include the leader sequence of a "Pnf" PVC effector protein (e.g., the leader of SEQ ID NO:78) fused to the effector portion of an hvnA (gene Plu1649) PVC effector protein (e.g., amino acids 51-295 of SEQ ID NO:46), but is not intended to refer to the leader sequence of a "Pnf" PVC effector protein (e.g., the leader of SEQ ID NO:78) fused to the effector portion of a Pnf PVC effector protein (e.g., amino acids 51-340 of SEQ ID NO:32).

[0021] On the other hand, the fusion / effector fusion may include, for example, a non-effector moiety, such as the leader sequence of the "Pnf" PVC effector protein (e.g., the leader of SEQ ID NO: 78) fused to a non-Photorhabdus protein, such as Cre recombinase. Thus, the leader sequence is useful for packaging a wide range of, for example, heterologous (non-wild-type) drugs into PVC needle complexes, opening up the possibility for the first time to use PVC needle complexes as a modular, versatile delivery system for delivering not only natural effectors but also "non-natural" payloads to cells. Thus, it is possible to produce PVC needle complexes with a payload of choice.

[0022] Another aspect of the present invention is a method of making a PVC needle composite containing a payload (e.g., in other words, a method of making a packaged PVC needle composite), comprising: a. contacting (e.g., in a host cell) a PVC-needle complex with an effector fusion comprising a PVC-effector leader sequence fused to a payload; b. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide); c. Effector fusions are distinguishable from wild-type PVC effector proteins A method is provided.

[0023] One aspect of the present invention is a method of producing a PVC needle composite containing a payload (e.g., in other words, a method of producing a packaged PVC needle composite), comprising: a. contacting (e.g., in a host cell) a PVC needle complex with a fusion, the fusion comprising a PVC effector leader sequence fused to a payload, wherein the leader sequence and payload form a fusion that is distinct from the PVC effector protein (e.g., a wild-type PVC effector protein); b. The payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide). A method is provided.

[0024] In one embodiment, the contacting can be performed in a cell (e.g., a bacterial host cell), in a cell lysate, or in a purified cell lysate (preferably, intracellularly). 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 in a cell (e.g., a bacterial host cell), in a cell lysate, a cell-free expression system, or in a purified cell lysate (preferably, in a cell, more preferably, a bacterial host cell).

[0025] The cassette (operon) encoding the PVC-needle complex can be operably linked to a first promoter, and the 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 expression systems in which the operon encoding the PVC is present in a first vector / plasmid (optionally operably linked to the first promoter) and the sequence encoding the effector fusion (leader sequence fused to the payload) is present in a second (preferably different) plasmid (optionally linked to the second promoter).

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

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

[0028] The payload can be, for example, a therapeutic payload such that the PVC needle composite is useful in medical treatment.

[0029] In a further aspect, the present invention provides a (packaged) PVC needle composite for use in a method of treatment comprising: a. comprises (e.g., is packaged with) an effector fusion comprising (or consisting essentially of) a PVC effector leader sequence fused to a payload; b. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide); c. Effector fusions are distinguishable from wild-type PVC effector proteins Provides PVC needle composite.

[0030] A further aspect of the invention is a (packaged) PVC needle composite for use in a method of treatment comprising: a. Carries a fusion comprising (or consisting essentially of) a PVC effector leader sequence fused to a payload (e.g., packaged with); b. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide); c. The fusion is distinct from the PVC effector protein (e.g., wild-type PVC effector protein). Provides PVC needle composite.

[0031] In one aspect, the present invention provides a method of treating a subject, comprising administering a (packaged) PVC needle composite to a subject (e.g., a patient); a. The PVC needle complex comprises (e.g., is packaged with) an effector fusion comprising (or consisting essentially of) a PVC effector leader sequence fused to a payload; b. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide); c. Effector fusions are distinguishable from wild-type PVC effector proteins A method is provided.

[0032] In other words, one aspect of the present invention is a method of treating a subject, comprising administering a (packaged) PVC needle composite to the subject (e.g., patient); a. The PVC needle complex is a PVC effector-leader sequence fused to a payload. holding (e.g., packaged with) a fusion comprising (or consisting essentially of) the sequence; b. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide); c. The fusion is distinct from the PVC effector protein (e.g., wild-type PVC effector protein). A method is provided.

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

[0034] The subject can be a mammalian subject, preferably a human subject.

[0035] The terms "PVC needle composite carrying an effector fusion" and "PVC needle composite comprising an 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 sequence and payload that remain in contact (e.g., fused) after packaging into a PVC needle complex (e.g., the leader sequence has not been cleaved from the payload), as well as combinations of PVC effector leader sequence and payload that are no longer in direct contact (e.g., no longer fused after cleavage of the leader sequence from the payload).

[0037] As used herein, the term "treat" or "treating" encompasses prophylactic treatment (e.g., to prevent the onset of a disease) as well as neutralizing treatment (treatment of a subject already suffering from a disease). Preferably, the term "treat" or "treating" as used herein refers to neutralizing treatment. The term "treat" or "treating" encompasses treating both the disease and its symptoms. In some embodiments, "treat" or "treating" refers to the symptoms of a disease.

[0038] Thus, the PVC needle complexes can be administered to a subject in a therapeutically or prophylactically effective amount.

[0039] A "therapeutically effective amount" is any quantity of (packaged / loaded) PVC needle complex that, when administered alone or in combination (e.g., with another therapeutic agent, administered concurrently or sequentially and acting additively or synergistically) to a subject to treat a disease (or a symptom thereof), is sufficient to effect such treatment of the disease, or a symptom thereof.

[0040] A "prophylactically effective amount" is any amount of (packaged / loaded) PVC needle composite 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 a disease (or symptoms thereof). In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of a disease. "Inhibiting" onset means reducing the likelihood of a disease (or its symptoms) developing, or preventing onset entirely.

[0041] In a related embodiment, a (packaged) PVC needle complex comprising (e.g., carrying / packaged with) an effector fusion; a. the effector fusion comprises a PVC effector leader fused to a payload comprising (or consisting essentially of) a sequence (or in other words, the effector fusion is formed by a PVC effector leader sequence and a payload); b. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof; c. Effector fusions are distinguishable 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 carrying (e.g., packaged with) a fusion; a. the fusion comprises (or consists essentially of) a PVC effector leader sequence fused to a payload (or in other words, the fusion is formed by a PVC effector leader sequence and a payload); b. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide); c. The fusion is distinct from the PVC effector protein (e.g., wild-type PVC effector protein). A (packaged) PVC needle composite is provided.

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

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

[0045] In a further embodiment of the present invention, there is provided a method for controlling pests, comprising: a. contacting the pest, or a target area containing the pest, with a (packaged) PVC needle complex containing (e.g., carrying / packaged with) an effector fusion; b. the effector fusion comprises (or consists essentially of) a PVC effector leader sequence fused to a payload (or in other words, the effector fusion is formed by a PVC effector leader sequence and a payload); c. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably a polypeptide); d. Effector fusions are distinguishable from wild-type PVC effector proteins A method is provided.

[0046] One aspect of the present invention is a method for controlling pests, comprising: a. contacting the pest, or a target area containing the pest, with (e.g., packaged with) a PVC needle composite carrying a fusion product; b. the fusion comprises (or consists essentially of) a PVC effector leader sequence fused to a payload (or in other words, the fusion is formed by a PVC effector leader sequence and a payload); c. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably a polypeptide); d. The fusion is distinct from the PVC effector protein (e.g., wild-type PVC effector protein). A method is provided.

[0047] The terms "PVC needle composite carrying an effector fusion" and "PVC needle composite comprising an 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., predicted or suspected to be present).

[0049] Thus, in one embodiment, the target area can be contacted before and / or when the pest is present. The target area can be near (e.g., in close proximity to) the pest. Alternatively, the target area can be an area that a user desires to protect from the pest. For example, the target area can include plants and / or plant products.

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

[0051] Examples of such pests include one or more insects, mites, woodlice, pill bugs, centipedes, mollusks, millipedes, protozoans, fungi (fungi), helminths, and / or blood-borne parasites. Pests may be at any stage of development, for example, juvenile and / or adult pests (e.g., adults).

[0052] The present invention can be used to target a variety of agricultural, industrial, household and horticultural pests.

[0053] In one embodiment, the pest is an insect, a mite, a woodlouse, a pill bug, a centipede, a mollusk and / or a millipede. Suitably, the pest may be an insect and / or a mite (preferably an insect).

[0054] Examples of suitable insects include insects from the orders Lepidoptera, Coleoptera, Diptera, Blattella, Hymenoptera, Termitida, Orthoptera, Thymidia, and / or Dermaptera. In one embodiment, the Lepidoptera insects can be one or more moths and / or butterflies. Suitable moths include the tobacco hornworm moth (Manduca Sexta) and / or the wax moth (Galleria mellonella).

[0055] In one embodiment, the insect of the order Coleoptera is a European chafer larva, a northern masked chafer larva, a southern chafer larva, or a The insects may be one or more of southern masked chafer larvae, Japanese beetle larvae, chafer larvae, nut weevils, strawberry weevils, clay-colored weevil, Colorado potato beetle, and / or click beetles. In another embodiment, the insect of the order Diptera may be one or more of leatherjackets (e.g., juvenile crane flies), onion flies, rhododendrons, carrot rust flies, fungus gnats, and / or mosquitoes. In another embodiment, the insect of the order Blattella may be one or more cockroaches, preferably selected from 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 malodorous house ant, a pavement ant, an Argentine ant, a house ant, a tan crazy ant, a harvester ant, a fire ant, a Southern fire ant, a yellow rat ant, or a yellow rat ant. In another embodiment, the insect may be one or more of an ant and / or a fire ant. may be a yellow jacket.

[0057] In one embodiment, the insect of the order Isoptera may be a termite. Preferably, the termite may be one or more of a wetwood termite, a drywood termite, and / or a subterranean termite. In another embodiment, the insect of the order Orthoptera may be one or more of a cricket, a grasshopper, and / or a locust. In one embodiment, the insect of the order Thymidera may be a silverfish. In another embodiment, the insect of the order Dermaptera may be an earwig.

[0058] Examples of suitable mollusks include slugs and / or snails.

[0059] In one embodiment, the pest is a protist, hi 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 spp., 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 pest 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 strigosozonata).

[0061] In one embodiment, the fungus is a Candida spp., including 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. carpophy 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. insectorum, C. inter 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. tolerans, C. tropicalis, C. tsuchiyae, C. sinolaborantium, C. sojae, C. subhashii, C. viswanathii, C. u C. utilis, C. ubatubensis, and / or C. zempf The Candida spp. may be one or more selected from C. zemplinina, C. albicans, and 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 Annelida, Platyhelminthes, Nematoda, and / or Acanthocephala. In one embodiment, the helminth is The parasitic flatworm may be one or more selected from the classes Cestoda, Trematoda, and / or Monogenea. wherein the helminth is a parasitic nematode, such as a roundworm (Ascaris), a filaria, a hookworm, a pinworm (Enterobius), and / or a whipworm. (Trichuris trichiura).

[0063] In one embodiment, the pest is a blood-borne parasite, such as Trypanosoma spp. (e.g., Trypanosoma brucei and / or T. cruzi), Babesia spp. (e.g., Babesia microti), Leishmania spp., Plasmodium spp. (e.g., P. falciparum), and / or Toxoplasma spp. (e.g., Toxoplasma gondii) .

[0064] PVC needle composites for pest control are preferably environmentally safe (eg, environmentally safe pesticidal compositions).

[0065] Other advantageous uses include, for example, delivering payload to cells during laboratory research.Such cells can be part of in vitro cell system, or can be cells of animals (for example, research animal models).In addition, or alternatively, cells can be contained in ex vivo system, for example, organoid.

[0066] Another aspect of the present invention is an in vitro (and / or ex vivo) method of delivering a payload into a cell, comprising: a. contacting a cell with a (packaged) PVC needle complex containing (e.g., carrying / packaged with) an effector fusion; b. the effector fusion comprises (or consists essentially of) a PVC effector leader sequence fused to a payload (or in other words, the effector fusion is formed by a PVC effector leader sequence and a payload); c. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably a polypeptide); d. Effector fusions are distinguishable from wild-type PVC effector proteins A method is provided.

[0067] One aspect of the present invention is an in vitro (and / or ex vivo) method of delivering a payload into a cell, comprising: a. contacting a cell with (e.g., packaged with) a PVC needle complex carrying a fusion product; b. the fusion comprises (or consists essentially of) a PVC effector leader sequence fused to a payload (or in other words, the fusion is formed by a PVC effector leader sequence and a payload); c. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably a polypeptide); d. The fusion is distinct from the PVC effector protein (e.g., wild-type PVC effector protein). A method is provided.

[0068] In one aspect, the invention provides an effector fusion comprising (or consisting essentially of) a PVC effector leader sequence fused to a payload (or in other words, an effector fusion formed by a PVC effector leader sequence and a payload); a. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof; B. Distinguishable from wild-type PVC effector proteins Effector fusions are provided.

[0069] One aspect of the invention is a fusion comprising (or consisting essentially of) a PVC effector leader sequence fused to a payload (or in other words, a fusion formed by a PVC effector leader sequence and a payload); a. the payload is one or more selected from a polypeptide, a nucleic acid, or a combination thereof (preferably, a polypeptide); b. Distinguishable from a PVC effector protein (e.g., a wild-type PVC effector protein) A fusion product is provided.

[0070] In one embodiment, the fusion / effector fusion is an isolated fusion / effector fusion (eg, an isolated, non-naturally occurring fusion / effector fusion).

