Bacterial-derived lipid compositions and uses thereof

Abstract

Disclosed herein is a bacterial-derived lipid composition comprising (a) a bacterial component comprising one or more lipids extracted from a bacterial source, and (b) an ionizable lipid. The disclosure also includes a method of making a bacterial-derived lipid composition, comprising reconstituting (a) the bacterial component comprising one or more lipids extracted from a bacterial source in the presence of (b) an ionizable lipid, and loading the bacterial-derived lipid composition with one or more heterologous functional agents to produce the bacterial-derived lipid composition.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 282,304, filed November 23, 2021, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Delivery of heterofunctional agents (such as therapeutic or immunological agents) can be limited by the extent to which the agent can penetrate cellular barriers and thereby effectively affect the organism. Thus, there is a continuing need in the art to develop new delivery systems that can effectively deliver heterofunctional agents and promote cellular uptake of the agents. Summary of the Invention

[0003] In one aspect, provided herein is a bacterial-derived lipid composition comprising: (a) a bacterial component comprising one or more lipids extracted from a bacterial source; and (b) an ionizable lipid.

[0004] In another aspect, provided herein is a bacterial-derived lipid composition comprising a plurality of lipid-reconstituted bacterial components, the lipid-reconstituted bacterial components being produced by a process comprising: (a) providing a plurality of purified bacterial lipids; (b) treating the plurality of purified bacterial lipids to produce a lipid membrane; (c) reconstituting the lipid membrane in an organic solvent selected from the group consisting of acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethyl sulfoxide:methanol, and dimethylformamide:methanol, thereby producing a lipid solution; and (d) treating the lipid solution of step (c) in a microfluidic device comprising an aqueous phase, thereby producing the bacterial-derived lipid composition.

[0005] In another aspect, provided herein is a method for producing a bacterial-derived lipid composition. The method comprises (a) (b) reconstituting bacterial components comprising one or more lipids extracted from a bacterial source in the presence of an ionizable lipid to produce a bacterial-derived lipid composition. The method further comprises loading the bacterial-derived lipid composition with one or more heterologous functional agents. The ionizable lipid may be one of the following: (i) at least two ionizable amines; (ii) at least three lipid tails, each of the lipid tails being at least six carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and heteroorganic group separated by a chain of at least two atoms; and (v) having two or more characteristics of an N:P ratio of at least 10;

[0006] In some alternative embodiments, the ionizable lipid has the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of the lipid tails being at least six carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and heteroorganic group separated by a chain of at least two atoms; and (v) an N:P ratio of at least 3;

[0007] In another aspect, provided herein is a method for delivering a bacterially derived lipid composition to a target cell, the method comprising introducing into the target cell (a) a bacterial component comprising one or more lipids extracted from a bacterial source, and (b) a bacterially derived lipid composition comprising an ionizable lipid.

[0008] In some embodiments, the reconstitution step comprises reconstituting membranes comprising purified bacterial lipids of bacterial component (a) in the presence of ionizable lipids (b) to produce a bacterial-derived lipid composition.

[0009] In some embodiments, the bacterial source is selected from the group consisting of Escherichia coli, Acinetobacter, Agrobacterium, Anabaena, Aquifex, Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, The bacterial strain is selected from the genera Polaromonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wallinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exiguobacterium, Geobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium, and Thermoanaerobacterium. In one embodiment, the bacterial source is Escherichia (e.g., E. coli). In one embodiment, the bacterial source is Salmonella (e.g., Salmonella typhimurium).

[0010] In some embodiments, the bacterial component comprises isolated bacterial extracellular vesicles.

[0011] In some embodiments, the bacterial components are modified by reconstituting membranes comprising the bacterial components in the presence of ionizable lipids.

[0012] In some embodiments, the bacterial components are modified by reconstituting membranes containing purified bacterial lipids of the bacterial components with ionizable lipids.

[0013] In some embodiments, the ionizable lipid is (i) at least two ionizable amines; (ii) at least three lipid tails, each of the lipid tails being at least six carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and heteroorganic group separated by a chain of at least two atoms; and (v) having one or more characteristics selected from the group consisting of an N:P ratio of at least 10;

[0014] In some alternative embodiments, the ionizable lipid is (i) at least two ionizable amines; (ii) at least three lipid tails, each of the lipid tails being at least six carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and heteroorganic group separated by a chain of at least two atoms; and (v) having one or more of the characteristics selected from the group consisting of an N:P ratio of at least 3.

[0015] In some embodiments, the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315. In one embodiment, the ionizable lipid is C12-200.

[0016] In some embodiments, the ionizable lipid is [ka] (I), wherein R is C-C 14 It is an alkyl group.

[0017] In some embodiments, the bacterially derived lipid composition further comprises a sterol. Thus, the reconstitution (or reconstitution) of the bacterial components is carried out in the presence of an ionizable lipid and a sterol.

[0018] In some embodiments, the bacterial-derived lipid composition further comprises a polyethylene glycol (PEG)-lipid conjugate. Thus, the reconstitution (or reconstitution) of bacterial components is carried out in the presence of an ionizable lipid and a PEGylated lipid (or PEG-lipid conjugate).

[0019] In some embodiments, the bacterial-derived lipid composition further comprises a sterol and a polyethylene glycol (PEG)-lipid conjugate. Thus, the reconstitution (or reconstitution) of bacterial components is carried out in the presence of an ionizable lipid, a sterol, and a PEGylated lipid (or a PEG-lipid conjugate).

[0020] In some embodiments, the sterol is cholesterol or sitosterol.

[0021] In some embodiments, the PEG-lipid conjugate is C14-PEG2k, C18-PEG2k or DMPE-PEG2k. In some embodiments, the PEG-lipid conjugate is PEG-DMG or PEG-PE. In some embodiments, the PEG-DMG is PEG2000-DMG or PEG2000-PE.

[0022] In some embodiments, the bacterial-derived lipid composition comprises: about 20 mol % to about 50 mol % of an ionizable lipid; about 20 mol% to about 60 mol% bacterial components (e.g., extracted bacterial lipids); about 7 mol % to about 45 mol % sterol, and It contains about 0.5 mol % to about 3 mol % polyethylene glycol (PEG)-lipid conjugate.

[0023] In some embodiments, the bacterial-derived lipid composition comprises: about 30 mol % to about 40 mol % of an ionizable lipid; about 20 mol% to about 50 mol% bacterial components (e.g., extracted bacterial lipids); about 12 mol % to about 43 mol % sterols, and It contains about 1.5 mol % to about 3 mol % polyethylene glycol (PEG)-lipid conjugate.

[0024] In some embodiments, the bacterial-derived lipid composition comprises: about 35 mole % ionizable lipids; about 50 mol % bacterial components (e.g., extracted bacterial lipids); about 12.5 mole % sterols, and It contains about 2.5 mole % polyethylene glycol (PEG)-lipid conjugate.

[0025] In one embodiment, the bacterially derived lipid composition comprises a molar ratio of about 35:50:12.5:2.5 of ionizable lipid:bacterial lipid:sterol:PEG-lipid.

[0026] In one embodiment, the bacterially derived lipid composition comprises a molar ratio of about 35:20:42.5:2.5 of ionizable lipid:bacterial lipid:sterol:PEG-lipid.

[0027] In some embodiments, the bacterial-derived lipid composition comprises: Lipids extracted from Escherichia coli (e.g., Escherichia coli) or Salmonella (e.g., Salmonella typhimurium), C12-200, Cholesterol, and Contains DMPE-PEG2k.

[0028] In some embodiments, the bacterial-derived lipid composition comprises: Polar lipids extracted from E. coli, C12-200, Cholesterol, and The bacterial-derived lipid composition may comprise C12-200:E. coli polar lipid:cholesterol:DMPE-PEG2k in a molar ratio of about 35:50:12.5:2.5, or about 35:20:42.5:2.5.

[0029] In some embodiments, the bacterial-derived lipid composition is a lipophilic moiety selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions. In one embodiment, the bacterial-derived lipid composition is a liposome selected from the group consisting of cationic liposomes, nanoliposomes, proteoliposomes, unilamellar liposomes, multilamellar liposomes, ceramide-containing nanoliposomes, and multivesicular liposomes.

[0030] In some embodiments, the bacterial-derived lipid composition is a lipid nanoparticle.

[0031] In some embodiments, the particles of the bacterial-derived lipid composition have a size of less than about 200 nm. In one embodiment, the particles of the bacterial-derived lipid composition are The particles of the bacterial-derived lipid composition have a size of less than about 150 nm. In one embodiment, the particles of the bacterial-derived lipid composition have a size of less than about 100 nm. In one embodiment, the particles of the bacterial-derived lipid composition have a size of about 55 nm to about 95 nm. In one embodiment, the particles of the bacterial-derived lipid composition have a size of about 85 nm to about 95 nm. In one embodiment, the particles of the bacterial-derived lipid composition have a size of about 85 nm to about 90 nm.

[0032] In some embodiments, the particles of the bacterial-derived lipid composition have an average polydispersity index (PDI) in the range of about 0.1 to about 0.5. In some embodiments, the particles of the bacterial-derived lipid composition have an average PDI in the range of about 0.1 to about 0.4. In some embodiments, the particles of the bacterial-derived lipid composition have an average PDI in the range of about 0.2 to about 0.3.

[0033] In some embodiments, the bacterial-derived lipid composition comprises one or more heterologous functional agents. In some embodiments, the heterologous functional agent is encapsulated by the bacterial-derived lipid composition. In some embodiments, the heterologous functional agent is embedded on the surface of the bacterial-derived lipid composition. In some embodiments, the heterologous functional agent is conjugated to the surface of the bacterial-derived lipid composition.

[0034] In some embodiments, the heterologous functional agent is a polynucleotide. In some embodiments, the polynucleotide is selected from mRNA, siRNA or siRNA precursor, microRNA (miRNA) or miRNA precursor, plasmid, Dicer substrate small interfering RNA (dsiRNA), small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), piRNA (piRNA), ribozyme, deoxyribozyme (DNAzyme), aptamer, circular RNA (circRNA), guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In some embodiments, the polynucleotide is mRNA. In some embodiments, the mRNA is derived from a DNA molecule or an RNA molecule (e.g., a self-replicating RNA molecule). In some embodiments, the polynucleotide is an siRNA or a precursor thereof. In some embodiments, the polynucleotide is a plasmid.

[0035] In some embodiments, the bacterial-derived lipid composition has an encapsulation efficiency of at least about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or greater than 99%. In one embodiment, the bacterial-derived lipid composition has an encapsulation efficiency of at least about 90%.