[0071] The invention encompasses a nucleic acid comprising a nucleotide sequence encoding a fusion / effector fusion, and / or an expression vector comprising said nucleic acid. Also encompassed are host cells comprising said nucleic acid and / or expression vector.

[0072] As described above, the present inventors were the first to discover and actually utilize the leader sequence described herein.

[0073] Thus, another aspect of the present invention provides an isolated PVC effector leader sequence, eg, the isolated PVC effector leader sequence is capable of packaging a payload into a PVC needle complex.

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

[0075] The isolated PVC effector leader sequence can be recombinant, synthetic, and / or purified. The isolated nucleic acid encoding the PVC effector leader sequence can be recombinant, synthetic, and / or purified. and / or may be purified.

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

[0077] Photorhabdus is a genus of bacteria in the family Enterobacteriacae. and three previously recognized species: P. luminescens, P. asymbiotica, and P. temperata. Important strains include P. asymbiotica subsp. australis and P. luminescens subsp. laumondii. Currently available genome sequences are available on GenBank. Photorhabdus asymbiotica ATCC43949 Complete genome - GenBank accession number FM162591.1; Photorhabdus laumondii subsp. Species laumondii (Photorhabdus laumondii subsp. laumondii) strain TT01 chromosome, complete genome - GenBank accession number: CP024901.1).

[0078] References to "Photorhabdus luminescens subsp. laumondii" are used interchangeably herein with "Photorhabdus luminescens subsp. laumondii TT01," "Photorhabdus laumondii subsp. laumondii strain TT01," and "P. luminescens TT01." It is possible.

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

[0080] Each of these species contains at least one operon known as the Photorhabdus virulence cassette (PVC) operon, which encodes a PVC-needle complex that may be referred to herein as a "nanosyringe." Photorhabdus is typically found originally as an insecticidal bacterium after regurgitation from (symbiotic) insect pathogenic Heterorhabditis sp. nematodes (e.g., food and insectivores). Considering the need for PVC needle complexes to inhibit insects (to avoid competition for resources from insects), it is understood that PVC needle complexes function primarily to deter insects. Indeed, it has been shown that isolated PVC needle complexes (carrying / packaged with natural effector toxins, e.g., Pnf) can be used to kill insect larvae; see Example 2. The Photorhabdus virulence cassette represents one of at least four well-characterized toxin delivery systems for Photorhabdus. Other major classes of Photorhabdus proteinaceous insecticidal toxins include "toxin complexes (Tc)," "binary PirAB toxins," and "makes caterpillars floppy" (Mcf) toxins.

[0081] The term "Photorhabdus virulence cassette" (PVC) (used interchangeably herein with the term "PVC operon") refers to a distinct operon in the Photorhabdus genome that contains genes encoding polypeptide subunits that, when expressed, assemble to provide the macromolecular PVC needle complex. The molecular architecture of these cassettes is described, for example, in The Molecular Biology of Photorhabdus Bacteria (Springer International Publishing, 1999), incorporated herein by reference. The PVC (operon) is well characterized and described in (Issue AG 2017, ISBN: 978-3-319-52714-7, Chapter 10, pages 159-177). The PVC (operon) typically contains approximately 16 genes (pvc1 to pvc16) encoding structural proteins that assemble to provide the "PVC needle complex," followed by one or more genes at the 3' end that typically encode PVC effector genes with toxic activity (and that are typically homologs of typical T3SS-like effectors). The Photorhabdus genome typically contains multiple such cassettes (e.g., at least four), often associated with different effector payloads, or even multiple effector payloads.

[0082] Three classes of PVC structural operons (classes I, II, and III) have been observed in the genomes of Photorhabdus and members of other genera. PVCs within each class are similar in terms of the number and type of genes encoding structural proteins they contain (see Figure 1(B)). More specifically, class I PVCs (which may be referred to herein as "prototypical PVCs") contain 16 conserved genes (pvc1-16). Class II PVCs lack the pvc13 host cell-binding fiber, and (without being bound by theory) we believe that pvc3 may be a minor specialized sheath subunit that attaches the pvc13 fiber protein onto the PVC-needle complex (nanosyringe). Thus, this class may be "nonspecific" and inject payloads into multiple (potentially any) cell types. Class III is similar to class I, but has an additional Pvc0 gene at the beginning of the operon (of unknown function) and two additional genes encoded between pvc13 and pvc14 that resemble "invasion"-type protein genes. This class is typically found in human clinical isolates of Photorhabdus, and we have shown that optimal transcription of PVC class III can occur when the strain (harboring a PVC operon encoding the PVC class III operon) is grown at 37°C and exposed to human serum, suggesting that this class may be a mammalian-adapted form of the PVC-needle complex.

[0083] An example cassette (PVC) is shown in Figure 1(D) and contains Photorhabdus asymbiotica ATCC 43949 (available from ATCC, accession no. 1 shows a map of the model "Class I" PVC operon of the PVC gene (ATCC 43949), , associated with the downstream effector gene "PAU_03332" (encoding a Pnf protein effector, e.g., SEQ ID NO: 32). This model operon is designated PaATCC43949PVCpnf. This operon contains 16 structural genes (pvc1 to 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 genes pvc1 to 16 correspond to genes PAU_03353 to PAU_03338 in the sequence of GenBank accession number FM162591.1, and are represented by SEQ ID NO: 9. It is represented by an array of 3.

[0084] An example PVC operon (e.g., encoding structural genes but not PVC effectors) is provided in SEQ ID NO:93 (encoding the operon shown schematically in Figure 1(D)), and other examples are SEQ ID NO:94 and SEQ ID NO: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 conjugates from any one of Classes I to III can be used for a variety of applications. However, certain classes of PVC needle conjugates are particularly suitable for delivery to specific cell types. For example, a PVC needle conjugate for delivery of a payload to mammalian cells may be preferably a member of Class III. A PVC needle conjugate for delivery of a payload to insect cells (e.g., insects) may be preferably a member of Class I. member (e.g., P. asymbiotica PVCpnf, encoded by SEQ ID NO: 93, expressed in E. coli from a cosmid clone). could be.

[0086] Therefore, as will be understood by those skilled in the art, the term "PVC-needle complex" (used herein interchangeably with the terms "PVC-needle complex delivery system" and "nanosyringe") refers to a macromolecular protein complex containing polypeptide subunits encoded by the PVC (operon) of the bacterium Photorhabdus. The PVC-needle complex is assembled into a nanosyringe structure with a physical structure superficially similar to that of antibacterial R-type pyocins (see Example 3). Functional and molecular studies have demonstrated that the PVC-needle complex can be packaged (loaded) with a PVC effector protein (i.e., the PVC effector protein is packaged within or on it), released from the bacterium, and then inject the PVC effector into target cells, resulting in toxicity of the PVC effector protein.

[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. PVC-like elements are not limited to Photorhabdus; a well-characterized homologous (to the PVC operon) operon is present on the pADAP plasmid of the entomopathogenic bacterium Serratia entomophila. Furthermore, a similar and (at least partially) homologous PVC-like "injectosome" needle complex system is used by the bacterium Pseudoalteromonas luteoviolacea (e.g., the marine worm Kasanekan). It is used to control metamorphosis in Hydroides elegans. Operon-encoded structures with homologous sequences exist in other Enterobacteriaceae (e.g., Yersinia spp.) and are similar to the leader sequences described herein. Each of these (PVC-like) structures is encompassed by the term "PVC needle composite" as used herein.

[0088] Thus, the PVC-needle complex is a "nanosyringe" complex with a polypeptide encoded by an effector gene packaged (loaded) within or at its end (tip), thus representing the "payload" or "reactive site" of the PVC-needle complex. The present inventors have demonstrated that the PVC-needle complex itself (still loaded with the payload) is freely released (e.g., secreted) from Photorhabdus cells, interacts with the membrane of a target cell, and injects the payload into the cytoplasm of that cell. Indeed, the present inventors have successfully expressed and loaded PVC-needle complexes in a heterologous expression system, isolated / purified them, and used them to inhibit (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) to a sequence selected from SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95 (e.g., SEQ ID NO: 93).

[0090] In one embodiment, the PVC needle composite is encoded by a sequence selected from SEQ ID NO:93, SEQ ID NO:94, and SEQ ID NO:95 (eg, SEQ ID NO:93).

[0091] Leader / signal sequences are typically peptides and are present at the N-terminus of the majority of (newly) expressed proteins that are destined for the secretory pathway (e.g., the protein Many proteins require signal sequences for entry into the Golgi or endoplasmic reticulum.

[0092] The term "leader sequence" (used interchangeably herein with the terms "leader peptide," "signal sequence," "targeting signal," "localization signal," "localization sequence," and "transport peptide") as used herein in the context of "PVC effector leader sequence" refers to a polypeptide sequence that functions to direct a PVC effector into the interior or end (tip) of a PVC-needle complex; thus, the leader sequence functions to package the PVC effector into 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 can be an assembled PVC-needle complex. The term "PVC-needle complex" can refer to a fragment of a PVC-needle complex (e.g., the leader sequence contacts the fragment, and optionally, the PVC-needle complex assembles around the leader sequence-payload "effector fusion").

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

[0094] In one embodiment, the leader sequence comprises (or consists essentially of) amino acid residues 1-50 of a PVC effector (e.g., a PVC effector protein). Reference to "amino acid residues 1-50" encompasses "amino acid residues 2-50" that exclude the N-terminal methionine, e.g., are truncated. The leader sequence can be an N-terminal 50 amino acid fragment of a PVC effector (e.g., a fragment comprising or consisting essentially of ≦45, ≦35, ≦25, or ≦15 amino acids), provided that the fragment is capable of packaging a payload into a PVC-needle complex.

[0095] In one embodiment, a leader sequence of the invention (e.g., an isolated leader sequence) comprises (or consists essentially of) an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to one or more sequences selected from SEQ ID NOs: 47-92 (preferably SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO: 92), e.g., provided that the leader sequence is capable of packaging a payload into a PVC-needle complex. In a preferred embodiment, the leader sequence comprises (or consists essentially of) an amino acid sequence having at least 60% sequence identity to one or more sequences selected from SEQ ID NOs: 47-92 (preferably SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO: 92), e.g., provided that the leader sequence is capable of packaging a payload into a PVC-needle complex. In a more preferred embodiment, the leader sequence comprises (or consists essentially of) one or more amino acid sequences selected from SEQ ID NO: 47 to SEQ ID NO: 92 (preferably SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO: 92). In one embodiment, the leader sequence comprises (or consists essentially of) an amino acid sequence selected from SEQ ID NO: 47 to SEQ ID NO: 92 (preferably SEQ ID NO: 50, SEQ ID NO: 68, SEQ ID NO: 71, SEQ ID NO: 76, SEQ ID NO: 78, or SEQ ID NO: 92).

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

[0098] Without being bound by theory, it is believed that the leader sequences share a "chemical composition consensus" based on amino acid properties. More specifically, the leader sequences contain a similar charge pattern, which includes two negatively charged regions followed by a positively charged region (e.g., [-ve][+ve][-ve][+ve]) (see Figure 9). This is consistent with the leader sequences of type 2 secretion system toxins, which contain a charge / property pattern of [+ve][hydrophobic][+ve][C]. A further theory postulates that the leader sequences share a typical "helix-turn-helix" structure. Another theory is that the leader sequences form a structure (e.g., encoded by gene PAU_03339 (pvc15) in the model operon of Figure 1(D)) that is recognized by an ATPase enzyme present within or at the end (e.g., tip) of the PVC-needle complex.

[0099] The term "PVC effector" (used interchangeably with the terms "PVC operon-encoded effector" and "PVC effector protein") refers to an effector polypeptide encoded by the Photorhabdus PVC operon, more particularly (and typically) found immediately downstream (3') of the structural genes of said operon (preferably immediately or immediately downstream of pvc16, and typically within 5 kb). The term "PVC effector" preferably encompasses homologs thereof. Thus, a leader sequence can also be from a polypeptide encoded by a gene that is homologous to a gene encoding a PVC effector; see Table 1 for such homologs. In practice, identification of PVC effectors is aided by detecting homology of genes downstream of pvc16 with known toxin polypeptides (e.g., genes encoding said toxin polypeptides). As will be understood by those skilled in the art, the term "homologue" preferably refers to a gene that is derived from the same ancestral gene and shares similar functions; such a gene (or the polypeptide encoded thereby) is homologous to a gene encoding a PVC effector. Homologues can be from the genome of Photorhabdus species or from species other than Photorhabdus species. Examples of suitable homologues are outlined in Table 1.

[0100] We have identified the three most common (best characterized) strains of Photorhabdus as well as P. asymbiotica Thai strain PB68.1. The genes encoding the PVC effectors of these PVC needle complexes of PVC have been elucidated and characterized in detail. This was done based on the proximity of the gene linkage to the 3' end of the PVC structural genes of the operon and an analysis of the predicted function of the protein sequence of the effector (e.g., a homolog of a known effector / toxin protein). More specifically, PVC effectors (e.g., genes encoding PVC effectors) are typically identified as open reading frames (OPFs) (typically with several or no intervening genes) that share homology with genes encoding known toxin polypeptides (e.g., homologs outlined in Table 1) and are typically located within a distance of 1 to 5 kilobases (kb) (e.g., within 1 kb) downstream of the last structural gene of the PVC operon (e.g., pvc16). Typically, there are no "non-toxin-like" ORFs between the end of the operon (encoding the PVC needle complex) and the PVC effector genes. Although other small predicted genes (e.g., one or two) may be present in these regions, these other genes are not assigned as PVC effectors (due to lack of homology with known effector / toxin genes mentioned above).