[0036] In some embodiments, the bacterial-derived lipid nanoparticles have a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 50:1 to about 10:1. In some embodiments, the bacterial-derived lipid nanoparticles have a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 44:1 to about 24:1. In some embodiments, the bacterial-derived lipid nanoparticles have a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 40:1 to about 28:1. In some embodiments, the bacterial-derived lipid nanoparticles have a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 38:1 to about 30:1. In some embodiments, the bacterial-derived lipid nanoparticles have a total lipid:heterofunctional agent (e.g., polynucleotide) weight ratio of about 37:1 to about 33:1.

[0037] In some embodiments, the bacterial-derived lipid compositions are formulated for delivery to an animal or human. In some embodiments, the bacterial-derived lipid compositions are formulated for delivery to a plant.

[0038] In some embodiments, the bacterial-derived lipid composition is produced by a method comprising lipid extrusion, hi some embodiments, the bacterial-derived lipid composition is produced by a method comprising processing a solution comprising lipids from the bacterial-derived lipid composition in a microfluidic device comprising an aqueous phase, thereby producing the bacterial-derived lipid composition.

[0039] In some embodiments, the heterologous functional agent is formulated into the bacterial-derived lipid composition via the aqueous phase. In some embodiments, the aqueous phase and the lipid solution (organic phase) are mixed in a volume ratio of 3:1.

[0040] In some embodiments, the aqueous phase comprises a polynucleotide.

[0041] In some embodiments, the bacterial-derived lipid composition, e.g., the aqueous phase, further comprises a HEPES or TRIS buffer. The HEPES or TRIS buffer may have a pH of about 7.0 to about 8.5. The HEPES or TRIS buffer may have a concentration of about 7 mg / mL to about 15 mg / mL. The aqueous phase may further comprise about 2.0 mg / mL to about 4.0 mg / mL of NaCl.

[0042] In some embodiments, the bacterial-derived lipid composition, e.g., the aqueous phase, comprises water, PBS, or a citrate buffer. In one embodiment, the aqueous phase comprises a citrate buffer having a pH of about 3.2.

[0043] In some embodiments, the bacterial-derived lipid composition further comprises one or more cryoprotectants. The one or more cryoprotectants may be sucrose, glycerol, or a combination thereof. In some embodiments, the bacterial-derived lipid composition comprises sucrose, e.g., at a concentration of about 70 mg / mL to about 110 mg / mL. In some embodiments, the bacterial-derived lipid composition comprises glycerol, e.g., at a concentration of about 50 mg / mL to about 70 mg / mL. In one embodiment, the bacterial-derived lipid composition comprises a combination of sucrose (e.g., at a concentration of about 70 mg / mL to about 110 mg / mL) and glycerol (e.g., at a concentration of about 50 mg / mL to about 70 mg / mL).

[0044] In some embodiments, the bacterial-derived lipid composition is a lyophilized or freeze-dried composition. The lyophilized or freeze-dried bacterial-derived lipid composition may include one or more lyoprotectants. The lyophilized bacterial-derived lipid composition may include a poloxamer, potassium sorbate, sucrose, or any combination thereof. In one embodiment, the lyophilized bacterial-derived lipid composition includes a poloxamer (e.g., about 0.01 to about 1.0% w / w poloxamer), and in one embodiment, the poloxamer is poloxamer 188.

[0045] In some embodiments, the bacterial-derived lipid composition is a lyophilized composition. In some embodiments, the lyophilized bacterial-derived lipid composition comprises about 0.01 to about 1.0% w / w of a heterologous functional agent (e.g., a polynucleotide). In some embodiments, the lyophilized bacterial-derived lipid composition comprises about 1.0 to about 5.0% w / w of lipid. In some embodiments, the lyophilized bacterial-derived lipid composition comprises about 0.5 to about 2.5% w / w of TRIS buffer. In some embodiments, the lyophilized bacterial-derived lipid composition comprises about 0.75 to about 2.75% w / w of NaCl. In some embodiments, the lyophilized bacterial-derived lipid composition comprises about 85 to about 95% w / w of a sugar, e.g., sucrose. In some embodiments, the lyophilized bacterial-derived lipid composition comprises about 0.01 to about 1.0% w / w of a poloxamer (e.g., about 0.01 to about 1.0% w / w of a poloxamer), e.g., poloxamer 188. In some embodiments, the lyophilized bacterial-derived lipid composition comprises about 1.0 to about 5.0% w / w potassium sorbate.

[0046] definition As used herein, the terms "effective amount," "effective concentration," or "effective concentration" refer to an amount of a bacterial-derived lipid or nucleic acid composition sufficient to produce a recited result or to reach a target level (e.g., a predetermined level or threshold level) in or on a target organism.

[0047] As used herein, the term "therapeutic agent" refers to an agent that can act on an animal, e.g., a mammal (e.g., a human), an animal pathogen, or a pathogen vector, such as an antifungal, antibacterial, virucide, antiviral, insecticide, nematicide, antiparasitic, or insect repellent.

[0048] As used herein, the term "heterologous" refers to an agent that is exogenous to the organism to which the bacterial-derived lipid composition is delivered.

[0049] As used herein, the term "functional agent" refers to an agent (e.g., an agricultural formulation (e.g., an insecticide, fertilizer, herbicide, plant-modifying agent) or therapeutic agent (e.g., an antifungal, antibacterial, virucide, antiviral, insecticide, nematicide, antiparasitic, or insect repellent)) that can be or be associated with a bacterial-derived lipid composition (e.g., loaded onto or onto a bacterial-derived lipid composition (e.g., encapsulated by, embedded in, or conjugated to a bacterial-derived lipid composition) using in vivo or in vitro methods) and affect an enumerated outcome (e.g., increasing or decreasing the fitness of a plant, plant pest, plant symbiont, animal (e.g., human) pathogen, or animal pathogen vector). In some embodiments, the heterologous functional agent is a polynucleotide.

[0050] As defined herein, the terms "nucleic acid" and "polynucleotide" are interchangeable and refer to RNA or DNA, linear or branched, single or double stranded, or a hybrid thereof, regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 150, 200, 250, 500, 1000, or more nucleic acids). The term also encompasses RNA / DNA hybrids. Nucleotides are typically linked in nucleic acids by phosphodiester bonds, but the term "nucleic acid" also encompasses nucleic acid analogs having other types of linkages or backbones (e.g., phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoramidate, morpholino, locked nucleic acid (LNA), glycerol nucleic acid (GNA), threos nucleic acid (TNA), and peptide nucleic acid (PNA) linkages or backbones, among others). Nucleic acids may be single-stranded, double-stranded, or contain portions of both single-stranded and double-stranded sequences. Nucleic acids may contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including, for example, adenine, thymine, cytosine, guanine, uracil, and modified or non-standard bases (including, for example, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine, and 5-hydroxymethylcytosine).

[0051] As used herein, the terms "peptide," "protein," or "polypeptide" encompass any chain of naturally occurring or non-naturally occurring amino acids (either D- or L-amino acids), regardless of length (e.g., at least 2, 3, 4, 5, 6, 7, 10, 12, 14, 16, 18, 20, 25, 30, 40, 50, 100, or more amino acids), the presence or absence of post-translational modifications (e.g., glycosylation or phosphorylation), or the presence of, for example, one or more non-aminoacyl groups (e.g., sugars, lipids, etc.) covalently attached to the peptide, including, for example, naturally occurring proteins, synthetic, or recombinant polypeptides and peptides, hybrid molecules, peptoids, or peptidomimetics.

[0052] As used herein, the "percent identity" between two sequences is determined by the BLAST 2.0 algorithm, as described in Altschul et al. (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0053] As used herein, the term "unmodified bacterial component" refers to a composition comprising a bacterial component (e.g., isolated bacterial extracellular vesicles, or extracted bacterial lipids) that lacks a heterologous cell uptake agent that can increase cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the bacterial component.

[0054] As used herein, the term "modified" or "modification" with respect to a bacterial component refers to a bacterial component, as well as a bacterial-derived lipid composition containing one or more heterologous agents (e.g., one or more exogenous lipids, e.g., ionizable lipids, sterols, and / or PEGylated lipids) that can increase cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) of the bacterial-derived lipid composition, or a portion or component thereof, increase delivery of a heterologous functional agent (e.g., a pesticide or therapeutic agent) to a cell by the bacterial-derived lipid composition, and / or increase loading (e.g., loading efficiency or loading capacity) of a heterologous functional agent (e.g., a pesticide or therapeutic agent) compared to the unmodified bacterial component. The bacterial component may be modified in vitro or in vivo.

[0055] As used herein, the term "cellular uptake" refers to the uptake of a bacterial-derived lipid composition, or a portion or component thereof (e.g., a polynucleotide carried by a bacterial-derived lipid composition), by a cell, such as an animal cell, plant cell, bacterial cell, or fungal cell. For example, uptake can include the transfer of a portion of a bacterial-derived lipid composition, or a component thereof, from the extracellular environment to a cell membrane, cell wall, extracellular matrix, or across a cell membrane, cell wall, extracellular matrix, or into the intracellular environment of a cell. Cellular uptake of a bacterial-derived lipid composition can occur via active or passive cellular mechanisms. Cellular uptake includes aspects in which the entire bacterial-derived lipid composition is taken up by a cell, for example, by endocytosis. In some embodiments, one or more polynucleotides are exposed to the cytoplasm of a target cell after endocytosis and endosomal escape. In some embodiments, a bacterial-derived lipid composition comprising an ionizable lipid (e.g., a bacterial-derived lipid composition comprising an ionizable lipid and a sterol and / or PEGylated lipid) has an increased rate of endosomal escape compared to unmodified bacterial components (e.g., extracted bacterial lipids). Cellular uptake also includes aspects in which the bacterially derived lipid composition fuses with the membrane of the target cell. In some embodiments, the one or more polynucleotides are exposed to the cytoplasm of the target cell after membrane fusion. In some embodiments, the bacterially derived lipid composition has an increased rate of fusion with the membrane of the target cell (e.g., is more fusogenic) compared to bacterial components that are not modified with ionizable lipids.

[0056] As used herein, the term "cell permeation agent" refers to an agent that alters the properties (e.g., permeability) of the cell wall, extracellular matrix, or cell membrane of a cell (e.g., an animal cell, a plant cell, a bacterial cell, or a fungal cell) in a manner that promotes increased cellular uptake compared to cells not contacted with the agent.