[0101] To assign putative PVC effector genes (e.g., ORFs within a distance of 5 kb, e.g., 1 kb, downstream of the last structural gene of the PVC operon) as PVC effector genes, we used a combination of BlastP and HHPRED (https: / / toolkit.tuebingen.mpg.de / # / tools / hhpred). Putative PVC effector genes were compared with known toxin codes. They were assigned as PVC effector genes based on direct homology to toxin genes, similarity to toxin protein families, proximity to the PVC operon (e.g., within 1–5 kb downstream of pvc16, the last structural gene of the PVC operon), and / or domain similarity of predicted secondary structure to known toxins.

[0102] Thus, a PVC effector (gene) can be identified (within the Photorhabdus genome) by (i) identifying pvc16 (e.g., via sequence homology to a known pvc16), (ii) identifying an ORF 3' to pvc16, preferably ≦5 kb downstream of pvc16), and (iii) confirming that the ORF encodes a PVC effector through identification of sequence homology to a known gene encoding a toxin polypeptide (e.g., a toxin protein listed in the column of Table 1 labeled "Homolog").

[0103] By way of example, the PVC effector gene PAU_03337 (referred to herein as "sepC" due to its homology to the virulent sep gene) is located 325 base pairs (bp) downstream of pvc16 (PAU_03338) of the PVC operon, referred to herein as PVCpnf (e.g., that of SEQ ID NO: 93), found in P. asymbiotica ATCC 43949. 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. asinbii, accessible via GenBank accession number FM162591.1. This can be explained by reference to the P. asymbiotica ATCC 43949 complete genome (see also, e.g., 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: and PAU_03338 is annotated to be located in the genome as follows: complementary strand (3914573..3915454). No other ORFs (encoding effectors or otherwise) are found between the two genes.

[0105] An additional PVC effector gene associated with the PVC operon designated herein as PVCpnf (e.g., of SEQ ID NO: 93), namely PAU_03332 (referred to herein as "pnf"), is located 3535 bp downstream of pvc16 (PAU_03338).

[0106] The PVC effector gene PAU_02095 (referred to herein as "Rhs-like toxin effector" due to its homology to virulent Rhs toxin genes) is located 3961 bp downstream of pvc16 (PAU_02099) in the PVC operon, referred to herein as PVClopT (e.g., that of SEQ ID NO: 94), found in P. asymbiotica ATCC 43949. That is, the start codon of PAU_02095 is It begins 3961 bp downstream of the end of the stop codon of PAU_02099.

[0107] In a further example, the PVC effector of gene PAU_02009 (referred to herein as "cif" due to its predicted 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 to herein as PVCcif, found in P. asymbiotica ATCC43949.

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

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

[0110] Collectively, 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 sequences can be cloned and fused to various payloads to be packaged into PVC needle complexes; see Examples 3 and 4. Thus, a PVC effector (as translated) 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., "effector portion") or "payload."

[0111] While the Photorhabdus genome sequence continues to be revised, this consolidated list of PVC effector genes represents a comprehensive description of such effectors and is based on currently available sequence data for the most common (best characterized) Photorhabdus strains, providing those of skill in the art with an understanding of the term "PVC effector" and the sequences of these PVC effectors (and, e.g., how to search / mine for additional PVC effectors in alternative (genomic) sequences). As noted above, the inventors have discovered that PVC effector proteins contain leader sequences necessary (and sufficient) to direct the packaging / loading of 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 exemplary amino acid sequences of (or highly similar to) the PVC effectors described herein, the sequences of which can be accessed via GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ).

[0116] Locus tags (starting with "PAU" or "Plu") are available via GenBank as above. The locus tags correspond to the locus tags assigned to the effectors in the available genome sequences. "PAT" (referring to strain P. asymbiotica Thai strain PB68.1) and "PAK" (referring to strain P. asymbiotica Thai strain PB68.1) Locus tags beginning with P. asymbiotica (referring to P. asymbiotica Kingscliff) Upon identification, the PVC effector genes within the genome of the strain were assigned by the inventors (in a manner consistent with the locus tags of publicly available sequences).

[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: 14), Column number 14), PAT_02309 (SEQ ID NO: 15), PAT_02310 (SEQ ID NO: 16), PAT_02956 (SEQ ID NO: 17), PAT_02957 (SEQ ID NO: 18), PAT_03171 (SEQ ID NO: 19), PAT_03172 (SEQ ID NO: 20), PAT_03177 (SEQ ID NO: 21), PAU_02009 (SEQ ID NO: 22), PAU_02010 (SEQ ID NO: 23), PAU_02095 (SEQ ID NO: 24), PAU_02096 (SEQ ID NO: 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 91 (SEQ ID NO: 38), Plu1712 (SEQ ID NO: 39), Plu1713 (SEQ ID NO: 40), Plu1714 (SEQ ID NO: 41), Plu2400 (SEQ ID NO: 42), Plu2401 (SEQ ID NO: 43), Plu2514 (SEQ ID NO: 44), Plu2515 (SEQ ID NO: 45), Plu1649 (SEQ ID NO: 46), or a combination thereof (with the SEQ ID NO of the encoded PVC effector protein in parentheses).

[0119] In one embodiment, the PVC effector is selected from the group consisting of PAU_02009 (SEQ ID NO: 22), PAU_02010 (SEQ ID NO: 23), PAU_02095 (SEQ ID NO: 24), PAU_02096 (SEQ ID NO: 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), Plu16 Plu1649 (SEQ ID NO:46), or a combination thereof. These gene names correspond to the "locus tags" of the PVC effector genes in the Photorhabdus genome sequence accessible via GenBank, as described above. The T and PAK locus tags were generated by us so that the nomenclature is consistent with the PAU and Plu locus tags of the publicly available genome sequence.

[0120] Thus, a PVC effector may 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 (SEQ ID NO: 4), PAU_02009 (SEQ ID NO: 22), PAU_02096 (SEQ ID NO: 25), PAU_02806 (SEQ ID NO: 30), PAU_03332 (SEQ ID NO: 32), Plu1651 (SEQ ID NO: 34), Plu1649 (SEQ ID NO: 46), or a combination thereof (with the SEQ ID NO: 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 (SEQ ID NO: 30), PAU_03332 (SEQ ID NO: 32), Plu1651 (SEQ ID NO: 34), Plu1649 (SEQ ID NO: 46), or a combination thereof (with the SEQ ID NO of the encoded PVC effector in parentheses).

[0123] The PVC effector may have a sequence having 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 having 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 present inventors have identified the leader sequences of the gogB1 (PAU_02806) and Pnf (PAU_03332) PVC effectors as being 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 comprises (or consists essentially of) one or more amino acid sequences selected from SEQ ID NO:1 to SEQ ID NO:46 (e.g., SEQ ID NO:22 to SEQ ID NO:46), or a combination thereof. For example, the PVC effector can comprise (or consist essentially of) 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.

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

[0127] The term "packaging" (used interchangeably with the terms "trans-packaging" and "loading") refers to the directing of a payload by a leader sequence of the present invention (to which the payload is attached / 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 a target cell. Thus, the payload can be packaged within the PVC needle complex or can be packaged at the end (or tip) of the PVC needle complex (e.g., at least a portion of the payload can be outside the PVC needle complex).

[0128] The term "payload" (used herein synonymously with the term "reactive site") refers to a molecule that is packaged within or at the end (tip) of an assembled PVC needle complex and subsequently delivered (e.g., injected) into a (target) cell. In wild-type Photorhabdus, the payload is a PVC effector (more particularly, the effector portion of said PVC effector) that is 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) encodes an adenylate cyclase effector. See the model PVC operon in Figure 1(D) with PAU_03332 (listed as PVCpnf21) (e.g., SEQ ID NO: 32), which encodes a Pnf effector; and PAU_03332 (listed as PVCpnf21) (e.g., SEQ ID NO: 33), which encodes a Pnf effector.

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

[0130] Effector fusions are not limited to fusion complexes that include a leader sequence fused to a toxic payload (e.g., the leader could be fused to a therapeutic payload). Thus, the term "effector" as used in the context of "effector fusion" refers to a payload packaged in a PVC needle complex (which could provide various effects, e.g., toxigenic 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 interchangeably with the terms "leader sequence-payload fusion" and / or "leader sequence-payload conjugate."

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

[0133] Analysis of the size (e.g., polypeptide length) and structure of various native PVC effector payloads encoded by Photorhabdus indicates the existence of a wide range of different PVC effector lengths and structures, demonstrating that the applicability of the PVC needle conjugate delivery system of the present invention is not limited by the size or properties of the intended payload. Taken together, no specific secondary structure, biophysical properties, or cargo length are required, confirming that PVC needle conjugates can be utilized as versatile, multifunctional delivery vehicles.

[0134] The payload can be one or more selected from a polypeptide (e.g., a polypeptide payload), a nucleic acid (e.g., a nucleic acid payload), or a combination 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 pathways), anti-apoptotic peptides (e.g., BH4), and nicotinamide adenine dinucleotide quinone internal oxidoreductase (Ndi1). Examples of suitable nucleic acid-modifying enzymes include a PHOX complex subunit, myotubularin, a nucleic acid (preferably, DNA)-modifying enzyme, or a combination thereof. Examples of suitable 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 can be, for example, tBid (SEQ ID NO: 109) and / or BaxBH3 peptide (aa 59-73) (SEQ ID NO: 111).

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

[0137] Nucleic acid payloads can be conjugated / crosslinked to the leader sequences of the present invention. For example, copper-free click chemistry (e.g., strain-promoted alkyne azide cycloaddition (SPAAC)) can be used to crosslink nucleic acids to leader sequences. Examples of nucleic acid payloads include primers, mRNAs, nucleic acid analogs, aptamers, small interfering RNAs (siRNAs), microRNA therapeutic inhibitors (anti-miRs), microRNA therapeutic mimics (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., via a spacer) to the payload. The leader sequence can be fused covalently or non-covalently to the payload. In a preferred embodiment, the leader sequence is covalently fused to the payload. For example, the fusion / effector fusion can be a (recombinant) fusion protein comprising (or consisting essentially of) a PVC effector leader sequence fused to a (polypeptide) payload.

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

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

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

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

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

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

[0145] In one embodiment, the host cell is a Photorhabdus cell, and optionally, the Photorhabdus cell comprises a PVC operon operably linked to an inducible promoter (see, e.g., Example 3). The PVC operon can be endogenous to the Photorhabdus cell (e.g., the PVC operon can be PVCu4). Suitably, the Photorhabdus cell can be available from the ATCC under accession number ATCC 29999.

[0146] Sequences (e.g., leader sequences and / or nucleic acid sequences) of the present invention include sequences removed from their naturally occurring environment, recombinant or cloned (e.g., DNA) isolates, and chemically synthesized analogs or biologically synthesized analogs in 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 amounts of the leader sequences and / or polynucleotides can be produced by replication and / or expression in a suitable host cell. A natural or synthetic DNA fragment encoding a desired fragment is typically incorporated into a recombinant nucleic acid construct, typically a DNA construct, capable of introduction into and replication within a prokaryotic or eukaryotic cell. Usually, the DNA construct is suitable for autonomous replication in a unicellular host, e.g., yeast or bacteria, but introduction and integration into the genome of cultured bacteria, insects, mammals, plants, or other eukaryotic cell systems may also be intended.

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

[0149] The term "isolated" in the context of the present invention, when applied to a leader sequence or nucleic acid sequence, indicates that the leader sequence and / or polynucleotide sequence has been removed from its natural genetic environment and is therefore free of other extraneous or undesired coding sequences (but may include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators), and is in a form suitable for use in genetically engineered protein production systems. Such isolated molecules are ones that have been separated from their natural environment.

[0150] sequence homology Any of a variety of sequence alignment methods, including but not limited to global, local, and hybrid methods such as segmental approaches, can be used to determine percent identity. Protocols for determining percent identity are routine procedures within the skill of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding the scores of each residue pair and by imposing gap penalties. Non-limiting methods include CLUSTAL W (see, 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 (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Proteins. See ... Quence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs that are shared by all of the input sequences. Non-limiting examples of methods include Match-box (see, 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 (see, e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214(1993)); Align-M (see, e.g., Ivo Van Walle et al., Align-MA: A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435(2004)).

[0151] Thus, percent sequence identity is determined by conventional methods. See Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, the two amino acid sequences were aligned to obtain the following: (amino acids are indicated by standard single-letter codes), a gap opening penalty of 10, a gap extension penalty of 1, and the nucleotide sequence of Henikoff and Henikoff (supra). The alignment score is optimized using the "blosum 62" scoring matrix (http: / / www.blosum62.org / ).

[0152] The "percent 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. Thus, percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of percent sequence identity can also take into account the number of gaps that need to be introduced to optimize the alignment of two or more sequences, and the length of each gap. Sequence comparison and percent identity determination 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] The percent identity is then calculated as:

number

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

[0155] Conservative amino acid substitutions Basic: arginine, lysine, histidine Acidic: glutamic acid, aspartic acid Polarity: Glutamine, Asparagine Hydrophobic: leucine, isoleucine, valine Aromatic: phenylalanine, tryptophan, tyrosine Small molecules: glycine, alanine, serine, threonine, methionine

[0156] In addition to the 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 polypeptides of the invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids can be substituted for polypeptide amino acid residues. The polypeptides of the invention can also include non-naturally occurring amino acid residues.

[0157] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used to suppress nonsense mutations using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of the plasmid containing the nonsense mutation is carried out in a cell-free system containing E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, for example, Robertson et al. (See, e.g., Ellman et 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 is carried out in Xenopus oocytes. This is accomplished by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996). In a third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired unnatural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The unnatural amino acid is incorporated into the polypeptide in place of its natural counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0158] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can be substituted for amino acid residues in the polypeptides of the invention.