[0057] Described herein is a bacterial-derived lipid composition comprising lipid-reconstituted bacterial components. The bacterial components are derived from lipid structures (e.g., lipid bilayers, monolayers, multilayers, e.g., vesicular lipid structures) derived from bacterial sources (e.g., enriched, isolated, or purified), and the lipid structures are disrupted (e.g., disrupted by lipid extraction) as described herein and reconstituted or reconstituted in a liquid phase (e.g., a liquid phase containing cargo) using standard methods, for example, by methods including lipid film hydration and / or solvent injection, to produce lipid-reconstituted bacterial components. If desired, the method may further include sonication, freeze / thaw treatment, and / or lipid extrusion, for example, to reduce the size of the reconstituted bacterial-derived lipid composition. Alternatively, the bacterial-derived lipid composition may be produced using a microfluidic device (e.g., NanoAssemblr® IGNITE™ microfluidic device (Precision NanoSystems)).

[0058] As used herein, the term "bacterial extracellular vesicles," "bacterial EVs," or "EVs" refers to enclosed lipid bilayer structures naturally present in bacteria. Optionally, bacterial EVs contain one or more bacterial EV markers. As used herein, the term "bacterial EV marker" refers to components naturally associated with bacteria, such as bacterial proteins, bacterial nucleic acids, bacterial small molecules, bacterial lipids, or combinations thereof.

[0059] As used herein, the term "cationic lipid" refers to a positively charged amphipathic molecule (e.g., a lipid or lipidoid) that contains a cationic group (e.g., a cationic head group).

[0060] As used herein, the term "ionizable lipid" refers to an amphipathic molecule (e.g., a lipid or lipidoid, e.g., a synthetic lipid or lipidoid) that contains a group (e.g., a head group) that can be ionized, e.g., dissociated to generate species having one or more charges, under given conditions (e.g., pH).

[0061] Surprisingly, it has been found that ionizable lipids comprising alkyl chains with multiple unsaturated sites, for example, at least two or three unsaturated sites, are particularly useful for forming lipid particles with increased membrane fluidity.Many ionizable lipids suitable for use herein and related analogs are described in U.S. Patent Publication Nos. 20060083780 and 20060240554, U.S. Patent Nos. 5,208,036, 5,264,618, 5,279,833, 5,283,185, 5,753,613 and 5,785,992, and PCT Publication No. 96 / 10390, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0062] In some embodiments, ionizable lipids are ionizable, so that they can dissociate according to pH and exist in a positively charged form.The ionization of ionizable lipids affects the surface charge of lipid nanoparticles that contain ionizable lipids under different pH conditions.The surface charge of lipid nanoparticles can in turn affect their plasma protein absorption, blood clearance and tissue distribution (Semple, SC, et al., Adv. Drug Deliv Rev 32:3-17(1998)) and their ability to form endosomolytic non-bilayer structures (Hafez, IM, et al., Gene Ther 8:1188-1196(2001)), which can affect the intracellular delivery of nucleic acids.

[0063] In some embodiments, the ionizable lipid is a lipid that is, for example, generally neutral at physiological pH (e.g., pH about 7), but can carry a net charge(s) at acidic or basic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH about 7, but can carry a net charge(s) at acidic pH. In one embodiment, the ionizable lipid is a lipid that is generally neutral at pH about 7, but can carry a net charge(s) at basic pH.

[0064] In some embodiments, ionizable lipids do not include cationic or anionic lipids, which generally carry a net charge(s) at physiological pH (eg, pH about 7).

[0065] As used herein, the term "lipidoid" refers to a molecule that has one or more characteristics of a lipid.

[0066] As used herein, the term "stable bacterial-derived lipid formulation" refers to a bacterial-derived lipid formulation that remains stable over a period of time (e.g., at least 24 hours, at least 48 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 30 days, at least 60 days, or at least 90 days), optionally within a defined temperature range (e.g., at least 24°C (e.g., at a temperature of at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C, or 23°C), or at least 24°C (e.g., at a temperature of at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C). at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 93 5%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or optionally, within a defined temperature range (e.g., at least 24°C (e.g., at least 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C), at least 20°C (e.g., at least 20°C, 21°C, 22°C, or 23°C), at least 4°C (e.g., at least 5°C, 10°C, or 15°C), at least -20°C (e.g., at least -20°C, -15°C, -10°C, , -5°C, or 0°C), or -80°C (e.g., at least -80°C, -70°C, -60°C, -50°C, -40°C, or -30°C)), compared to the initial activity of the bacterial-derived lipid composition (e.g., upon production or formulation), the activity (e.g., cell wall permeation activity and / or activity of mRNA formulated within the bacterial-derived lipid composition) is at least 5% (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,It refers to a bacterial-derived lipid composition that retains 100% or less of the lipids.

[0067] As used herein, the term "formulated for delivery to an animal" refers to a bacterial-derived lipid composition comprising a pharmaceutically acceptable carrier. As used herein, a "pharmaceutically acceptable" carrier or excipient is, for example, a carrier or excipient that is suitable for administration to an animal (e.g., a human) without undue adverse side effects to the animal (e.g., a human). [Brief explanation of the drawings]

[0068] [Figure 1] FIG. 1 is a scheme illustrating the workflow for preparing bacterial-derived lipid compositions by modifying extracted bacterial lipids with ionizable lipids.

[0069] [Figure 2] FIG. 2 shows the molar ratios of various components that make up an exemplary bacterial-derived lipid composition (BacLC) compared to the molar ratios of various components that make up conventional lipid nanoparticle compositions (LNPs), as described in Examples 2 and 5.

[0070] [Figure 3A] FIG. 3A shows the size and polydispersity of particles of an exemplary bacterial-derived lipid composition (E. coli BacLC) compared to particles of conventional lipid nanoparticle compositions (LNPs) prepared according to Examples 2 and 5. [Figure 3B] FIG. 3B shows the encapsulation efficiency of particles of the E. coli BacLC / mRNA formulation compared to particles of a comparative formulation (LNP / mRNA) prepared according to Example 5.

[0071] [Figure 4]Figure 4 shows the number of IFNg-producing antigen-specific T cells in 100 μL of blood in mice 12 days after a single dose of intramuscular delivery of BacLC E. coli (Avanti) / SARS-CoV-2 (E. coli (Avanti) BacLC, containing 1 μg of S mRNA) in mice compared to mice with intramuscular delivery of a comparative formulation (LNP / mRNA, containing 1 μg of S mRNA) prepared according to Example 5. The control was PBS.

[0072] [Figure 5] Figure 5 shows the levels of antibodies (IgG) specific to the S1 antigen of SARS-CoV-2 in the plasma of mice 12 days after a single dose of intramuscular delivery of BacLC E. coli (Avanti) / SARS-CoV-2 (E. coli (Avanti) BacLC, containing 1 μg of S mRNA) in mice compared to mice with intramuscular delivery of a comparative formulation (LNP / mRNA, containing 1 μg of S mRNA) prepared according to Example 5. The control was PBS.

[0073] [Figure 6] Figure 6 shows the number of SARS-CoV-2 S-specific T cells producing the cytokine IFNg per 10 splenocytes in mice 28 days after a single dose of intramuscular delivery of BacLC E. coli (Avanti) / SARS-CoV-2 (E. coli (Avanti) BacLC, containing 1 μg of S mRNA) in mice compared to mice with intramuscular delivery of a comparative formulation (LNP / mRNA, containing 1 μg of S mRNA) prepared according to Example 5. The control was PBS.

[0074] [Figure 7]Figure 7 shows the number of SARS-CoV-2 S-specific T cells producing the cytokine IFNg per 10 splenocytes in mice 12 days after single-dose intramuscular delivery of BacLC E. coli / SARS-CoV-2 (containing 10 μg of E. coli BacLC, S mRNA), BacLC E. coli (Avanti) / SARS-CoV-2 (containing 10 μg of E. coli (Avanti) BacLC, S mRNA), and BacLC Salmonella / SARS-CoV-2 (containing 10 μg of Salmonella BacLC, S mRNA), prepared according to Example 5. The control was PBS.

[0075] [Figure 8] Figure 8 shows the levels of antibodies (IgG) specific to the receptor binding domain (RBD) of SARS-CoV-2 in the plasma of mice 12 days after single-dose intramuscular delivery of BacLC E. coli / SARS-CoV-2 (containing 10 μg of E. coli BacLC, S mRNA), BacLC E. coli (Avanti) / SARS-CoV-2 (containing 10 μg of E. coli (Avanti) BacLC, S mRNA), and BacLC Salmonella / SARS-CoV-2 (containing 10 μg of Salmonella BacLC, S mRNA), prepared according to Example 5. The control was PBS.

[0076] [Figure 9] Figure 9 shows the levels of antibodies (IgG) specific to the receptor binding domain (RBD) of SARS-CoV-2 in the plasma of mice 28 days after single-dose intramuscular delivery of BacLC E. coli / SARS-CoV-2 (containing 10 μg of E. coli BacLC, S mRNA), BacLC E. coli (Avanti) / SARS-CoV-2 (containing 10 μg of E. coli (Avanti) BacLC, S mRNA), and BacLC Salmonella / SARS-CoV-2 (containing 10 μg of Salmonella BacLC, S mRNA), prepared according to Example 5. The control was PBS.

[0077] [Figure 10A]Figures 10A-10B show the levels of antibodies (IgA) specific to the receptor binding domain (RBD) (Figure 10A) or S-protein (Figure 10B) of SARS-CoV-2, respectively, in the plasma of mice 28 days after single-dose intramuscular delivery of BacLC E. coli / SARS-CoV-2 (containing 10 μg of BacLC, S mRNA), BacLC(Avanti) / SARS-CoV-2 (containing 10 μg of E. coli (Avanti) BacLC, S mRNA), and BacLC Salmonella / SARS-CoV-2 (containing 10 μg of Salmonella BacLC, S mRNA), prepared according to Example 5. The control was PBS. [Figure 10B] Same as above.

[0078] [Figure 10C] Figures 10C-10D show the levels of antibodies (IgA) specific to the receptor binding domain (RBD) (Figure 10C) or S-protein (Figure 10D) of SARS-CoV-2 in the bronchoalveolar lavage fluid (BALF) of mice 28 days after single-dose intramuscular delivery of BacLC E. coli / SARS-CoV-2 (containing 10 μg of E. coli BacLC, S mRNA), BacLC E. coli (Avanti) / SARS-CoV-2 (containing 10 μg of E. coli (Avanti) BacLC, S mRNA), and BacLC Salmonella / SARS-CoV-2 (containing 10 μg of Salmonella BacLC, S mRNA), prepared according to Example 5. The control was PBS. [Figure 10D] Same as above.

[0079] [Figure 11] 11 shows the number of influenza HA-specific T cells producing the cytokine IFNg per 10 splenocytes in mice 14 days after a single dose of intramuscular delivery of BacLC E. coli (Avanti) / Influenza (E. coli (Avanti) BacLC, containing 10 μg of HA mRNA) compared to mice with intramuscular delivery of a comparative formulation (LNP) prepared according to Example 5. The control was PBS.

[0080] [Figure 12] 12 shows the levels of influenza hemagglutinin (HA)-specific antibodies (IgG) in the plasma of mice 14 days after a single dose of intramuscular delivery of BacLC E. coli (Avanti) / Influenza (E. coli (Avanti) BacLC, containing 10 μg of HA mRNA) compared to mice with intramuscular delivery of a comparative formulation (LNP) prepared according to Example 5. The control was PBS.

[0081] [Figure 13A] Figures 13A-13C show the frequency of tdTomato lymphocytes (Figure 13A), myeloid cells (Figure 13B), and non-immune cells (Figure 13C), respectively, in the spleens of Ai9 mice 6 days after a single dose of intramuscular delivery of BacLC E. coli (Avanti) / mRNA (E. coli (Avanti) BacLC, containing 10 μg of CRE mRNA) compared to mice with intramuscular delivery of a comparative formulation (LNP) prepared according to Example 5. The control was PBS. [Figure 13B] Same as above. [Figure 13C] Same as above.

[0082] [Figure 14A] Figures 14A-14C show the frequency of tdTomato+ lymphocytes (Figure 14A), myeloid cells (Figure 14B), and non-immune cells (Figure 14C), respectively, in the lymph nodes of Ai9 mice 6 days after a single dose of intramuscular delivery of BacLC E. coli (Avanti) / mRNA (E. coli (Avanti) BacLC, containing 10 μg of CRE mRNA) compared to mice with intramuscular delivery of a comparative formulation (LNP) prepared according to Example 5. The control was PBS. [Figure 14B] Same as above. [Figure 14C] Same as above.

[0083] [Figure 15A]Figures 15A-C show the whole body (Figure 15A), liver (Figure 15B), and spleen (Figure 15C) radiance of mice 4-6 hours after intravenous administration of doses of BacLC E. coli / mRNA (E. coli BacLC, containing 10 μg of FLuc:EPO mRNA), BacLC E. coli (Avanti) / mRNA (E. coli (Avanti) BacLC, containing 10 μg of FLuc:EPO mRNA), and BacLC Salmonella / mRNA (Salmonella BacLC, containing 10 μg of FLuc:EPO mRNA) prepared according to Example 5. Figure 15D shows the spleen-to-liver radiance ratio 4 to 6 hours after mice were intravenously administered doses of BacLC E. coli / mRNA (E. coli BacLC, containing 10 μg of FLuc:EPO mRNA), BacLC E. coli (Avanti) / mRNA (E. coli (Avanti) BacLC, containing 10 μg of FLuc:EPO mRNA), and BacLC Salmonella / mRNA (Salmonella BacLC, containing 10 μg of FLuc:EPO mRNA). [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above.

[0084] [Figure 16A] Figures 16A-C show mesenteric lymph node (Figure 16A), stomach (Figure 16B), and mesenteric fat pad (Figure 16D) radiance 4-6 hours after mice were intravenously administered doses of BacLC E. coli / mRNA (E. coli BacLC, containing 10 μg of FLuc:EPO mRNA), BacLC E. coli (Avanti) / mRNA (E. coli (Avanti) BacLC, containing 10 μg of FLuc:EPO mRNA), and BacLC Salmonella / mRNA (Salmonella BacLC, containing 10 μg of FLuc:EPO mRNA) prepared according to Example 5. [Figure 16B] Same as above. [Figure 16C] Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0085] The present invention provides bacterial lipid compositions containing (a) bacterial components, including one or more lipids extracted from bacterial sources, and (b) ionizable lipids that can increase cellular uptake of the bacterial components. These bacterial lipid compositions can be formulated with one or more heterologous functional agents, such as polynucleotides, and can be used as delivery vehicles for these heterologous functional agents (e.g., polynucleotides).

[0086] bacterial components The bacterial component includes one or more lipids extracted from a bacterial source.

[0087] The bacterial component may have a lipid (e.g., lipid bilayer, monolayer, or multilayer) structure comprising a bacterial extracellular vesicle (EV), or a segment, portion, or extract (e.g., lipid extract) thereof.

[0088] The bacterial component includes isolated bacterial extracellular vesicles. Bacterial EVs are naturally occurring enclosed lipid bilayer structures within bacteria. Bacterial EVs are derived from bacteria containing bacterial lipids and may contain bacterial proteins and / or bacterial nucleic acids and / or carbohydrate moieties contained in nanoparticles. Bacterial EVs may contain 1, 2, 3, 4, 5, 10, or more than 10 different lipid species.

[0089] As used herein, the term "bacteria" refers broadly to the domain of prokaryotes, including gram-positive and gram-negative organisms. Suitable bacterial sources include Escherichia coli, Acinetobacter, Agrobacterium, Anabaena, Aquifex, Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Pora The genera include Romonas, Prochlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wallinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exiguobacterium, Geobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium, and Thermoanaerobacterium.

[0090] In one embodiment, the bacterial source is Escherichia (e.g., E. coli). In one embodiment, the bacterial source is Salmonella (e.g., Salmonella typhimurium).

[0091] In some embodiments, the bacterial source belongs to the Actinobacteria or Proteobacteria, such as the families Burkholderiaceae, Xanthomonadaceae, Pseudomonadaceae, Enterobacteriaceae, Mycobacteriaceae, and Rhizobiumaceae.

[0092] In some embodiments, the bacterial source is Agrobacterium spp., Sinorhizobium (=Ensifer) spp., Mesorhizobium spp., Bradyrhizobium spp., Azotobacter spp., Phyllobacter spp.), Sinorhizobium (=Ensifer) spp., Mesorhizobium spp., Azorhizobium spp., Bradyrhizobium spp., or Rhizobium spp.

[0093] In some embodiments, the bacterial source is selected from the group consisting of Acidovorax avenae subsp., Burkholderia spp., Liberibacter spp., Corynebacterium spp., Erwinia spp., Pseudomonas syringae subsp., Streptomyces spp., Xanthomonas campestris pv. musacearum, Xanthomonas campestris pv. pruni (=Xanthomonas arboricola pv. pruni), Xanthomonas fragariae, Xanthomonas translucens, translucens subsp. (=Xanthomonas campestris pv. hordei), Xanthomonas oryzae subsp., Xanthomonas oryzae pv. oryzae (=Xanthomonas campestris pv. oryzae), or Xanthomonas oryzae pv. oryzae (=Xanthomonas campestris pv. oryzae), or Xanthomonas oryzae pv. oryzicola (=Xanthomonas campestris pv. oryzicola).

[0094] Further examples of bacterial species and / or strains that may be used include those listed in Tables 1-2 of U.S. Patent Application Publication No. 2020 / 0254028, the entire contents of which are incorporated herein by reference.

[0095] The bacterial component may include bacterial EVs, or segments, parts, or extracts thereof. In some embodiments, the bacterial EVs are about 5 to 1000 nm in diameter. For example, the bacterial component may be about 5 to 50 nm, about 50 to 100 nm, about 100 to 150 nm, about 150 to 200 nm, about 200 to 250 nm, about 250 to 300 nm, about 300 to 350 nm, about 350 to 400 nm, about 400 to 450 nm, about 450 to 500 nm, about 500 to 550 nm, about 550 to 600 nm, about 600 to 650 nm, about 650 to 700 nm, or about The present invention may include bacterial EVs, or segments, parts, or extracts thereof, having an average diameter of 700-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, about 950-1000 nm, about 1000-1250 nm, about 1250-1500 nm, about 1500-1750 nm, or about 1750-2000 nm. In some cases, the bacterial component may comprise bacterial EVs, or segments, portions, or extracts thereof, having an average diameter of about 5-950 nm, about 5-900 nm, about 5-850 nm, about 5-800 nm, about 5-750 nm, about 5-700 nm, about 5-650 nm, about 5-600 nm, about 5-550 nm, about 5-500 nm, about 5-450 nm, about 5-400 nm, about 5-350 nm, about 5-300 nm, about 5-250 nm, about 5-200 nm, about 5-150 nm, about 5-100 nm, about 5-50 nm, or about 5-25 nm. In certain cases, the bacterial component may comprise bacterial EVs, or segments, portions, or extracts thereof, having an average diameter of about 50-200 nm, about 50-300 nm, about 200-500 nm, or about 30-150 nm. A variety of methods standard in the art (e.g., dynamic light scattering) can be used to measure the particle diameter of bacterial EVs, or segments, portions, or extracts thereof.

[0096] In some cases, the bacterial component is at least 77 nm2 (e.g., at least 77 nm 2 , at least 100 nm 2 , at least 1000 nm 2 , at least 1 x 10 4 nm 2 , at least 1 x 10 5 nm 2 , at least 1 x 10 6 nm 2 , or at least 2 × 10 6 nm 2 In some cases, the bacterial component may comprise a bacterial EV, or a segment, portion, or extract thereof, having an average surface area of ​​77 nm 2 ~3.2×10 6 nm 2 (e.g., 77-100 nm 2 , 100~1000nm 2 , 1000 to 1 × 10 4 nm 2 , 1×10 4 ~1×10 5 nm 2 , 1×10 5 ~1×10 6 nm 2 , or 1 × 10 6 ~3.2×10 6 nm 2 ) or a segment, part, or extract thereof.

[0097] In some cases, the bacterial component is at least 65 nm 3 (e.g., at least 65 nm 3 , at least 100 nm 3 , at least 1000 nm 3 , at least 1 x 10 4 nm 3 , at least 1 x 10 5 nm 3 , at least 1 x 10 6 nm 3 , at least 1 x 10 7 nm 3 , at least 1 x 10 8 nm 3 , at least 2 × 10 8nm 3 , at least 3 × 10 8 nm 3 , at least 4 × 10 8 nm 3 , or at least 5 × 10 8 nm 3 In some cases, the bacterial component may comprise a bacterial EV, or a segment, portion, or extract thereof, having an average volume of 65 nm 3 ~5.3×10 8 nm 3 (e.g., 65 to 100 nm 3 , 100~1000nm 3 , 1000 to 1 × 10 4 nm 3 , 1×10 4 ~1×10 5 nm 3 , 1×10 5 ~1×10 6 nm 3 , 1×10 6 ~1×10 7 nm 3 , 1×10 7 ~1×10 8 nm 3 , 1×10 8 ~5.3×10 8 nm 3 ) or a segment, part, or extract thereof.