[0159] Essential amino acids in the polypeptides 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). Sites of biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutations of putative contact site amino acids. See, e.g., 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. Essential amino acids Identity can also be inferred from analysis of homology with related components of the polypeptide of the invention (eg translocation or protease components).

[0160] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, 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). Briefly, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible 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. Pat. No. 5,223,409; Huse, WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., J. Med. Chem. Soc. 1999; 1999; 2000). et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0161] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, 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). Briefly, these authors disclose a method for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible 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. Pat. No. 5,223,409; Huse, WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., J. Med. Chem. Soc. 1999; 1999; 2000). et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0162] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. 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 those skilled in the art with general knowledge of many of the terms used in this disclosure. To provide.

[0163] The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0164] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0165] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some instances, the term "amino acid sequence" is synonymous with the term "peptide." In some instances, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional one-letter and three-letter codes for amino acid residues may be used. Amino acids as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN) are used interchangeably. It is also understood that a polypeptide can be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.

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

[0167] Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded from the range, and if either, neither, or both limits are included in the smaller range, each range is also encompassed within the disclosure, subject to the specifically excluded limit in the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0168] As used herein and in the appended claims, the singular "a" is used interchangeably with "an" or "an" in the following description. "," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to an "effector" includes a plurality of such effectors, reference to "the effector" includes reference to one or more effectors and equivalents thereof known to those skilled in the art, and so forth.

[0169] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application and should not be construed as an admission that such publications constitute prior art against the appended claims.

[0170] Embodiments of the invention will now be described, by way of example only, with reference to the following figures and examples. [Brief explanation of the drawings]

[0171] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] A schematic representation of one PVC operon layout (a gene cluster present in a variable region of the original genome) encoding the PVC needle complex is shown. [Figure 1B] Schematic representation of class I, II, and III PVC operon layouts. Homologous subunit types between these classes are shown with similar shading (grayscale). [Figure 1C] Illustration of the assembled PVC needle complex. Use the numbering shown to correlate gene clusters in (A) with the location of their encoded proteins in the structure in (C) (e.g., the cap "16" cluster in A is indicated as "16" in the left-most cap region in (B)). [Figure 1D] Map of the model class I PaATCC43949PVCpnf operon (e.g., encoded by SEQ ID NO: 93) showing two effector genes in the payload region (Rhs-like adenylate cyclase, and PAU_03332). [Figure 2] This figure shows an outline of the cloning procedure for the preparation of PVC-needle complex expression plasmids based on overlapping PCR. The PCR fragment (with overlapping regions) is provided from the template gDNA of P. asymbiotica ATCC 43949 (available from ATCC under accession number ATCC 43949) with relevant primers targeting the PVC operon. [Figure 3A] Transmission electron micrographs of in vitro samples of PVC-needle complexes (e.g., prepared from cells harboring the expression vector) are shown. The PVC-needle complexes assemble into distinct "nanosyringe" structures, consistent with their role as contractile structures. [Figure 3B]A 3D rendering model of the PVC needle composite derived from a high-resolution single particle cryo-EM tomography structure is shown. [Figure 4A] Transmission electron micrographs of PVC-needle complexes containing Pnf payloads after immunogold staining with anti-Pnf (immunogold) antibodies are shown, confirming the association of Pnf-payload toxin with the PVC-needle complexes (designated PVCpnf). PVCpnf-needle complexes were prepared from the supernatant of an E. coli cosmid clone encoding the PVCpnf operon. Payload toxin proteins were localized using an anti-peptide antibody against the Pnf (TGQKPGNNEWKTGR, SEQ ID NO: 96) epitope. Pnf toxin could only be detected at the ends of disrupted or contracted needle complexes, providing evidence that the toxin is contained within the complex (arrow). [Figure 4B] Western blot analysis confirms that Pnf protein (toxin) can only be detected using anti-peptide antibodies when PVC-needle complexes are chemically or physically disrupted. These preparations were taken from PaATCC43949 supernatants. The inability to detect Pnf in the clarified supernatant confirms that all proteins are associated with the PVC-needle complex-enriched preparations. Lanes 1 + 5: sonicated sample; 2 + 6: 1 M NaCl treatment; 3 + 7: 1% SDS treatment; 4 + 8: 1 M urea treatment. Note that PVC-needle complexes are considered stable in 1 M NaCl. [Figure 5A] Cryo-SEM images of ex vivo hemocytes (insect macrophage / neutrophil equivalents) from 5th instar tobacco hornworms (Manduca sexta) injected with a native concentrated preparation of PaATCC43949 PVCpnf needle complexes (nanosyringes) heterologously produced by the E. coli cosmid clone. Note the abundant lineation corresponding to the PVC needle complexes (nanosyringes) (small arrows) and membrane ruffling effects (large arrows), consistent with the mode of action of the Pnf payload toxin, which are absent from the control treatment. Scale bar = 50 μm. 25 kV; magnification 40K. [Figure 5B]Cryo-SEM image of ex vivo hemocytes (insect macrophage / neutrophil equivalents) from a 5th instar tobacco hornworm (Manduca sexta) injected with a heat-inactivated, concentrated preparation of PaATCC43949PVCpnf-needle complex (nanosyringe) heterologously produced by an E. coli cosmid clone. Scale bar = 50 μm. 25 kV; magnification 50K. [Figure 6A] Experimental results are presented demonstrating that the (toxic) cellular phenotype following contact with PVC-needle complexes is due to intracellular toxin delivery. Pnf-loaded PVC-needle complexes were injected into insects (Galleria mellonella insect larvae) and showed potent activity within 15 minutes for a given dose (described in the Examples). Note that mortality / morbidity is typically associated with a "melanized" immune response in those dead / dying insects. [Figure 6B] Experimental results are shown demonstrating that the (toxic) cell phenotype following contact with PVC-needle complexes is due to intracellular toxin delivery. Control, denatured (via boiling) Pnf-loaded PVC-needle complexes injected into animals showed no activity. [Figure 6C] Experimental results demonstrating that the (toxic) cellular phenotype following 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 against animals (left) or the HeLa cell line (right). [Figure 6D] Experimental results are presented demonstrating that the (toxic) cell phenotype following contact with PVC-needle conjugates 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 in the cells. [Figure 6E]Experimental results are presented demonstrating that the (toxic) cellular phenotype following contact with PVC-needle complexes is due to intracellular toxin delivery. Pnf (payload) delivered into the cytoplasm of HeLa cells by intracellular expression after transfection with the appropriate plasmid showed potent activity / toxicity as evidenced by multinucleation in the cells. [Figure 6F] Experimental results demonstrating that the (toxic) cell phenotype following contact with the PVC-needle complex is due to intracellular toxin delivery are shown. The effect of PVCpnf+Pnf on the respiration rate of THP1-derived human macrophages as measured by a resazurin plate reader assay. Note that the heat-denatured and empty PVCpnf nanosyringes did not exhibit any significant adverse effects. These same samples were tested by injection into Galleria larvae. The PVCpnf+Pnf samples exhibited approximately >50% mortality within minutes (darkened larvae in the bottom two panels), while all heat-denatured and empty PVCpnf-injected insects remained healthy (undarkened larvae in the top two panels). [Figure 7A] Predicted (in silico) secondary structures of a range of endogenous payloads (toxins) associated with different PVC operons are shown, demonstrating the wide variety of structural types. [Figure 7B] Amino acid lengths of various payloads (toxins) plotted against predicted isoelectric points. [Figure 8A] Figure 1 shows evidence that the leader sequence of the present invention (e.g., 50 amino acids) is necessary and sufficient for (trans)packaging of payload proteins / peptides into PVC needle complexes (nanosyringes) expressed in Photorhabdus. 1-6: Schematic maps of chimeric effector protein expression constructs (trans-expressed in arabinose-inducible pBAD30 vectors) including those expressing Pnf and non-native cre recombinase and Myc tags. The C-terminal Myc tag epitope is indicated as a black arrow. [Figure 8B]Figure 1 shows evidence that the leader sequence of the present invention (e.g., 50 amino acids) is necessary and sufficient for (trans)packaging of payload proteins / peptides into PVC needle complexes (nanosyringes) expressed in Photorhabdus. Western blot using anti-Myc mouse antibody. Samples are from purified PVC (u4) needle complexes (nanosyringes) overexpressed from chromosomally engineered P. luminescens TT01 carrying transpackaging expression constructs 1-6 shown in (A). Blank pBAD30 plasmid was used as a negative control and showed no signal. Arrows indicate the correct band sizes for the predicted products. [Figure 9] An alignment of the leader sequences is shown to demonstrate the existence of a chemical composition consensus among the leader sequences based on amino acid characteristics. More specifically, the leader sequences contain a similar charge pattern of two negatively charged regions each followed by a positively charged region: [-ve][+ve][-ve][+ve]. [Figure 10A]Western blot analysis of PVC-needle complexes and payloads from particulate preparations (cesium chloride gradient and monolith FPLC preparations as described in Materials and Methods) is shown. In [1] (pBADPVCpnf, where PVC16 in the nanosyringe is FLAG-tagged, providing PVC16::FLAG detectable with an anti-FLAG Ab), a signal from the tagged cap protein of "PVCPnf" (PVC-needle complexes with Pnf payload) is visible, confirming the presence of PVC-needle complexes in the purified fraction. In [2] (pBADPVCpnf+Cre::Myc, detectable with an anti-Myc Ab, where Cre has an N-terminal fusion of the Pnf leader, e.g., SEQ ID NO: 78), a signal from the Myc-tagged payload protein packaged in large quantities in the same sample as (1), confirming the presence of the Cre payload in the purified PVC-needle complexes (nanosyringes). [3] (PVCU4+Cre::Myc, detectable with anti-Myc Ab, Cre has an N-terminal fusion to the Pnf leader, e.g., SEQ ID NO: 78) Different PVC needle complex chassis ("PVCU4") purifications were probed for Myc-tagged Cre, revealing the corresponding band for packaged (packaged Myc-tagged Cre), which is highlighted on the blot for clarity. [Figure 10B] Transmission electron micrographs of PVC-needle complexes show that both wild-type (with a Pnf payload) and atypical (non-native) recombinase (Cre) payloads do not affect the morphology of the PVC-needle complexes in any of the chassis examined, ensuring that they are not aberrantly assembled. [Figure 10C](C) provides additional / supplementary data to that in (A). More specifically, (C) provides further evidence via Western blot analysis of the (trans)packaging of Cre recombinase into purified PVCpnf expressed in E. coli. The Western blot demonstrates that for a given amount of anti-FLAG antibody Western signal (a specific probe for the nanosyringe due to incorporation of PVC16::FLAG), a significantly higher amount of Cre payload is detected (using an anti-Myc tag antibody). Numbers indicate two-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 that the sample was further anion-exchanged through a "monolith" column. "Post-elution," "interphase," and "sub-interphase" indicate the liquid fractions in which signal is detected from the purification process. [Figure 10D] Western blot analysis of Cre trans-packaged into PVCpnf in E. coli. Payloads are probed for their incorporated "Myc" tag (C-terminal fusion) after purification of the nanosyringe-payload complex. Western blot analysis of particle preparations confirms that all four leaders were able to efficiently trans-package the exogenous Cre enzyme. [Figure 10E] Phylogenetic tree demonstrating that the exemplified leader sequences are well distributed throughout and therefore diversity is at or near maximally contiguous (see Example 4.2). [Figure 11]Western blot analysis of PVC-needle complexes expressed without (1) and with (2) coexpression of (Myc-tagged) Pnf from separate plasmids simultaneously probed with anti-FLAG and anti-Myc antibodies is shown. In lane labeled 1, PVC-needle complexes (nanosyringes) were expressed and purified in E. coli without the presence of a "payload plasmid" (an expression plasmid encoding a payload protein linked to a leader sequence). This results in a band corresponding only to the FLAG tag present on the syringe (PVC-needle complex) itself. For lane 2, the same approach was undertaken, but using a culture that also contained a (separate) plasmid carrying a tagged payload (Myc-Pnf). Bands corresponding to the FLAG and Myc tags are visible, 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] Western blot analysis of a transpackaging experiment in the P. luminescens TT01 PVCu4 overexpression strain. The results demonstrate transpackaging of myc-tagged Pvc17 (Plu1651whole::Myc). [Figure 13A]Figure 1 shows further Western blot analysis of trans-packaging experiments (described in the Examples) in the P. luminescens TT01 PVCunit4 overexpression strain. The results demonstrate that transpackaging of Myc-tagged Pvc17 (Plu1651::Myc) and Myc-tagged Pvc17 alone using the Pnf leader (PAU_03332 leader) and that the leader is required. (A) Lane 1 shows packaging of a leader fused to a Myc tag (PAU_03332::Myc); lane 3 shows lack of packaging in the absence of the leader sequence (Myc alone is not packaged); lane 4 shows lack of packaging of HvnA (the natural effector) in the absence of the leader sequence; lane 6 shows packaging of Myc-tagged PAU_03332::Plu1649, a chimera 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 to the C-terminus). The high intensity bands in lanes 1 and 6 demonstrate that the Pnf(PAU_03332) leader is particularly effective for packaging payloads. [Figure 13B] (B) Western blot analysis of a trans-packaging experiment (described in the Examples) in the P. luminescens TT01 PVCunit4 overexpression strain. The results demonstrate that Myc-tagged Pvc17 (Plu1651::Myc) and the Myc tag alone transpackage using the Pnf leader (PAU_03332 leader), and that the leader is required. (B) Lane 1 shows packaging of Plu1651 with a C-terminal Myc tag using an anti-Myc antibody Western blot. [Figure 14]Further Western blot analysis (second lane, ladder not included) demonstrates extremely high levels of trans-packaging of Myc-tagged Pnf (PAU_03332::Myc) using the PAU_02806 (GogB) leader. The first lane demonstrates the use of the Plu1649 leader (Myc-tagged Plu1649::PAU_03332) to package the PAU_03332 effector. The band appears weaker due to the relative intensity of the band in the second lane. The experiment included filter-sterilizing 50 mL of culture and adding urea to a final concentration of 8 M to degrade the PVC. Samples were collected from 10 mL of supernatant. [Figure 15] Further Western blot analysis demonstrating trans-packaging of Plu1651 (pvc17) with a C-terminal Myc tag as described in Figure 13 into PVCunit4 expressed from Photorhabdus is shown. Raw represents a particulate preparation from the supernatant, while Be, B2, and IP represent different "cuts" from cesium chloride gradient purification. [Figure 16A] (Example 6) provides a schematic illustration of the mechanism of action of Cre in mouse organoid experiments, and how a positive control (TAM) promotes Cre activation. The white arrow indicates the location of cells expressing the tdTom fluorescent reporter gene. [Figure 16B] Demonstration of delivery of active trans-packaged Cre recombinase into murine bile duct organoids by purified PVCpnf expressed from E. coli. Open circles indicate the localization of a group of cells expressing a fluorescent reporter gene. The top image shows a direct grayscale conversion of an image obtained via light microscopy. The bottom image shows the corresponding image of positive cells with enhanced false color, which is provided solely to aid in the identification of differences between affected and surrounding unaffected cells within the former grayscale conversion. [Figure 17]Dot blot analysis of nanosyringe expression both with and without payload (the Cas9-like protein MAD7) is shown. Some leaky expression of IPTG-induced MAD7 is seen before induction (T1), as is typical for this expression system. As expected, there is no Myc signal from the PVC-only sample at any time point, and the MAD7 signal grows throughout expression over a ∼24-hour period. A strong Myc signal is maintained after purification via 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 after induction and persisting after purification, as expected, because this promoter system reduces leaky expression. We conclude that the nanosyringe and MAD7 are compatible with each other for expression, and that MAD7, the largest protein tested to date, can be packaged into the nanosyringe system. [Figure 18] Western dot blot analysis (7 and 8) confirms trans-packaging of the pro-apoptotic tBid protein domain and BaxBH3 peptide (both with the leader sequence of SEQ ID NO: 78 fused to their N-terminus) into purified PVC pnf expressed from E. coli. Nanosyringes containing its cognate toxin, "Pnf," are shown as purified by two different methods (5 and 6) as a positive control. The blots in the lower panel represent the same examples as in panels 7 and 8 above. These blots were generated from separate purifications of the same construct, demonstrating the reproducibility of the purification. This experiment demonstrated that the "tBid protein domain and BaxBH3 peptide" packed sample (nanosyringe) used, for example, in the apoptotic delivery system of Example 9 can be successfully prepared. [Figure 19A]TUNEL staining microscopy analysis from cells exposed to packaged nanosyringes for only 20 minutes is shown. First (left) bar = DNase I-treated cells (+ control); second bar = no DNase I 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 (right) bar = cells exposed to nanosyringes packaged with Bax_BH3 domain (via the leader sequence of SEQ ID NO: 78 fused to the N-terminus). [Figure 19B] Representative photomicrographs 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 (no DNase I treatment) controls for 20 minutes at room temperature, and then TUNEL staining was performed to determine the apoptotic response. In the original (non-grayscale) photomicrographs: Cells negative for an apoptotic response show blue or light brown staining. Blue staining (methyl green) or light brown staining indicates healthy cells with an absent apoptotic signal. Dark brown staining indicates cells undergoing apoptosis. [Example]

[0172] Example Materials and Methods Cloning Plasmids encoding the PVC-needle complex were prepared using standard molecular techniques known in the art. Briefly, P. asymbiotica ) Genomic DNA from ATCC 43949 (accession number ATCC 43949) (available from Sigma-Aldrich, Inc.) was used in PCR (with appropriate primers) to amplify multiple (e.g., four) overlapping regions of the PVC operon. The entire operon was prepared using overlap / extension PCR and fused into a suitable expression vector (again, using overlapping PCR) as detailed in Figure 1 (SEQ ID NO:101 to SEQ ID NO:102). Use primer 06).

[0173] Briefly, four overlapping PVC fragments (generated using primers SEQ ID NO:101 (F1) and SEQ ID NO:105 (R1); SEQ ID NO:102 (F2) and SEQ ID NO:106 (R2); SEQ ID NO:103 (F3) and SEQ ID NO:107 (R3); and SEQ ID NO:104 (F4) and SEQ ID NO:108 (R4), respectively) covering the PVC operon (e.g., that of SEQ ID NO:93) were generated. The target cloning vector was cut at the desired insertion site. These five DNA fragments were then assembled by overlapping PCR (using primers SEQ ID NO:101 and SEQ ID NO:108), and the resulting fragments were ligated into the cloning vector. The products were 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 linked to an inducible promoter (e.g., arabinose-inducible and / or IPTG-inducible) as known in the art, commonly found in pBAD family plasmids (inducible via arabinose) (Invitrogen, Cat. No. V43001) and pVTRa (inducible via IPTG) (Biomedal, SL ) vector (although any combination of compatible expression vector systems should suffice).

[0175] The PVC-needle complex can be expressed independently of the payload (toxin), and vice versa: separate expression vectors (e.g., with different inducible promoters) can carry the PVC-needle complex and the payload, respectively.

[0176] Expression (e.g., lab-scale expression) / purification of PVC-needle complex in E. coli A typical process for purifying PVC needle complexes from a 1 L culture of an E. coli expression strain (transformed with the appropriate expression vector / cosmid) is as follows: 1. Prepare an overnight culture of bacteria (transformed with the PVC needle complex expression vector) by picking a colony from the plate and inoculating it into 100 ml of LB medium. Grow the culture at 37°C with shaking. a. Typically, the media can be routinely supplemented with 0.2% d-glucose to aid in repression of the gene construct for optimal cell health. b. The media is also supplemented with the relevant antibiotic for maintenance of the expression (PVC needle complex) vector, and if a payload vector is also used, the relevant antibiotic for that vector. 2- The next day, a 1 L flask is inoculated via a 1:100 dilution from the overnight culture. The medium for the 1 L flask is the same as the overnight medium, but typically does not contain glucose. 3- The culture is grown to approximately mid or late exponential phase (OD600nm of approximately 0.8) at which point the plasmid is induced. For PVC needle complex (nanosyringe) plasmids, expression is typically induced by adding 0.2% arabinose. For payload plasmids (e.g., plasmids encoding payloads, e.g., Pnf), IPTG concentrations can be optimized, typically on a protein basis, with a typical starting number of 0.1 mM being preferred. 4- After induction, the cultures are returned to the incubator and incubated at 18°C until the next day. 5- Harvest the culture by centrifugation in an appropriate centrifuge / bottle / rotor at 5000 x g for 30 minutes. 6-The cell pellet is then dissolved to release the PVC needle complex (nanosyringe). a. The following lysis methods can be used: (i) Overnight lysozyme incubation. (ii) Sonication in a needle sonicator (with or without first treating with lysozyme). (iii) Cell disruptor / homogenizer. 7-Optionally, DNase and protease inhibitors can be added to the lysate. 8- Cell debris is removed by centrifugation at 50,000 xg at 4°C for 20 minutes in a high speed centrifuge. Concentrate the lysate through a 9-100,000 kDa MWCO centrifuge column to reduce the volume and remove small proteins. Once the volume is reduced to a manageable volume, centrifuge several times and replace the retentate with an appropriate sample buffer, e.g., TM (20 mM Tris-HCl, 8 mM MgCl, pH 7.4) and dialyze.

[0177] The subsequent purification process via a cesium chloride density gradient is as follows: 1. Prepare CsCl density solution as follows: (a) 1.7 g / mL CsCl in HO; (B) 1.5 g / mL CsCl in HO; (C) 1.45 g / mL CsCl in HO 2. A gradient (bottom to top of the tube) is then set up in the ultracentrifuge tube, e.g., (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 as not to blend the boundary with the previous density layer. 3. The equilibrated tubes are then subjected to ultracentrifugation at 35,000 RPM in an SW40Ti swinging bucket rotor equivalent to 155,000 x g for 2 hours at 4°C. 4. The correct gradient fraction is the area directly above the "blue-white" halo that appears. Extract the fraction via puncturing the tubing with a syringe and needle. 5. A PVC needle conjugate of good purity is thus obtained and can be stored in buffer at 4° C. Preferably, it is dialyzed again into TM buffer to remove CsCl.

[0178] Following CsCl gradient purification, or alternatively, PVC can be extracted via monolith anion exchange chromatography as follows (note that all steps can be performed manually using a peristaltic pump or syringe apparatus, or via F / HPLC): 1. Unless already done, dialyze the sample extract into a binding mobile phase (typically TM buffer) with low salt (20 mM NaCl). 2. Equilibrate the column according to the manufacturer's guidelines, briefly: a. At least 5 column volumes (CV) of dH2O; b. At least 5 CV of binding buffer (TM, with low salt); c. At least 5 CV of elution buffer (high salt, TM with >= 1 M NaCl); d. Another round of at least 10 CV of binding buffer. 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 TM buffer containing 5.1 M NaCl (alternatively, if using an FPLC instrument, use gradient elution). 6. PVC needle complexes are present in the elution fractions. If a fraction collector is used, subsequent SDS-PAGE or similar may be required to identify the correct fractions.

[0179] The column (e.g., that in step 2) was a CIMmultus™ quaternary amine anion exchange column. The column was from a BIA Separations doco, e.g., 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 exchange) column can be used.

[0181] Alternatively, for use with the Photorhabdus expression system, the PVC-needle complexes can be purified from the cell pellet and / or alternatively from the supernatant, with the following additions / modifications: 1. After cell harvest from the standard protocol above, transfer the supernatant to a pyrex bottle and optionally Optionally, it can be concentrated via a 100,000 MWCO column if necessary. a. DNase (0.25 U / mL) and protease inhibitors can be optionally added. 2. Add NaCl to a final concentration of 0.5 M, and also add 80 g / L PEG 6000. Mix the solution overnight at 4° C. 3. Centrifuge the solution at 8000 xg for 30 minutes at 4°C to pellet the PEG6000. 4. Resuspend the pellet in a small volume (approximately 5 mL) of TM buffer (or similar) and incubate with shaking at room temperature for 2 hours. 5. Pellet by centrifugation at 13,000 x g for 10 minutes and collect the supernatant into a new tube. Continue with the purification method of choice.

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

[0183] P. luminescens TT01 PVCunit4 (gene plu16 Construction of an arabinose-inducible overexpression strain for 67 (chassis encoded by plu1652) Chromosomal recombination was used to place a selected PVC (operon) (the operon encoding the PVCunit4 needle complex was used here as an example) under the control of an arabinose-inducible transcription promoter to prepare a Photorhabdus strain overexpressing 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 facilitate PVC needle complex overexpression, PVC effector expression, PVC effector trans-packaging, and secretion of the complete complex solely through the addition of arabinose sugars.

[0184] Construction of recombinant Photorhabdus PVC overexpression strain P. luminescens strain DJC (also known as strain TT0) was used as a template. Genomic DNA from 1) was used to amplify the promoter region of PVCunit4 using primers PVCpromF (5'-TATCATATGTCTACAACTCCAGAACAAATTGCTG-3', SEQ ID NO: 97) and PVCpromR (5'-ATCTCTAGAACAGATATTCCAGCCAGC-3', SEQ ID NO: 98). A suitable P. luminescens strain is available from the ATCC under accession number ATCC29999. The PCR product was digested with NdeI and XbaI and transformed into the suicide vector pC using E. coli DH5α λ-pir (Biomedal SL) as the carrier strain. The resulting plasmid was transferred to the E. coli donor strain S17.1λ-pir (Biomedal SL) for conjugation into Photorhabdus. and the donor strain of P. luminescens DJC and rifampicin resistance. Overnight cultures of the (RifR) isolates were diluted in LB supplemented with 10 mM MgSO4 and grown to mid-exponential phase (OD600 approx. 0.5). 3 ml of each culture was then harvested, washed twice, and resuspended in 100 μl of LB supplemented with 10 mM MgSO4. 80 μl of P. luminescens DJC RifR was resuspended in 20 1 μl of donor bacteria (resulting in a 4:1 recipient to 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. Transconjugants were selected by plating aliquots onto plates containing rifampicin (50 μg / ml) and chloramphenicol (25 μg / ml), and the plates were incubated at 30°C for 3 days. Possible transconjugants were restreaked 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 Photorhabdus Overnight culture of P. luminescens DJC PVCunit4::pCEP The culture was diluted in 2 x 250 ml of LB 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 an additional 26 hours. The cells were pelleted by centrifugation (7000 g, 30 minutes), and the supernatant was collected. DNase I was added to the supernatant at a concentration of 0.25 U / ml to degrade any extracellular DNA. After a 30-minute incubation at room temperature, 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 for 30 minutes at 4°C. The pellet was resuspended in 8 ml of TM buffer (20 mM Tris-HCl, 20 mM MgCl, pH 7.4) and incubated for 2 hours at room temperature with gentle shaking. Any debris was removed by centrifugation at 13,000 g for 10 minutes, and the supernatant containing the PVC-needle complexes was applied to a CsCl density gradient and centrifuged at 35,000 rpm in a Beckman Coulter Optima L-90K or XPN-80K ultracentrifuge. The tube was centrifuged for 2 hours at 20°C. TM buffer containing CsCl was layered at ρ = 1.7 (2 ml), 1.5 (3 ml), and 1.45 (3 ml) from the bottom of the tube to create a CsCl density gradient. The fractions containing the PVC-needle complex were collected, and the CsCl was removed using an Ultracel-100K device (Amicon). The buffer was then replaced with TMS (20 The solution was replaced with CI Multus (trademark) ... The PVC-needle conjugate was further purified using a quaternary amine 2 μm pore anion exchange column (BIAseparations). The column was washed with TMS buffer containing 200 mM NaCl, and the PVC-needle conjugate was eluted in TMS containing 1 M NaCl. NaCl was removed by buffer exchange using an Ultracel-100K device, and the sample was then eluted for final purification. The sample was applied to a CIMmultus™ DEAE 2 μm pore column (BIA separations). The column was washed with TMS containing 200 mM NaCl, and the sample was eluted in TMS containing 500 mM NaCl.