[0098] In some cases, the bacterial component may include intact bacterial EVs. Alternatively, the bacterial component may include a segment, portion, or extract of the total surface area of ​​a bacterial EV vesicle (e.g., a segment, portion, or extract comprising less than 100% of the total surface area of ​​the vesicle (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 10%, 5%, or 1%)). The segment, portion, or extract may be of any shape, such as a circumferential segment, a spherical segment (e.g., a hemisphere), a curved segment, a straight segment, or a flat segment. When the segment is a spherical segment of a vesicle, the spherical segment may represent one resulting from the division of the spherical vesicle along a pair of parallel lines or one resulting from the division of the spherical vesicle along a pair of non-parallel lines. Thus, the bacterial component may contain multiple intact bacterial EVs, multiple bacterial EV segments, parts, or extracts, or a mixture of intact EVs and EV segments. Those skilled in the art will understand that the ratio of intact to segmented bacterial EVs will depend on the particular isolation method used. For example, crushing or blending bacteria, or parts thereof, may produce a bacterial component containing a higher percentage of bacterial EV segments, parts, or extracts than non-destructive extraction methods such as vacuum infiltration.

[0099] When the bacterial component comprises a segment, part, or extract of a bacterial EV, the EV segment, part, or extract has an average surface area that is smaller than the average surface area of ​​an intact vesicle, e.g., 77 nm 2 , 100 nm 2 , 1000nm 2 , 1×10 4 nm 2 , 1×10 5 nm 2 , 1×10 6 nm 2 , or 3.2 × 10 6 nm 2 In some cases, the bacterial components may have an average surface area less than the average volume of an intact vesicle, e.g., less than 65 nm 3 , 100 nm 3, 1000nm 3 , 1×10 4 nm3, 1 × 10 5 nm 3 , 1×10 6 nm 3 , 1×10 7 nm 3 , 1×10 8 nm 3 , or 5.3 × 10 8 nm 3 The bacterial EV may comprise a bacterial EV, or a segment, part or extract thereof, having the following structure:

[0100] The bacterial component comprises one or more lipids extracted from a bacterial source. In some embodiments, the bacterial component may comprise at least 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more than 99% of the lipids extracted from the bacterial source. The bacterial component may comprise bacterial EV segments and / or extracted lipids or mixtures thereof.

[0101] Production of bacterial components The bacterial component may be produced from bacterial EVs, or segments, parts, or extracts thereof (e.g., lipid extracts) that naturally occur in bacteria. An exemplary method for producing the bacterial component includes (a) providing an initial sample from bacteria, and (b) isolating a crude bacterial fraction from the initial sample, the crude bacterial fraction having a reduced level of at least one contaminant or undesirable component from the bacteria compared to the level in the initial sample. The method may further include an additional step (c) comprising purifying the crude bacterial fraction, thereby producing a pure bacterial component, the pure bacterial component having a reduced level of at least one contaminant or undesirable component from the bacteria compared to the level in the crude EV fraction.

[0102] In some cases, the bacterial component can be obtained by (a) providing an initial sample from bacteria; (b) isolating a crude bacterial fraction from the initial sample, the crude bacterial fraction having a reduced level of at least one contaminant or undesirable component from bacteria compared to the level in the initial sample (e.g., a level that is at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% reduced); and (c) purifying the crude bacterial fraction to thereby produce a pure bacterial component having a reduced level of at least one contaminant or undesirable component from the bacteria compared to the level in the crude EV fraction (e.g., a level that is at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 70%, 80%, 90%, 95%, 96%, 98%, 99%, or 100% reduced).

[0103] Bacterial components (e.g., bacterial lipids) can be produced by whole cell extraction. For example, cells are first disrupted, and then intracellular and membrane / wall-associated lipids and extracellular hydrocarbons can be separated from the cell mass, such as by centrifugation. In some embodiments, intracellular lipids produced in bacteria are extracted after lysing bacterial cells. Additional methods for extracting bacterial lipids can be found in U.S. Patent No. 8,592,188, which is incorporated herein by reference in its entirety.

[0104] Bacterial components can be produced from bacterial sources by various methods. For example, bacterial EVs can be separated from bacteria by either disruptive (e.g., crushing or blending bacteria) or non-destructive (washing or vacuum infiltration) methods. For example, bacteria can be vacuum infiltrated, crushed, blended, or a combination thereof, and EVs can be isolated from the bacteria. For example, the isolation step can include vacuum infiltrating the bacteria (e.g., with vesicle isolation buffer). Alternatively, the isolation step can include crushing or blending the bacteria to release EVs.

[0105] Upon isolation of bacterial EVs, bacterial components can be separated or collected into a crude bacterial fraction (e.g., an apoplastic fraction). For example, the separation step can involve using centrifugation (e.g., differential centrifugation or ultracentrifugation) and / or filtration to separate the bacterial components into a crude bacterial fraction, separating the bacterial-containing fraction from larger contaminants, including bacterial tissue debris or bacterial cells. Thus, the crude bacterial fraction will have a reduced number of large contaminants compared to the initial sample from bacteria. Depending on the method used, the crude bacterial fraction may additionally contain reduced levels of bacterial organelles compared to the initial sample from bacteria.

[0106] In some cases, the isolation step may include centrifugation (eg, differential centrifugation or ultracentrifugation) and / or filtration.

[0107] The crude bacterial fraction can be further purified by additional purification methods. For example, the crude bacterial fraction can be purified by ultracentrifugation, for example, using a density gradient (iodixanol or sucrose), and / or other approaches to remove aggregated components (e.g., precipitation or size exclusion chromatography). The resulting pure bacterial component may have reduced levels of contaminants or other undesirable components from the bacterial source (e.g., protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof) compared to one or more fractions produced during a previous separation step, or compared to a pre-established threshold level, e.g., a commercially available release standard. For example, a pure bacterial component may have a reduced level (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%), or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) compared to the level in the initial sample, or be substantially free of contaminants or other undesirable components (e.g., protein aggregates, nucleic acid aggregates, protein-nucleic acid aggregates, free lipoproteins, lipid-protein structures, nuclei, cell wall components, organelles, or combinations thereof).

[0108] For example, protein aggregates may be removed from bacterial components. For example, bacterial components can be taken through a range of pH (e.g., measured using a pH probe) to precipitate protein aggregates in solution. The pH can be adjusted to, for example, pH 3, pH 5, pH 7, pH 9, or pH 11, for example, by adding sodium hydroxide or hydrochloric acid. Once the solution is at the specified pH, it can be filtered to remove particles. Alternatively, bacterial components can be aggregated using the addition of a charged polymer, such as Polymin-P or Plastol 2640. Briefly, Polymin-P or Plastol 2640 is added to the solution and mixed using an impeller. The solution can then be filtered to remove particles. Alternatively, aggregates can be solubilized by increasing the salt concentration. For example, NaCl can be added to the bacterial components, for example, to 1 mol / L. The solution can then be filtered to isolate the bacterial components. Alternatively, aggregates can be solubilized by increasing the temperature. For example, the bacterial components can be heated for 5 minutes under mixing until the solution reaches a uniform temperature, e.g., 50°C. The bacterial component mixture can then be filtered. Alternatively, soluble contaminants from the bacterial component solution can be separated by a size-exclusion chromatography column according to standard procedures; bacterial lipids elute in the first fraction, while proteins and ribonucleoproteins, as well as some lipoproteins, elute later. The efficiency of protein aggregate removal can be determined by measuring and comparing protein concentrations before and after removal of protein aggregates via BCA / Bradford protein quantitation.

[0109] Alternatively, bacterial lipids extracted from bacterial sources can be obtained from commercial sources.

[0110] Any of the production methods described herein can be supplemented with any quantitative or qualitative method known in the art to characterize or identify bacterial components (e.g., bacterial lipids extracted from bacterial sources) at any step of the production process.For example, bacterial components (e.g., bacterial lipids extracted from bacterial sources) can be characterized by a number of methods known in the art that allow visualization, quantitative, or qualitative characterization (e.g., composition determination), such as microscopy (e.g., transmission electron microscopy), dynamic light scattering, nanoparticle tracking, spectroscopy (e.g., Fourier transform infrared analysis), or mass spectrometry (protein and lipid analysis), by various analytical methods to estimate yield, concentration, purity, composition, or size.

[0111] During the production process, bacterial components (e.g., bacterial lipids extracted from a bacterial source) can optionally be prepared so that the bacterial components are at an increased concentration (e.g., about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, or about 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, or more than 100-fold) compared to the level in a control or initial sample. The bacterial component may constitute from about 0.1% to about 100% of the bacterial-derived lipid composition, such as from about 0.01% to about 100%, from about 1% to about 99.9%, from about 0.1% to about 10%, from about 1% to about 25%, from about 10% to about 50%, from about 50% to about 99%, or from about 75% to about 100%.

[0112] Bacterial lipid composition - lipid modification of bacterial components The bacterial-derived lipid composition comprises (a) a bacterial component, and (b) an ionizable lipid. The ionizable lipid and / or other exogenous lipid is used to modify the bacterial component.

[0113] Modification refers to modifying bacterial components containing lipid structures (e.g., lipid bilayers, monolayers, multilayers, e.g., vesicular lipid structures) derived from a bacterial source (e.g., enriched, isolated, or purified), where the lipid structures are disrupted (e.g., disrupted by lipid extraction) as described herein and reconstituted or reconstituted in a liquid phase (e.g., a liquid phase containing cargo) using standard methods, e.g., reconstituted by methods including lipid film hydration and / or solvent injection, to produce a bacterial-derived lipid composition. In some embodiments, the bacterial components are modified by reconstituting membranes containing the bacterial components in the presence of ionizable lipids.

[0114] In some embodiments, the bacterial components are modified by reconstituting membranes containing purified bacterial lipids of the bacterial components with ionizable lipids.

[0115] Alternatively, bacterially derived lipid compositions may be generated using a microfluidic device (such as the NanoAssemblr® IGNITE™ microfluidic device (Precision NanoSystems)).

[0116] In some embodiments, the bacterial-derived lipid composition is produced by a process comprising the steps of: (a) providing bacterial components (e.g., bacterial components purified as described above); (b) treating the bacterial components to produce a lipid membrane; (c) reconstituting the lipid membrane in an organic solvent or combination of solvents, thereby producing a lipid solution; and (d) treating the lipid solution of step (c) in the presence of ionizable lipids in a microfluidic device comprising an aqueous phase, thereby producing the bacterial-derived lipid composition.

[0117] In some cases, processing bacterial components to produce lipid films includes extracting lipids using the Bligh-Dyer method (Bligh and Dyer, J Biolchem ​​Physiol, 37:911-917, 1959), which is incorporated herein by reference in its entirety. The extracted lipids may be provided as a stock solution, for example, a solution in chloroform:methanol. Producing lipid films may include, for example, evaporating the solvent with a stream of inert gas (e.g., nitrogen).

[0118] The method may further include, if desired, sonication, freeze / thaw treatment, and / or lipid extrusion, for example, to reduce the size of the reconstituted bacterial-derived lipid composition.

[0119] bacterial lipids A bacterially derived lipid composition may comprise 10% to 100% of the lipids derived from lipid structures from a bacterial source, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the lipids derived from lipid structures from a bacterial source (e.g., E. coli). A bacterially derived lipid composition may comprise all or a fraction of the lipid species present in the lipid structures from a bacterial source (e.g., E. coli), for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of the lipid species present in the lipid structures from a bacterial source. The bacterial-derived lipid composition may contain none, a fraction thereof, or all of the protein species present in the lipid structure derived from bacterial sources, for example, it may contain 0%, less than 1%, less than 5%, less than 10%, less than 15%, less than 20%, less than 30%, less than 40%, less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, less than 100%, or 100% of the protein species present in the lipid structure derived from bacterial sources.In some cases, the lipid bilayer of the bacterial-derived lipid composition does not contain protein.In some cases, the lipid structure of the bacterial-derived lipid composition contains a reduced amount of protein compared to the lipid structure derived from bacterial sources.

[0120] In some embodiments, the bacterial lipids of the bacterial-derived lipid composition are extracted from Escherichia (e.g., Escherichia coli) or Salmonella (e.g., Salmonella typhimurium).

[0121] exogenous lipids The bacterial components may be modified to contain a heterologous agent (e.g., a cell penetration agent) that can increase cellular uptake (e.g., animal cell uptake (e.g., mammalian cell uptake, e.g., human cell uptake), plant cell uptake, bacterial cell uptake, or fungal cell uptake) compared to the unmodified bacterial component. For example, the modified bacterial component may include (be loaded, e.g., encapsulated, or conjugated with) a cell penetration agent such as an ionizable lipid, or may be formulated with a plant cell penetration agent (e.g., suspended or resuspended in a solution containing the cell penetration agent). Each of the modified bacterial components may comprise at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipid.

[0122] The bacterial-derived lipid composition may contain one or more exogenous lipids, for example, lipids exogenous to bacteria (e.g., derived from a source other than the bacterial source from which the bacterial component is produced).The lipid composition of the bacterial-derived lipid composition may contain 0%, less than 1%, or at least 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more than 95% exogenous lipids.In some cases, the exogenous lipids (e.g., ionizable lipids) are added in an amount of 25% or 40% (w / w) of the total lipids in the preparation.In some cases, the exogenous lipids are added to the preparation before step (b), for example, mixed with the bacterial lipids extracted before step (b).

[0123] Exemplary exogenous lipids include ionizable lipids.

[0124] The exogenous lipids may also include cationic lipids.

[0125] In some cases, the exogenous lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), DLin-MC3-DMA (MC3), dioleoyl-3-trimethylammonium propane (DODAP), DC-cholesterol, DOTAP, ethyl PC, GL67, DLin-KC2-DMA (KC2), MD1 (cKK-E12), OF2, EPC, ZA3-Ep1 The ionizable lipid or cationic lipid may be selected from 0, TT3, LPO1, 5A2-SC8, Lipid 5 (Moderna), cationic sulfonamide amino lipids, amphipathic zwitterionic amino lipids, DODAC, DOBAQ, YSK05, DOBAT, DOBAQ, DOPAT, DOMPAQ, DOAAQ, DMAP-BLP, DLinDMA, DODMA, DOTMA, DSDMA, DOSPA, DODAC, DOBAQ, DMRIE, DOTAP-cholesterol, GL67A, and 98N12-5, and combinations thereof.

[0126] In some embodiments, the exogenous lipid may be an ionizable lipid or cationic lipid selected from C12-200, MC3, DODAP, DC-cholesterol, DOTAP, ethyl PC, GL67, KC2, MD1, OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5 (Moderna), cationic sulfonamide amino lipids, and amphipathic zwitterionic amino lipids, and combinations thereof. In some embodiments, the ionizable lipid is selected from C12-200, MC3, DODAP, and DC-cholesterol, or combinations thereof. In some cases, the ionizable lipid is an ionizable lipid. In some embodiments, the ionizable lipid is 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200) or (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, DLin-MC3-DMA (MC3). In some cases, the exogenous lipid is a cationic lipid. In some embodiments, the cationic lipid is DC-cholesterol or dioleoyl-3-trimethylammoniumpropane (DOTAP).

[0127] In some cases, the bacterially derived lipid composition comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipids.

[0128] In some cases, the bacterially-derived lipid composition comprises at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 90% ionizable lipids, e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% ionizable lipids, e.g., about 30%-75% ionizable lipids (e.g., about 30%-75% ionizable lipids). In some embodiments, the bacterially-derived lipid composition comprises 25% C12-200 by mole. In some embodiments, the bacterial-derived lipid composition comprises 35% C12-200 by mole. In some embodiments, the bacterial-derived lipid composition comprises 50% C12-200 by mole. In some embodiments, the bacterial-derived lipid composition comprises 40% MC3 by mole. In some embodiments, the bacterial-derived lipid composition comprises 50% C12-200 by mole. In some embodiments, the bacterial-derived lipid composition comprises 20% or 40% DC-cholesterol by mole. In some embodiments, the bacterial-derived lipid composition comprises 25% or 40% DOTAP by mole.

[0129] The agent may increase the uptake of the bacterial-derived lipid composition as a whole, or may increase the uptake of a portion or component of the bacterial-derived lipid composition (e.g., a heterologous functional agent) carried by the bacterial-derived lipid composition. The degree of increased cellular uptake may vary depending on the bacteria to which the composition is delivered, the bacterial-derived lipid composition, and other modifications made to the bacterial-derived lipid composition. For example, the bacterial-derived lipid composition may have at least 1%, 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increased cellular uptake (e.g., animal cell uptake, plant cell uptake, bacterial cell uptake, or fungal cell uptake) compared to the unmodified bacterial component. In some cases, the increased cellular uptake is at least 2-fold, 4-fold, 5-fold, 10-fold, 100-fold, or 1000-fold increased cellular uptake compared to the unmodified bacterial component.

[0130] In some embodiments, bacterial-derived lipid compositions modified with ionizable lipids encapsulate negatively charged polynucleotides more efficiently than bacterial-derived lipid compositions not modified with ionizable lipids. In some aspects, bacterial-derived lipid compositions modified with ionizable lipids have altered biodistribution compared to bacterial-derived lipid compositions not modified with ionizable lipids. In some aspects, bacterial-derived lipid compositions modified with ionizable lipids have altered (e.g., increased) fusion with the endosomal membrane of target cells compared to bacterial-derived lipid compositions not modified with ionizable lipids.

[0131] Ionizable lipids In some embodiments, the ionizable lipid has the following characteristics: (i) at least two ionizable amines (e.g., at least two, at least three, at least four, at least five, at least six, or more than six ionizable amines, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 ionizable amines); (ii) at least three lipid tails (e.g., at least three, at least four, at least five, at least six, or more than six lipid tails, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 lipid tails), each of the lipid tails independently at least six carbon atoms in length (e.g., at least six, at least seven, at least eight, at least nine, at least ten, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or more than 18 carbon atoms in length, e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 carbon atoms in length); (iii) an acid dissociation constant (pKa) of about 4.5 to about 7.5 (e.g., a pKa of about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5 (e.g., a pKa of about 6.5 to about 7.5 (e.g., a pKa of about 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5)); (iv) ionizable amine and heteroorganic groups, and (v) have at least one (e.g., one, two, three, four, or all five) of the following N:P (ionizable lipid amine:mRNA phosphate) ratios of at least 10:

[0132] In some embodiments, the BacLC has an N / P ratio of about 12 to about 17, e.g., an N / P ratio of about 15±1, or an N / P ratio of about 15±0.5. In some embodiments, the N / P ratio is about 15. Alternatively, the ionizable lipid is characterized by an N / P ratio of about 3 to about 10, e.g., an N / P ratio of about 6±1, or an N / P ratio of about 6±0.5. In some embodiments, the N / P ratio is about 6.

[0133] In some embodiments, the ionizable lipid is not selected from 1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5 (Moderna), and 98N12-5.

[0134] In some embodiments, the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LPO1, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

[0135] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group. In some embodiments, the heteroorganic group is hydroxyl. In some embodiments, the heteroorganic group comprises a hydrogen bond donor. In some embodiments, the heteroorganic group comprises a hydrogen bond acceptor. In some embodiments, the heteroorganic group is -OH, -SH, -(CO)H, -COH, -NH, -CONH, optionally substituted C-C alkoxy, or fluorine.

[0136] In some embodiments, the ionizable lipid is an ionizable amine and a heteroorganic group separated by a chain of at least two atoms.

[0137] In some embodiments, the ionizable lipid has the following formula I: [ka] (I), wherein R is C-C 14 It is represented by an alkyl group.

[0138] In some embodiments, the lipid film of the bacterial-derived lipid composition comprises at least 35% lipids of formula I, e.g., at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more than 90% lipids of formula I, e.g., 35% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, or 80% to 90% lipids of formula I.

[0139] In some cases, the bacterially derived lipid composition comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% ionizable lipids.

[0140] In some cases, the bacterially-derived lipid composition comprises at least 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or greater than 90% ionizable lipids, e.g., 1%-10%, 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, or 80%-90% ionizable lipids, e.g., about 25%-75% ionizable lipids (e.g., about 25%-75% ionizable lipids), all by mole percent.

[0141] The ionizable lipids described herein can include an amine core described herein substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6) lipid tails. In some embodiments, the ionizable lipids described herein include at least three lipid tails. The lipid tails are C8-C 18 Hydrocarbons (e.g., C6-C 18 Alkyl or C6-C 18 The amine core may be substituted at the nitrogen atom with one or more lipid tails (e.g., one hydrogen atom attached to the nitrogen atom may be replaced with a lipid tail).

[0142] In some embodiments, the amine core is [ka] It has the following structure.

[0143] In some embodiments, the amine core is [ka] It has the following structure.

[0144] In some embodiments, the amine core is [ka] It has the following structure.

[0145] In some embodiments, the amine core is [ka] It has the following structure.

[0146] In some embodiments, the amine core is [ka] It has the following structure.

[0147] In some embodiments, the amine core is [ka] It has the following structure.

[0148] In some embodiments, the amine core is [ka] It has the following structure.

[0149] In some embodiments, the amine core is [ka] It has the following structure.

[0150] The bacterially derived lipid composition may further comprise a cationic lipid.

[0151] Other lipids suitable for use in bacterially-derived lipid compositions, and methods for making and using them, include those described in International Patent Publication No. WO 2016 / 118725, which is incorporated herein by reference in its entirety.

[0152] In certain embodiments, bacterially derived lipid compositions and methods of making and using same comprise: [ka] and pharmaceutically acceptable salts thereof.

[0153] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. WO 2016 / 118724, which is incorporated herein by reference in its entirety.