[0186] This can be done with or without lysis of the cells (to release the PVC-needle complexes) (e.g., the PVC-needle complexes are believed to be secreted from viable cells and can therefore be recovered in the supernatant).

[0187] Transmission electron microscopy Pioloform coating 3 coated with a fine carbon layer for transmission electron microscopy (TEM) 00 mesh copper grids were used as substrates for protein fractionation. The preferred aqueous negative stain is 3% methylamine tungstate. The coated grids were exposed to UV light for 16 hours immediately prior to use to ensure proper wetting of the substrate. A 10 μl drop was applied to the TEM grid and the proteins were 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 filtered negative stain. The drop was partially removed with filter paper and the grid was then rinsed. The rods were allowed to air dry completely before they were observed under a JEOL 1200EX transmission electron microscope (JEOL, Tokyo, Japan) operated at 80 kV.

[0188] BioPORTER assay and actin stress fiber analysis For the BioPORTER assay (Genlantis), 80 μl of purified wild-type and mutant Pnf proteins (500 μg ml−1), or PBS as a negative control, was added to one BioPORTER tube. The samples were added to tubes (Genlantis) and resuspended in 920 μl of DMEM. The solution was added to HeLa cells grown in a 1000 ml plate and incubated for 4 hours. The protein or PBS mix was replaced with fresh complete medium, and cells were incubated for 20–48 h. To visualize cell morphology and actin cytoskeleton, cells were fixed in 4% PBS-formaldehyde for 15 min, permeabilized with 0.1% Triton X-100, and stained with tetramethylrhodamine B isothiocyanate (TRITC) phalloidin (Sigma) and D. Staining was performed with 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 have successfully excised (cloned) the required expression genes (e.g., contained within SEQ ID NO:93, SEQ ID NO:94, and / or SEQ ID NO:95) from the host bacterium, Photorhabdus, and devised a reliable and scalable expression system in laboratory E. coli as described above. Expression in trans on a separate plasmid was demonstrated, allowing for the incorporation of a payload (e.g., Pnf) into the syringe, creating a multi-plasmid (modular) platform.

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

[0191] Furthermore, electron microscopy analysis demonstrated that the purified complexes properly localized to the cell surface of cells, and that the PVC-needle complexes bearing 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 effect via intracellular delivery of effectors The polypeptide Pnf was identified as a PVC effector as follows: It was identified within the Photorhabdus asymbiotica ATCC 43949 complete genome - GenBank accession number: FM162591.1.

[0193] The last gene of the PVC operon (P. asymbiotica ATCC 43949 PVCpnf operon, having the sequence of SEQ ID NO: 93), i.e., 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). ORFs immediately 3' of pvc16 (e.g., within approximately 5 kb downstream of pvc16) were identified, with one such ORF (PAU_03332) being 3535 bp downstream of pvc16. The predicted function of the polypeptide (having the sequence of SEQ ID NO: 32) encoded by this putative effector ORF was obtained by a combination of BlastP and HHPRED (https: / / toolkit.tuebingen.mpg.de / # / tools / hhpred). This ORF was then compared with the previously identified ORFs. It could be assigned as a PVC effector based on direct homology with known bacterial toxins (eg, those of the CNF1 family from E. coli).

[0194] Pnf-loaded PVC needle composites were then prepared according to Example 1.

[0195] We 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 whole insects 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 injection of 10 μl of supernatant after centrifugation (pelleting) of an overnight culture (typically 1 L) of E. coli culture harboring a cosmid clone encoding a PVC-needle complex with Pnf (PVCPnf), e.g., the PVC encoded by SEQ ID NO:93 packaged with the PVC effector of SEQ ID NO:32.

[0197] Having demonstrated that the PVC-needle complex was responsible for the phenotype due to intracellular delivery (e.g., injection) of the Pnf payload, the toxic effect could only be reconstituted if the same protein (Pnf) was provided via an alternative route to access the cell cytoplasm (transfection and expression of an expression plasmid or delivery via a liposome preparation containing the protein), see Figure 6. Conversely, denatured (via boiling) PVC-needle complex preparations, toxin protein layered on tissue culture cells, or toxin protein injected into whole animals showed no activity.

[0198] 2.2. Evidence for delivery of the virulence effector enzyme Pnf into cultured human macrophages To complement the data outlined above, we performed additional experiments that provide further evidence for the delivery of the toxic effector enzyme Pnf into cultured human macrophages.

[0199] Concept: We tested purified PVCpnf expressed from E. coli (trans)packaged with native Pnf toxin on cultured human THP1-derived macrophages. Unlike the lethal effects of Pnf in insect models, previous liposome-mediated Pnf protein transfection experiments demonstrated a more subtle phenotype in human HeLa cells. In those experiments, cells exhibited actin stress fiber formation at 24 hours and multinucleation at 48 hours. Therefore, we tested the effect of purified PVCpnf (nanosyringes) carrying / packaged with Pnf PVC effectors on macrophage respiratory rate using a resazurin colorimetric assay.

[0200] method: Background on the Resazurin Assay. The blue compound resazurin was investigated for use in the assay to determine the activity of PVC on macrophages (MO). Resazurin is metabolically reduced in cellular mitochondria to produce the pink and highly fluorescent compound resorufin. The effect of PVC on macrophage metabolism can be determined by introducing resazurin into the culture medium. The number of macrophages affected by PVC 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 the use of resazurin for THP1-derived macrophages. Macrophage metabolism over 18 hours was assessed at different seeding densities to determine the optimal cell density for use in this assay with PVC. A 30 mL culture of THP-1 cells was 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 THP-1 cells were then activated with phorbol 12-myristate-13-acetate (PMA) immediately prior to plating. 200 μL of cells were plated in quadruplicate in a 96-well plate, and two-fold serial dilutions were performed to reach a final cell density of 1.5625 × 10 cells mL. 125 μL of the starting cell dilution was also plated in quadruplicate on the same plate for five-fold serial dilutions to reach a cell density of 0.32 × 10 cells mL. Four blank wells containing RPMI and PMA were also prepared. The plate was incubated at 37°C with 5% CO for 48 hours. The medium was aspirated from the wells and replaced with fresh RPMI, and the macrophages were incubated for an additional 24 hours. Resazurin tablets (VWR) were dissolved in RPMI (12.5 mg / mL), and 10 μL was added to each well. The cells were added sequentially to the plate (well concentration: 1.25 mg / mL). The fluorescence produced was measured every 30 minutes for 18 hours on a plate reader (excitation: 530-570 nm, emission: 580-620 nm, maintained at 37°C and 5% CO). The optimal cell density over time was then determined for use with PVC.

[0202] Use of the assay for PVC testing. THP-1 cells diluted to 1.25 × 10 cells mL were activated and seeded in 96-well plates, with each well containing 100 μL of cells at a final well density of 1.25 × 10 cells mL. Blank wells containing cells without PVC samples and wells containing medium and PMA alone were also prepared in quadruplicate. The plates were incubated at 37°C and 5% CO for 48 hours. The medium was then replaced with fresh RPMI, and 10 μL of each PVC sample was added. The plates were then incubated for an additional 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, emission: 580-620 nm, maintained at 37°C and 5% CO).

[0203] Results: Figure 6F shows that challenge with PVCpnf+Pnf indeed reduced macrophage respiration rates, while heat-denatured or empty PVCpnf nanosyringes had no significant adverse effects. Despite this, control cells without added sample still exhibited the best respiration rates. The effects on macrophages were correlated with an insect injection toxicity assay. In this case, the two PVCpnf+Pnf preparations caused mortality in over half of the insect cohort, while all heat-denatured and empty PVCpnf-injected insects remained healthy.

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

[0205] To demonstrate this, an expression construct (chromosomally engineered overexpression in P. luminescens TT01) was prepared in which Plu1649 ("hvn" in the figure) was overexpressed. The leader sequence (N-terminal amino acid residues 1-50) was removed so that the payload expressed by the PVC-needle complex (referred to as "hvnA" and having the sequence of SEQ ID NO: 46) (Myc-tagged for detection purposes) lacked the leader sequence (see Figure 8A - construct 1). After expression (of both the payload and the PVC-needle complex) and isolation (and running its components, including any packaged payload, on a gel), (Myc-tagged) Plu1649 ("hvnA") was not detectable within the PVC-needle complex via Western blot analysis, indicating that the payload (lacking the leader sequence) was not packaged into the complex (Figure 8B, layer (See lane 1), thus demonstrating that hvnA does not associate with the isolated complex. However, good packaging was observed for hvnA that retains the leader sequence, see lane 2 (note that the band appears weaker due to the relative intensity of the band in lane 3).

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

[0207] Example 4 4.1 Demonstration that leader sequences direct packaging of atypical / exogenous payloads (into PVC needle complexes) In an unexpected technical effect of the present invention, the inventors have discovered that fusing a leader sequence described herein to an exogenous (non-Photorhabdus) polypeptide (preferably at the N-terminus) enables packaging of the exogenous polypeptide into a PVC-needle complex, with the exogenous polypeptide remaining associated with the PVC-needle complex upon isolation / purification. See, for example, Figure 8B (lane 4), which demonstrates that a non-Photorhabdus "Myc" polypeptide (<10 kDa) is packaged into a PVC-needle complex when fused to a leader sequence, and lane 6, which demonstrates that a much larger non-Photorhabdus "Cre recombinase" polypeptide (>32 kDa) can also be successfully packaged into a PVC-needle complex when fused to a leader polypeptide of the present invention.

[0208] The present inventors conducted a detailed analysis of the size (e.g., polypeptide length) and structure of various native PVC effector payloads encoded by Photorhabdus (see Figure 7), which showed a wide range of different lengths and structures, demonstrating that the applicability of the PVC needle composite (nanosyringe) delivery system of the present invention is not limited by the size or properties of the target payload protein. Taken together, no specific secondary structure, biophysical properties, or cargo length are required, confirming that the PVC needle composite (nanosyringe) chassis can be utilized as a versatile, multifunctional delivery vehicle.

[0209] Furthermore, this packaging of exogenous polypeptides was independent of the PVC-needle complex chassis chosen; for example, it was achieved using both the "PVCpnf" chassis (SEQ ID NO: 93) and the "PVCU4" (e.g., PVCunit4) chassis (endogenous to Photorhabdus overexpression strains) (see Figure 10A). Importantly, we ensured that packaging of exogenous payloads in either chassis did not affect the morphology of the PVC-needle complexes, preventing them from assembling abnormally (see Figure 10B).

[0210] In the data presented herein, the payload proteins are supplied "in trans" to a separate gene construct. Surprisingly, the leader sequence is sufficient to target these separately synthesized proteins for packaging into the PVC needle complex vehicle (see Figure 11). This is true in Escherichia coli (E. coli) and in the host organism Photorhabdus sp. () when the chassis (PVC) gene itself is also present on the plasmid. As in Photorhabdus, the chassis gene is integrated into the chromosome.

[0211] Further illustration of high-level trans-packaging of Cre site-specific recombinase into PVCpnf nanosyringes expressed in E. coli is provided in Figure 10(C). More specifically, we constructed a laboratory E. coli expression strain harboring (i) an arabinose-inducible expression plasmid of the P. asymbiotica ATCC43949 PVCpnf operon (e.g., SEQ ID NO: 93) (with a C-terminal FLAG tag on Pvc16, e.g., immediately 3' of SEQ ID NO: 93) and (ii) a second IPTG-inducible expression plasmid containing Cre recombinase with an N-terminal fusion of the native Pnf effector 50-amino acid leader sequence (e.g., the leader of SEQ ID NO: 78) and a C-terminal Myc-tag epitope. The PVC operon and effector (Cre+ leader sequence) were co-induced for 24 hours, and 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 trans-packaged Myc-tagged Cre recombinase.

[0212] 4.2 Transpackaging using additional readers to demonstrate functionality for a larger, more diverse sequence space Complementing the data outlined in Example 3, FIG. 10D demonstrates (trans)packaging of Cre into PVCpnf (in E. coli) using the following four additional leader sequences (thus demonstrating functionality of a larger sequence space): - Lane 1: leader of PAU_02096 (leader sequence = SEQ ID NO: 71), experiment called "nanosyringe + lopt50::cre::Myc" in Figure 10D; - Lane 2: Leader of PAK_02075 (leader sequence = SEQ ID NO: 50), experiment referred to as "Nanosyringe + cnf50::cre::Myc" in Figure 10D; Lane 3: Leader of PAU_02009 (leader sequence = SEQ ID NO: 68), experiment called "Nanosyringe + cif50::cre::Myc" in Figure 10D; and - Lane 4: Leader of PAU_02806 (leader sequence = SEQ ID NO: 76), experiment named "Nanosyringe + gog50::cre::Myc" in Figure 10D.