[0154] In certain embodiments, bacterially derived lipid compositions and methods of making and using same comprise: [ka] and pharmaceutically acceptable salts thereof.

[0155] Other lipids suitable for use in the bacterially-derived lipid compositions and methods of making and using same include lipids having the formula 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[0156] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication Nos. 2013 / 063468 and 2016 / 205691, each of which is incorporated by reference in its entirety.

[0157] In some embodiments, bacterially derived lipid compositions and methods of making and using same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein R LEach instance of is independently an optionally substituted C6-C40 alkenyl.

[0158] In certain embodiments, bacterially derived lipid compositions and methods of making and using same comprise: [ka] and pharmaceutically acceptable salts thereof.

[0159] In certain embodiments, bacterially derived lipid compositions and methods of making and using same comprise: [ka] and pharmaceutically acceptable salts thereof.

[0160] In certain embodiments, bacterially derived lipid compositions and methods of making and using same comprise: [ka] and pharmaceutically acceptable salts thereof.

[0161] In certain embodiments, bacterially derived lipid compositions and methods of making and using same comprise: [ka] and pharmaceutically acceptable salts thereof.

[0162] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. 2015 / 184256, which is incorporated herein by reference in its entirety. In some embodiments, the bacterially-derived lipid compositions and methods for making and using them comprise lipids of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each X is independently O or S, each Y is independently O or S, each m is independently 0 to 20, each n is independently 1 to 6, and each R A are independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen, and each R B is independently hydrogen, optionally substituted C1-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen.

[0163] In certain embodiments, bacterially derived lipid compositions and methods of making and using same comprise: [ka] (Target 23), which is a lipid having the compound structure: (Target 23), and pharmaceutically acceptable salts thereof.

[0164] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. 2016 / 004202, which is incorporated herein by reference in its entirety.

[0165] In some embodiments, bacterially derived lipid compositions and methods of making and using same include: [ka] or a pharmaceutically acceptable salt thereof.

[0166] In some embodiments, bacterially derived lipid compositions and methods of making and using same include: [ka] or a pharmaceutically acceptable salt thereof.

[0167] In some embodiments, bacterially derived lipid compositions and methods of making and using same include: [ka] or a pharmaceutically acceptable salt thereof.

[0168] Other lipids suitable for use in the bacterially-derived lipid compositions and methods of making and using them include those described in U.S. Provisional Patent Application No. 62 / 758,179, which is incorporated herein by reference in its entirety.

[0169] In some embodiments, bacterially derived lipid compositions and methods of making and using same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein each R 1 and R 2 is independently H or a C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or arylene; and each L 1 are independently an ester, thioester, disulfide, or anhydride group, and each L 2 are independently C2-C10 aliphatic, and each X 1 are independently H or OH, and each R 3 are independently C6-C20 aliphatic.

[0170] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a lipid having the following formula: [ka] (Compound 1), or a pharmaceutically acceptable salt thereof.

[0171] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a lipid having the following formula: [ka] (Compound 2), or a pharmaceutically acceptable salt thereof.

[0172] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a lipid having the following formula: [ka] (Compound 3), or a pharmaceutically acceptable salt thereof.

[0173] Other lipids suitable for use in the bacterially-derived lipid compositions and methods of making and using them include those described in J. McClellan, MCKing, Cell 2010, 141, 210-217 and Whitehead et al., Nature Communications (2014) 5:4277, which are incorporated herein by reference in their entirety.

[0174] In certain embodiments, the lipids of the bacterially derived lipid compositions and methods of making and using same have the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0175] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. WO 2015 / 199952, which is incorporated herein by reference in its entirety.

[0176] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0177] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0178] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0179] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0180] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0181] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0182] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0183] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0184] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0185] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0186] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0187] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0188] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0189] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. 2017 / 004143, which is incorporated herein by reference in its entirety.

[0190] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0191] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0192] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0193] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0194] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0195] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0196] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0197] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0198] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0199] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0200] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0201] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0202] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0203] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0204] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0205] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0206] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0207] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. WO 2017 / 075531, which is incorporated herein by reference in its entirety.

[0208] In some embodiments, bacterially derived lipid compositions and methods of making and using same comprise a lipid of the following formula: [ka] or a pharmaceutically acceptable salt thereof, wherein L 1 or L 2 One of the following is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x, -SS-, -C(=O)S-, -SC(=O)-, -NR aC(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -, or -NR a C(=O)O-, and L 1 or L 2 The rest are -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x , -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, NR a C(=O)NR a -, -OC(=O)NR a -or-NR a C(=O)O- or a direct bond, and G 1 and G 2 are each independently a substituted C-C 12 Alkylene or C1-C 12 alkenylene, G 3 is C1-C 24 Alkylene, C1-C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene, and R a is H or C1-C 12 alkyl, and R 1 and R 2 are each independently C6-C 24 Alkyl or C6-C 24 alkenyl, and R 3 H, OR 5 , CN, -C(=O)OR 4 , -OC(=O)R 4 or -NR 5 C(=O)R 4 and R 4 is C1-C 12 alkyl, and R 5 is H or C1-C6 alkyl and x is 0, 1 or 2.

[0209] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. 2017 / 117528, which is incorporated herein by reference in its entirety. In some embodiments, the bacterially-derived lipid compositions and methods for making and using them comprise the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0210] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0211] In some embodiments, the bacterial-derived lipid compositions and methods of making and using same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0212] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publication No. WO 2017 / 049245, which is incorporated herein by reference in its entirety.

[0213] In some embodiments, the lipids of the bacterial-derived lipid compositions and methods of making and using same have the following formula: [ka] and pharmaceutically acceptable salts thereof. For any one of these four formulas, R4 is -(CH2) n Q and -(CH2) nCHQR, where Q is -OR, -OH, -O(CH2) n and heterocyclyl, wherein n is 1, 2, or 3.

[0214] In certain embodiments, bacterially derived lipid compositions and methods of making and using the same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0215] In certain embodiments, bacterially derived lipid compositions and methods of making and using the same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0216] In certain embodiments, bacterially derived lipid compositions and methods of making and using the same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0217] In certain embodiments, bacterially derived lipid compositions and methods of making and using the same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0218] Other lipids suitable for use in bacterially-derived lipid compositions and methods for making and using them include those described in International Patent Publications 2017 / 173054 and 2015 / 095340, which are incorporated herein by reference in their entireties. In certain embodiments, the bacterially-derived lipid compositions and methods for making and using them comprise the compound structure: [ka] and pharmaceutically acceptable salts thereof.

[0219] In certain embodiments, bacterially derived lipid compositions and methods of making and using the same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0220] In certain embodiments, bacterially derived lipid compositions and methods of making and using the same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0221] In certain embodiments, bacterially derived lipid compositions and methods of making and using the same comprise a compound having the structure: [ka] and pharmaceutically acceptable salts thereof.

[0222] In some embodiments, the bacterial-derived lipid compositions described herein are selected from the group consisting of bacterial lipid compositions disclosed in WO2016118724, WO2016118725, WO2016187531, WO2017176974, WO2018078053, WO2019027999, WO2019036030, WO2019089828, WO2019099501, WO2019099502, WO2019099601, WO2019099701, WO2019099828, WO2019099501, WO201909 ... The composition may comprise, be formulated as described, or comprise or be comprised in a composition described in WO 2020072605, WO 2020081938, WO 2020118041, WO 2020146805, or WO 2020219876, each of which is incorporated by reference in its entirety.

[0223] Other lipids and other drugs The exogenous lipid may be a cell-penetrating agent, may increase the delivery of the polypeptide to cells by the bacterial-derived lipid composition, and / or may increase the loading (e.g., loading efficiency or loading capacity) of the polypeptide. Further exemplary exogenous lipids include sterols and PEGylated lipids.

[0224] The bacterial-derived lipid composition may include other components (e.g., lipids, e.g., sterols, e.g., cholesterol, or small molecules) to further alter the functional and structural characteristics of the bacterial-derived lipid composition. For example, the bacterial-derived lipid composition may further include a stabilizing molecule that increases the stability of the bacterial-derived lipid composition (e.g., stable at room temperature for at least one day and / or stable at 4°C for at least one week).

[0225] In some embodiments, the bacterial-derived lipid composition further comprises a sterol, such as sitosterol, sitostanol, β-sitosterol, 7α-hydroxycholesterol, pregnenolone, cholesterol (e.g., ovine cholesterol or cholesterol isolated from plants), stigmasterol, campesterol, fucosterol, or any sterol analog (e.g., glycoside, ester, or peptide). In some examples, the exogenous sterol is added to the preparation before step (b), for example, by mixing with the bacterial lipid extracted before step (b). The exogenous sterol may be added in an amount of, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% (w / w) of the total lipids and sterols in the preparation.

[0226] In some embodiments, the sterol is cholesterol or sitosterol. In some cases, the bacterially derived lipid composition comprises at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more than 60% sterol (e.g., cholesterol or sitosterol), for example, a molar ratio of 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% sterol. In some embodiments, the bacterially derived lipid composition comprises about 35% to 50% sterol (e.g., cholesterol or sitosterol), for example, a molar ratio of about 36%, 38.5%, 42.5%, or 46.5% sterol. In some embodiments, the bacterially derived lipid composition comprises about 20% to 40% DC-cholesterol.

[0227] In some embodiments, bacterially-derived lipid compositions that are modified with sterols have altered stability (e.g., increased stability) compared to bacterially-derived lipid compositions that are not modified with sterols. In some aspects, bacterially-derived lipid compositions that are modified with sterols have a faster rate of fusion with the membrane of a target cell compared to bacterially-derived lipid compositions that are not modified with sterols.

[0228] In some cases, the bacterially-derived lipid composition comprises an exogenous lipid and an exogenous sterol.

[0229] In some embodiments, the bacterial-derived lipid composition further comprises a PEGylated lipid. The length of the polyethylene glycol (PEG) can vary from 1 kDa to 10 kDa, and in some aspects, PEGs having a length of 2 kDa are used. In some embodiments, the PEGylated lipid is C14-PEG2k, C18-PEG2k, or DMPE-PEG2k. In some cases, the bacterially derived lipid composition comprises at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.5%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 9 , 10%, 20%, 30%, 40%, 50%, or greater than 50% PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), for example, 0.1%-0.5%, 0.5%-1%, 1%-1.5%, 1.5%-2.5%, 2.5%-3.5%, 3.5%-5%, 5%-10%, 10%-20%, 20%-30%, 30%-40%, or 30%-50% molar ratio of PEGylated lipid. In some embodiments, the bacterial-derived lipid composition comprises a molar ratio of about 0.1% to 10% PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k), e.g., about 1% to 3% PEGylated lipid, e.g., about 1.5% or about 2.5%, all by mole.