[0213] These results also demonstrate the utility of leader sequences that exhibit greater sequence diversity for (trans)packaging of payloads. Indeed, to provide further validation, we performed a CLUSTALW sequence comparison of a panel of leader sequences to determine diversity. PVC effectors are identified as proteins encoding recognizable toxin-like domains encoded immediately downstream of the pvc16 structural gene. Each PVC operon may encode only a single effector or several different effector genes in tandem arrays. A phylogenetic tree is shown in Figure 10E, demonstrating the identity of the leader sequences exemplified herein for packaging payload proteins into nanosyringe complexes with either the P. asymbiotica ATCC43949 PVCpnf operon (solid arrow) or the P. luminescens TT01 PVCunit4 operon (dotted arrow), or both. It is produced by

[0214] As can be seen from the tree in Figure 10E, the exemplary leader sequences are well distributed throughout and therefore maximally continuous in diversity, or close to it.

[0215] Example 5 Tail fiber / binding domain modification PVC needle complexes are known to contain tail fibers (see the asterisk at the left end of the 3D rendering PVC structure image at the far right) that may enable cell type-specific targeting of the PVC complex. It has been successfully demonstrated that modifications of the fiber region (eg, substitution of amino acids in the wild-type sequence with alternative amino acids to the 20 standard amino acids) do not affect tail fiber expression.

[0216] Example 6 Demonstration of delivery of active (exogenous) enzyme / payload into ex vivo murine organoids using leader sequence-packaged PVC needle complexes Concept: To obtain data on the delivery of exogenous functional enzymes into mammalian tissues. We demonstrated the delivery of a trans-packaged bacteriophage-derived recombinase protein known as "Cre" into ex vivo mouse biliary organoids. The organoids were derived from a mouse strain in which expression of a chromosomally encoded red fluorescent protein (RFP) reporter is normally prevented by a termination signal flanked by loxP recognition sites for Cre-recombinase. In the presence of the recombinase, the termination signal is recombined, and the cells then continue to express the reporter protein. The general principle of this experimental demonstration is summarized in Figure 16A.

[0217] Methods: Bile duct organoid preparation: Primary murine bile ducts were isolated and expanded as organoids in Matrigel for 12 passages using "BD Expansion Medium" according to the protocol of Huch et al. (Regen Med. 2013 Jul;8(4):385-7. PMID: 23826690; DOI: 10.2217 / rme.13.39). Cells were then plated in 2D and cultured in BD Expansion Medium. Mouse genotype: LSL-Tom reporter in the Rosa26 locus + Axin2CreRT (inducible upon 4OHT treatment). Cells were cultured at a seeding density of 10,000 cells / well in non-coated polystyrene plates. Nanosyringes were prepared with 30% volume of syringe preparation in PBS + 70% culture medium. The total volume was 100 μl per well. The positive control represented 1:1000 (v / v) of 500 nM 4OHT (in ethanol) as a positive control for recombination. The negative control represented 1:1000 (v / v) ethanol dilution only. Cells were seeded, grown for 48 hours, nanosyringe added, and then cultured for another 24 hours before fixation (4% PFA fixation for 15 minutes at room temperature) and staining for microscopy. Staining: Primary antibody anti-RFP (1:1000) from Rockland. Secondary anti-rabbit 568 (used at 1:500 v / v). Samples were visualized on a laser confocal microscope.

[0218] Results: Figure 16B contains representative micrographs from these experiments, demonstrating that a signal for RFP protein could be detected in large numbers of cells treated with Cre-loaded PVC pnf nanosyringes. Because these are ex vivo organoids rather than simple cell monolayers, some randomness in the number of cells administered is expected, as is also observed in the positive control, which is a small molecule inducer (not a large protein complex). Because these are organoids, some level of cellular differentiation is expected to be present, which may alter the binding characteristics of the nanosyringe. An additional interesting observation from this preliminary run is that, while information regarding the total volume of nanosyringe applied to the system is not yet available, we demonstrate that the TAM small molecule inducer does not appear to have appreciably greater tissue penetration than the nanosyringe, suggesting that their distribution capabilities are not significantly hindered by their size.

[0219] Additional Interpretation: In summary, we have demonstrated the ability to deliver (e.g., administer) an exogenous enzyme to a cellular target. Furthermore, this "nanosyringe + Cre" experiment is a promising proof of concept for a bioengineering tool / aid by demonstrating its ability to provide DNA changes that result in transformed cells. Thus, this experiment specifically demonstrates the use of an exogenous payload (a non-bacterial viral protein) and a nucleic acid-modifying enzyme. It is clear that the Cre enzyme can be functionally delivered and translocate across the cell interior to the nucleus to affect the DNA-modifying changes.

[0220] Example 7 Trans-packaging of MAD7 site-specific recombinase (exogenous payload) into PVC pnf nanosyringes expressed in E. coli Concept: Based on the Cre data (Example 6) and other examples of packaged payloads provided herein, we demonstrated packaging of the Cas-like enzyme MAD7 into a nanosyringe via a leader sequence, which is the largest exogenous example of a payload described herein (MAD7 = 147.9 kDa).

[0221] Methods: Briefly, the chassis gene and the MAD7 gene (the latter tagged with a C-terminal Myc tag for detection as described herein and a leader sequence for nanosyringe uptake) were co-expressed (upon induction) in Escherichia coli (E. coli). Upon harvesting and purification of the nanosyringe complexes, payload packaging was probed via dot blot analysis (e.g., for detection of the Myc tag). The purification method described herein (using ultracentrifugation) can be used to select for (e.g., very) high molecular weight protein complexes / biological materials, allowing for the recovery of nanosyringes and any cargo (payload) they carry. "Loose" / unpackaged payload remains in solution and is not subjected to sufficient centrifugal force, and therefore is lost during purification unless it is contained (i.e., well packaged) within the significantly larger nanosyringe "shell." Successful packaging of MAD7 is demonstrated in Figure 17.

[0222] Example 8 Trans-packaging of an apoptosis-inducing payload into PVCpnf expressed in E. coli Using the E. coli PVCpnf leader::payload::Myc trans-packaging system (PVCpnf leader = SEQ ID NO: 78) depicted in Figure 10C, we demonstrated the ability to trans-package at least two pro-apoptotic human-derived protein sequences or peptides (e.g., the sequences of SEQ ID NO: 109 and SEQ ID NO: 111). A Pnf effector protein leader 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 in Example 7) confirmed the presence of these human-derived proteins in the 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 studies confirmed the ability to deliver trans-packaged human protein sequences (e.g., packaged according to Example 8) using PVC pnf nanosyringes produced in E. coli and induce apoptosis in ex vivo circulating PBMC cells from human donors. The assay consisted of TUNEL staining microscopy analysis from cells exposed to the packaged nanosyringes for only 20 minutes. The results are shown in Figure 19A and demonstrate delivery of the tBid p15 fragment and Bax BH3 domain (via successful induction of apoptosis). The tBid p15 fragment (SEQ ID NO: 109) is part of the normal human apoptosis regulatory pathway. Cellular effect: A pro-apoptotic 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 by caspase 8 from its latent cytoplasmic full-length pro-Bid form. BaxBH3 (aa 59-73) (SEQ ID NO: 111) is a minimal BH3 domain synthetic peptide containing the critical 15 residues of the defined Bax BH3 domain. Cellular effect: These 15 residues are required to bind to and functionally antagonize Bcl-xL. and contains sufficient information to specifically induce Bax / Bak, which is thought to terminate Bak / Bcl-2 interaction and release pro-apoptotic factors.

[0224] A more detailed examination of the delivery of proapoptotic human peptides into ex vivo peripheral blood mononuclear cells (PBMCs) is now described. The purpose of this study was to investigate whether proapoptotic peptide-loaded PVC nanosyringes could induce apoptosis in ex vivo human peripheral blood mononuclear cells. The nanosyringes were evaluated first for any immediate cytotoxicity using a trypan blue dye exclusion assay, and then for an apoptotic response by using a TUNEL assay.

[0225] Trypan blue exclusion test for cell viability: Trypan blue is a diazo dye commonly used to selectively stain dead tissues or cells, so that dead cells appear distinctly blue under a microscope, while viable cells or tissues with intact cell membranes remain unstained. Because viable cells are excluded from the stain, this staining method is also described as a dye exclusion method. Trypan blue is commonly used to assess tissue or cell viability. An appropriate number of cells (2 × 105) was exposed to a nanosyringe and an empty nanosyringe for 20 minutes. An appropriate 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. Blood cells from two independent human donors were tested for each compound at each concentration, and each sample was tested in duplicate.

[0226] Cell processing and preparation for microscopy: The viability of peripheral blood mononuclear cells (PBMCs) from two independent healthy human donors was determined after 20 minutes of treatment with two chimeric nanosyringes (e.g., loaded with exogenous pro-apoptotic peptides) at three test concentrations in two independent experiments. PBMCs were harvested by centrifugation and resuspended in culture medium at 1 x 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 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 / fixation. Two fixations were performed per slide to allow for duplicate staining. The cell suspension was allowed to air dry.

[0227] PBMC cell viability assay results: Trypan blue viability assays confirmed that the PVC preparation was not immediately toxic to PBMCs collected from healthy human donors (Table 2). Nanosyringe treatments demonstrated >60% viability and low toxicity at the highest dose concentrations (Table 2). We then subsequently examined the ability of the chimeric nanosyringe to induce apoptosis.

[0228] [Table 4]

[0229] Testing for 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 deoxynucleotidyl transferase (TdT) binds to the exposed 3'-OH ends of DNA fragments generated in response to apoptotic signaling factors. This, in turn, catalyzes the addition of biotin-labeled deoxynucleotides, which can be detected using streptavidin-horseradish peroxide (HRP) conjugates. Diaminobenzidine (DAB) reacts with the HRP-labeled sample to produce an insoluble brown substrate at the site of DNA fragmentation. Methyl green counterstaining allows visualization of normal and apoptotic cells.

[0230] The induction of apoptosis after exposure of human PBMCs to the nanosyringe was determined. A TUNEL assay kit (Abcam) was used to detect apoptotic cells. The assay was performed according to the manufacturer's instructions. The procedure was performed according to the manufacturer's instructions. Briefly, slides were coated with 100 μL of proteinase K solution for 5 minutes and then rinsed with 1× TRIS-buffered saline (TBS). Nanosyringe or DNase I positive kit control treatments were performed for 20 minutes at room temperature. Slides were rinsed with TBS. Slides were then incubated with TdT equilibration buffer for 30 minutes before the TdT labeling reaction mix was added. Slides were incubated at 37°C for 19 minutes. Slides were then washed with TBS, and stop buffer was applied and incubated for 5 minutes at room temperature. Slides were washed again with TBS, and blocking buffer was added for 10 minutes at room temperature. Detection was performed by applying the conjugate to the sample for 30 minutes. Slides were rinsed with TBS, and DAB solution was applied for 15 minutes. Slides were rinsed with dH2O and then counterstained with methyl green. Slides were dehydrated in 100% ethanol, followed by xylene, and mounted with glass coverslips. All staining was performed in duplicate. Apoptotic endpoints showing positive staining in the apoptosis detection assay are represented by a dark brown (DAB) signal. Lighter shades of brown and / or blue / green to green / brown shades indicate non-responsive negative cells for apoptosis.

[0231] Analysis was performed by selecting five random portions of cells on the slide, counting positively stained (dark brown) and negatively stained cells (blue or light brown), and determining the percentage of cells showing apoptotic bodies.

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

[0233] PBMC Apoptosis Assay Results: 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 nanosyringes induced an apoptotic signal. Positive (DNase I-treated) and negative (no DNase I-treated) controls were included. Results showed that both nanosyringes containing either tBID or Bax exhibited strong apoptotic signals in PBMCs (89% and 78% positive, respectively). The positive control exhibited a strong apoptotic signal (79%), while the negative control exhibited no apoptotic signal (100% negative). A significant loss in the number of adherent cells was also observed in the nanosyringe-treated samples, which was considered to indicate a rapid and comprehensive apoptotic response and failure to retain them after washing. Note that this effect was significantly more pronounced than the kit positive control, suggesting a more rapid response. Representative photomicrographs are shown in Figure 19B.

[0234] Conclusion: We conclude that tBID- and Bax-loaded nanosyringes can rapidly induce extensive apoptosis in human peripheral blood mononuclear cells. Furthermore, trypan blue dye exclusion assays confirmed that these chimeric nanosyringes did not cause rapid lethal lysis or extensive membrane damage to cells.

[0235] Example 10 Demonstrating the practical utility of leader sequences and PVC needle conjugates - Intracellular delivery of atypical (non-Photorhabdus) payloads (1) An anti-MDM (p53 inhibitor) antibody is conjugated to a leader sequence described herein and expressed together with a PVC-needle complex for packaging. The isolated PVC-needle complex (containing the antibody payload) is contacted with a tumor for intracellular delivery of the antibody (the tumor cells are characterized as having high MDM suppression of p53 activity due to MDM inhibition). The tumor is inhibited by the activity of the anti-MDM antibody. (2) Antitumor peptide vaccines are delivered intracellularly using PVC needle conjugates to activate MHC-1-dependent cytotoxic T cell lymphocyte (CTL) responses. Tyrosinase-related protein 2 (TRP2) peptide vaccines are delivered to CTLs for enhanced cross-presentation, resulting in antitumor effects against TRP2-expressing tumors. Tumors are suppressed by the activity of the peptide vaccine. (3) The PVC needle complex is used to deliver nuclear factor-kB inhibitors (used to control inflammatory disorders, e.g., rheumatoid arthritis) to cells (intracellularly), which subsequently demonstrate a reduction in the expression of pro-inflammatory cytokines. (4) The PVC-needle complex is used to deliver a T3SS payload (inhibiting the NF-kB and MAPK pathways) intracellularly, which is accomplished with isolated (purified) PVC-needle complexes, without the need for the PVC-needle complexes to remain associated with the bacterial cells from which they originate. (5) Using the PVC needle complex, cells are injected with anti-apoptotic peptides, such as BH4, Bcl-xL-protein, and / or peptide inhibitors of c-Jun N-terminal kinase. The inhibitors deliver (intracellularly) harmful agents that can protect the heart and brain from ischemic damage (restriction of blood supply to tissues, resulting in a lack of oxygen and glucose needed for cellular metabolism). For example, inhibition of Jun-kinase via the 20 amino acid binding motif of JUN kinase is sufficient. For example, the release of cytochrome c in cells is inhibited. (6) PVC needle conjugates were used to deliver nicotinamide adenine dinucleotide quinone internal oxidoreductase (Ndi1), a single-subunit yeast analog of complex I (which provides significant cardioprotective effects), to complex I-deficient mutant cells. The Ndi1 protein was precisely targeted to the matrix side of the inner mitochondrial membrane and restored NADH oxidase activity to complex I-deficient cells. (7) Using a PVC needle conjugate, we delivered one of two essential subunits of the PHOX complex (used in enzyme replacement therapy to restore ROS production in chronic granulomatous disease) to chronic granulomatous disease cells, and observed restoration of ROS production. (8) PVC needle complexes were used to deliver myotubularin (used to improve local and distal muscle performance in patients with X-linked myotubular myopathy) intracellularly (e.g., intramuscularly). Myotubularin dephosphorylation of phosphatidylinositol triphosphate and phosphatidylinositol (3,5) bisphosphate was observed. (9) A PVC needle complex is used to deliver the recombinase "Cre" (which can excise a defined gene cassette) into a mouse cell line whose genome contains loxP recombination sites flanking a termination signal upstream of the mCherry gene. The Cre payload excises the recombination site, removing the termination signal and allowing expression of the mCherry gene in the cell. (10) A PVC needle conjugate is used to deliver (intracellularly) an approximately 15 kDa nanobody (antibody fragment) with affinity for intracellular components. The nanobody-intracellular complex is detected. (11) PVC needle conjugates have been used to deliver atypical (non-Photorhabdus) polypeptide toxins intracellularly (e.g., into insect cells) for insect crop pests and animal parasites, and pest suppression has been observed. (12) A PVC needle complex is used to deliver nucleases (e.g., Cas9 and / or Mad7) containing guide RNA into target cells (intracellularly), where the nucleases perform site-specific gene inactivation.

[0236] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and bioengineering or related fields are intended to be within the scope of the following claims.

[0237] array Where the first Met amino acid residue or the corresponding first codon is shown in any of the following SEQ ID NOs, said residue / codon may be optional.

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

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

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

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

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

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

[0244] SEQ ID NO: 7 (PAK_2893) [ka]

[0245] SEQ ID NO: 8 (PAK_2894) [ka]

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

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

[0248] Sequence number 11 (PAT_00149) MIFKMLNLAVFYLLGNIFHYLICQKFICYFCSVLKSVTMFLTKVAVQIALYLNILPTMAGIAGLHAEVQALNNLFISGDRGTEKRENWKYIRNMLESTIFTQRLTAGQAGKDFAACHNCSGILSSPVNVITGKVESAGGNFFINIISI

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

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

[0251] SEQ ID NO: 14 (PAT_02308) [ka]

[0252] SEQ ID NO: 15 (PAT_02309) [ka]

[0253] SEQ ID NO: 16 (PAT_02310) [ka]

[0254] SEQ ID NO: 17 (PAT_02956) [ka]

[0255] SEQ ID NO: 18 (PAT_02957) [ka]

[0256] SEQ ID NO: 19 (PAT_03171) [ka]

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

[0258] SEQ ID NO: 21 (PAT_03177) [ka]

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

[0260] SEQ ID NO: 23 (PAU_02010) [ka]

[0261] SEQ ID NO: 24 (PAU_02095) [ka]

[0262] SEQ ID NO: 25 (PAU_02096) [ka]

[0263] SEQ ID NO: 26 (PAU_02097) [ka]

[0264] SEQ ID NO: 27 (PAU_02098) [ka]

[0265] SEQ ID NO: 28 (PAU_02230) [ka]

[0266] SEQ ID NO: 29 (PAU_02805) [ka]

[0267] SEQ ID NO: 30 (PAU_02806) [ka]

[0268] SEQ ID NO: 31 (PAU_02807) [ka]

[0269] SEQ ID NO: 32 (PAU_03332) [ka]

[0270] SEQ ID NO: 33 (PAU_03337) [ka]

[0271] SEQ ID NO: 34 (Plu1651) [ka]

[0272] SEQ ID NO: 35 (Plu1671) [ka]

[0273] SEQ ID NO: 36 (Plu1672) [ka]

[0274] SEQ ID NO: 37 (Plu1690) [ka]

[0275] SEQ ID NO: 38 (Plu1691) [ka]

[0276] SEQ ID NO: 39 (Plu1712) [ka]

[0277] SEQ ID NO: 40 (Plu1713) [ka]

[0278] SEQ ID NO: 41 (Plu1714) MKKTDEKYGQYEYKDEDITSYPIAWTNPDNGKIYIGINSPEYSHLNNKGESELNLAKIISTIIHESLHASSHQHKGLQSQTDTGADNLNYDEYVTDYFAREVYKQILPDKDYVANCFTKGLGGENKIWGGNIVEFMIQ

[0279] SEQ ID NO: 42 (Plu2400) [ka]

[0280] SEQ ID NO: 43 (Plu2401) [ka]

[0281] SEQ ID NO: 44 (Plu2514) [ka]

[0282] SEQ ID NO: 45 (Plu2515) [ka]

[0283] SEQ ID NO: 46 (Plu1649) [ka]

[0284] [Table 5]

[0285] SEQ ID NO: 93 (Photorhabdus asymbiotica strain ATCC 43949 PVCPnf operon, pvc1 to pvc16; see GenBank accession no. (corresponding to genes PAU_03353 to PAU_03338 in sequence No. FM162591.1) [ka] [ka] [ka] [ka] [ka]

[0286] SEQ ID NO: 94 (Photorhabdus asymbiotica strain ATCC 43949 PVClopT operon, pvc1 to pvc16; e.g., corresponding to genes PAU_02112 to PAU_02099 in the sequence of GenBank accession number FM162591.1) [ka] [ka] [ka] [ka]

[0287] SEQ ID NO: 95 (Photorhabdus asymbiotica strain ATCC 43949 PVC PaTox operon, pvc1 to pvc16) [ka] [ka] [ka] [ka] [ka]

[0288] SEQ ID NO: 96 (Pnf epitope) TGQKPGNNEWKTGR

[0289] SEQ ID NO: 97 (PVCpromF) TATCATATGTCTACAACTCCAGAACAAATTGCTG

[0290] SEQ ID NO: 98 (PVCpromR) ATCTCTAGAACAGATATTCCAGCCAGC

[0291] SEQ ID NO: 99 (ParaINF) GGCGTCACACTTTGCTATG

[0292] SEQ ID NO: 100 (ParaINF) TCGGTGGCAGTAAATTGTCC

[0293] SEQ ID NO: 101 (F1 primer) ATGTCTACAAGTACATCTCAAATTGCG

[0294] SEQ ID NO: 102 (F2 primer) GACTCCCTTGAGGGTACGG

[0295] SEQ ID NO: 103 (F3 primer) TTCTGATGAGAGTGATGGTAC

[0296] SEQ ID NO: 104 (F4 primer) TGAATAAAGAATTCAGTCAATATC

[0297] SEQ ID NO: 105 (R1 primer) TAGTGGCTGATGAAAGTCTG

[0298] SEQ ID NO: 106 (R2 primer) GGAAGCCAAAGATAATGAAGTG

[0299] SEQ ID NO: 107 (R3 primer) CATTTCTTCCCTATGGTTG

[0300] SEQ ID NO: 108 (R4 primer) TTAAATTCCTACAAGATTATCTTT

[0301] SEQ ID NO: 109 (tBid amino acid sequence) RSSHSRLGRIEADSESQEDIIRNIARHLAQVGDSMDRSIPPGLVNGLALQLRNTSRSEEDRNRDLATALEQLLQAYPRDMEKEKTMLVLALLLAKKVASHTPSLLRDVFHTTVNFINQNLRTYVRSLARNGMD

[0302] SEQ ID NO: 110 (E. coli sequence-optimized tBid base) [ka]

[0303] SEQ ID NO: 111 (BaxBH3 peptide (aa59-73)) LSESLKRIGDELDSN

[0304] SEQ ID NO: 112 (E. coli sequence-optimized BaxBH3 base) CTGTCGGAGAGTTTGAAGCGTATAGGTGACGAGCTGGACAGCAAT

Claims

1. 1. Use of a leader peptide of a Photorhabdus virulence cassette (PVC) effector protein to package a payload into a PVC needle complex, comprising: the leader peptide has an amino acid sequence that packages a polypeptide payload into a PVC needle complex; the polypeptide payload is a therapeutic polypeptide, a toxigenic polypeptide, and / or a nucleic acid-modifying enzyme; the leader peptide and the payload form an effector fusion that is distinct from a wild-type PVC effector protein; The use comprises contacting the PVC needle complex 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 of the sequences selected from SEQ ID NO: 47 to SEQ ID NO:

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 NO: 1 to SEQ ID NO:

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. 1. A method of manufacturing a PVC needle composite containing a payload, comprising: a. contacting a PVC-needle complex with an effector fusion comprising a leader peptide of a PVC-effector protein fused to a payload, wherein said 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, a toxigenic polypeptide, and / or a nucleic acid-modifying enzyme; c. The effector fusion is distinct from the wild-type PVC effector protein method.

9. 9. The method of claim 8, wherein the contacting is performed intracellularly in an in vitro cell system, in a cell lysate, or in a purified cell lysate.

10. 1. An in vitro and / or ex vivo method of delivering a payload into a cell, comprising: a. contacting a cell with a PVC needle complex containing an effector fusion; b. the effector fusion comprises 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 a PVC-needle complex; c. the polypeptide payload is a therapeutic polypeptide, a toxigenic polypeptide, and / or a nucleic acid-modifying enzyme; d. The effector fusion is distinct from the wild-type PVC effector protein method.

11. 1. An in vitro method for controlling pests comprising: a. contacting the pest, or a target area containing the pest, with a PVC needle conjugate containing an effector fusion; b. the effector fusion comprises 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 a PVC-needle complex; c. the polypeptide payload is a therapeutic polypeptide, a toxigenic polypeptide, and / or a nucleic acid-modifying enzyme; d. The effector fusion is distinct from the wild-type PVC effector protein method.

12. 1. A PVC needle composite for use in a method of treatment comprising: a. the PVC-needle complex comprises an effector fusion comprising 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, a toxigenic polypeptide, and / or a nucleic acid-modifying enzyme; c. The effector fusion is distinct from the wild-type PVC effector protein PVC needle composite.

13. A PVC needle composite comprising an effector fusion; a. the effector fusion comprises 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 a PVC-needle complex; b. the polypeptide payload is a therapeutic polypeptide, a toxigenic polypeptide, and / or a nucleic acid-modifying enzyme; c. The effector fusion is distinct 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 a payload; a. the leader peptide has an amino acid sequence that packages a polypeptide payload into a PVC needle complex; b. the polypeptide payload is a therapeutic polypeptide, a toxigenic polypeptide, and / or a nucleic acid-modifying enzyme; c. The effector fusion is distinct from the wild-type PVC effector protein Effector fusions.

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

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

18. 12. The method of any one of claims 8 to 11, wherein the leader peptide comprises an amino acid sequence having about 60% sequence identity with one or more sequences selected from SEQ ID NO: 47 to SEQ ID NO:

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 NO: 47 to SEQ ID NO:

92.

20. The effector fusion of claim 14, wherein the leader peptide comprises an amino acid sequence having about 60% sequence identity with one or more sequences selected from SEQ ID NO: 47 to SEQ ID NO:

92.

21. The method of 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 NO: 1 to SEQ ID NO:

46.

22. The PVC needle complex according to claim 12 or 13, wherein the PVC effector protein comprises one or more amino acid sequences selected from SEQ ID NO: 1 to SEQ ID NO:

46.

23. The effector fusion of claim 14, wherein the PVC effector protein comprises an amino acid sequence of one or more sequences selected from SEQ ID NO: 1 to SEQ ID NO:

46.

24. 12. The method of 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. 14. The PVC needle complex of 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. 15. The effector fusion of 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 of any one of claims 8 to 11, wherein the leader peptide is covalently fused to the payload.

28. 14. The PVC needle conjugate of claim 12 or 13, wherein the leader peptide is covalently fused to the payload.

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

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

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

32. 32. A host cell comprising the isolated nucleic acid molecule of claim 30 or the expression vector of claim 31.

33. one or more selected from mammalian cells, insect cells, yeast cells, bacterial cells, and / or plant cells; 33. The host cell of claim 32, wherein the bacterial cell is preferably an E. coli cell.

34. 33. The host cell of claim 32, which is a Photorhabdus cell.

35. 35. The host cell of claim 34, wherein the Photorhabdus cell comprises a Photorhabdus PVC operon operably linked to an inducible promoter.

Images