[0230] In some embodiments, bacterial-derived lipid compositions modified with PEGylated lipids have altered stability (e.g., increased stability) compared to bacterial-derived lipid compositions not modified with PEGylated lipids. In some embodiments, bacterial-derived lipid compositions modified with PEGylated lipids have altered particle size compared to bacterial-derived lipid compositions not modified with PEGylated lipids. In some embodiments, bacterial-derived lipid compositions modified with PEGylated lipids are less likely to be phagocytosed than bacterial-derived lipid compositions not modified with PEGylated lipids. The addition of PEGylated lipids may also affect stability in the GI tract and enhance particle penetration through mucus. PEG may also be used as a method of attaching targeting moieties.

[0231] In some embodiments, the bacterial-derived lipid composition is modified with an ionizable lipid (e.g., C12-200 or MC3) and one or both of a sterol (e.g., cholesterol or sitosterol) and a PEGylated lipid (e.g., C14-PEG2k, C18-PEG2k, or DMPE-PEG2k). In one embodiment, the bacterially-derived lipid composition comprises, all by mole percent, about 5% to 50% bacterially-derived lipid (e.g., about 10% to 20% bacterially-derived lipid, e.g., about 10%, 12.5%, 16%, or 20% bacterially-derived lipid), about 30% to 75% ionizable lipid (e.g., about 35% or about 50% ionizable lipid), about 35% to 50% sterol (e.g., about 36%, 38.5%, 42.5%, or 46.5% sterol), and about 0.1% to 10% PEGylated lipid (e.g., about 1% to 3% PEGylated lipid, e.g., about 1.5% or about 2.5% PEGylated lipid).

[0232] In some embodiments, the bacterial-derived lipid composition comprises a molar ratio of about 5% to 60% bacterial-derived lipid (e.g., about 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, or 50% to 60% bacterial-derived lipid, e.g., about 10%, 12.5%, 16%, 20%, 30%, 40%, 50%, or 60% bacterial-derived lipid), about 25% to 75% ionizable lipid (e.g., about 35% or about 50%). % ionizable lipid), about 10% to 50% sterol (e.g., about 10%, 12.5%, 14%, 16%, 18%, 20%, 36%, 38.5%, 42.5%, or 46.5% sterol), and about 0.1% to 10% PEGylated lipid (e.g., about 0.5% to 5% PEGylated lipid, e.g., about 1% to 3% PEGylated lipid, or about 1.5% or about 2.5% PEGylated lipid).

[0233] In some embodiments, the ionizable lipids, bacterially-derived lipids, sterols, and PEGylated lipids comprise about 25%-75%, about 20%-60%, about 10%-45%, and about 0.5%-5%, respectively, of the lipids in the bacterially-derived lipid composition.

[0234] In some embodiments, the ionizable lipids, bacterially-derived lipids, sterols, and PEGylated lipids comprise about 30%-75%, about 20%-50%, about 10%-45%, and about 1%-5%, respectively, of the lipids in the bacterially-derived lipid composition.

[0235] In some embodiments, the ionizable lipids, bacterially-derived lipids, sterols, and PEGylated lipids comprise about 35%-75%, about 20%-50%, about 10%-45%, and about 1%-5%, respectively, of the lipids in the bacterially-derived lipid composition.

[0236] In some embodiments, the ionizable lipid, bacterially-derived lipid, sterol, and PEGylated lipid are formulated in a molar ratio of about 35:50:12.5:2.5.

[0237] In some embodiments, the ionizable lipid, bacterially-derived lipid, sterol, and PEGylated lipid are formulated in a molar ratio of about 35:50:11.5:3.5.

[0238] In some embodiments, the ionizable lipid, bacterially-derived lipid, sterol, and PEGylated lipid are formulated in a molar ratio of about 35:20:42.5:2.5.

[0239] In some embodiments, bacterial-derived lipid compositions modified with ionizable lipids (and / or cationic lipids) and sterols and / or PEGylated lipids more efficiently encapsulate negatively charged cargo (e.g., nucleic acids) than bacterial-derived lipid compositions not modified with ionizable lipids (and / or cationic lipids) and sterols and / or PEGylated lipids. The bacterial-derived lipid compositions may have an encapsulation efficiency for cargo (e.g., heterologous functional agents such as nucleic acids, e.g., RNA or DNA) of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or greater than 99%, e.g., 5% to 30%, 30% to 50%, 50% to 70%, 70% to 80%, 80% to 90%, 90% to 95%, or 95% to 100%.

[0240] Cellular uptake of bacterially derived lipid compositions can be measured by various methods known in the art. For example, the bacterially derived lipid composition, or its components, can be labeled with a marker (e.g., a fluorescent marker) that can be detected in isolated cells to confirm uptake.

[0241] In some embodiments, the bacterial-derived lipid compositions provided herein comprise two or more different bacterial components, e.g., bacterial components derived from two or more different bacterial sources. In some embodiments, the bacterial-derived lipid compositions provided herein comprise two or more different types of modifications, e.g., different types and / or ratios of ionizable lipids, sterols, and / or PEGylated lipids.

[0242] In some cases, the organic solvent in which the lipid film is dissolved is dimethylformamide:methanol (DMF:MeOH). Alternatively, the organic solvent or solvent combination may be, for example, acetonitrile, acetone, ethanol, methanol, dimethylformamide, tetrahydrofuran, 1-butanol, dimethyl sulfoxide, acetonitrile:ethanol, acetonitrile:methanol, acetone:methanol, methyl tert-butyl ether:propanol, tetrahydrofuran:methanol, dimethyl sulfoxide:methanol, or dimethylformamide:methanol.

[0243] The aqueous phase may be any suitable solution, such as a citrate buffer (e.g., a citrate buffer having a pH of about 3.2), water, or phosphate buffered saline (PBS). The aqueous phase may further comprise a heterologous functional agent (e.g., an agricultural or therapeutic agent) or a small molecule.

[0244] The lipid solution and the aqueous phase may be mixed in the microfluidic device in any suitable ratio. In some examples, the aqueous phase and the lipid solution are mixed in a volume ratio of 3:1.

[0245] The bacterial-derived lipid composition may optionally contain additional agents, such as cell-penetrating agents, therapeutic agents, polynucleotides, polypeptides, or small molecules. The bacterial-derived lipid composition can carry or associate with additional heterologous functional agents in various ways to enable delivery of the heterologous functional agent to target cells, for example, by encapsulating the agent, incorporating the heterologous functional agent into the lipid bilayer structure, or associating the heterologous functional agent with the surface of the lipid bilayer structure (e.g., by conjugation). The heterologous functional agent can be incorporated into the bacterial-derived lipid composition either in vivo or in vitro (e.g., in tissue culture, in cell culture, or synthetically incorporated).

[0246] Zeta potential A bacterially derived lipid composition comprising an ionizable lipid (e.g., C12-200 or MC3) and optionally a cationic lipid (e.g., DC-cholesterol or DOTAP) may have a zeta potential of, for example, greater than -30 mV in the absence of cargo, greater than -20 mV, greater than -5 mV, greater than 0 mV, or about 30 mV in the absence of cargo. In some examples, the bacterially derived lipid composition has a negative zeta potential, for example, a zeta potential of less than 0 mV, less than -10 mV, less than -20 mV, less than -30 mV, less than -40 mV, or less than -50 mV in the absence of cargo. In some examples, the bacterially derived lipid composition has a positive zeta potential, for example, a zeta potential of greater than 0 mV, greater than 10 mV, greater than 20 mV, greater than 30 mV, greater than 40 mV, or greater than 50 mV in the absence of cargo. In some instances, the bacterially-derived lipid composition has a zeta potential of about zero.

[0247] The zeta potential of bacterial lipid compositions can be measured using any method known in the art.Zeta potential is generally measured indirectly, for example, by using a theoretical model to calculate the data obtained by methods and techniques known in the art, such as electrophoretic mobility or dynamic electrophoretic mobility.Electrophoretic mobility is typically measured by microelectrophoresis, electrophoretic light scattering, or tunable resistive pulse sensing.Electrophoretic light scattering is based on dynamic light scattering.Typically, zeta potential can be accessed by photon correlation spectroscopy or dynamic light scattering (DLS), also known as quasi-elastic light scattering.

[0248] Bacterial EV markers The bacterial components (e.g., bacterial lipids) in bacterial-derived lipid compositions and methods for their production and use can have a wide range of markers that identify the bacterial component being produced. As used herein, the term "bacterial EV marker" refers to a component that is naturally associated with bacteria and incorporated into or on bacterial EVs, such as a bacterial protein, bacterial nucleic acid, bacterial small molecule, bacterial lipid, or a combination thereof.

[0249] Drug loading The bacterial-derived lipid composition may include heterologous functional agents, such as those described herein, e.g., cell-penetrating agents and / or heterologous agricultural agents (e.g., insecticides, fertilizers, herbicides, plant modifying agents), heterologous therapeutic agents (e.g., antifungal agents, antibacterial agents, virucides, antivirucides, insecticides, nematicides, antiparasitics, or insect repellents).

[0250] Bacterial-derived lipid compositions can carry or associate with such agents in a variety of ways to enable delivery of such agents to target organisms (e.g., target animals, plants, bacteria, or fungi), for example, by encapsulating the agent, incorporating the agent into the lipid bilayer structure, or associating the agent with the surface of the lipid bilayer structure of the bacterial-derived lipid composition (e.g., by conjugation). In some cases, heterologous functional agents (e.g., cell-penetrating agents) are included in formulations made with bacterial-derived lipid composition formulations, as described herein.

[0251] Heterologous functional agents can be incorporated into or loaded onto bacterially-derived lipid compositions by any method known in the art that allows for a direct or indirect association between the bacterially-derived lipid composition and the agent. Agents can be incorporated into bacterially-derived lipid compositions in vivo or in vitro (e.g., in tissue culture or cell culture), or by both in vivo and in vitro methods.

[0252] In some cases, bacterially-derived lipid compositions are loaded in vitro. Heterologous functional agents may be loaded onto or within bacterially-derived lipid compositions (e.g., encapsulated therein) using physical, chemical, and / or biological methods (e.g., tissue culture or cell culture), including, but not limited to, methods. For example, agents may be introduced into bacterially-derived lipid compositions by one or more of electroporation, sonication, passive diffusion, agitation, lipid extraction, or extrusion. In some cases, agents are incorporated into bacterially-derived lipid compositions using a microfluidic device, e.g., a method in which lipids are provided in an organic phase and an agent is provided in an aqueous phase, and the organic and aqueous phases are combined in the microfluidic device to produce a bacterially-derived lipid composition containing a heterologous functional agent. The loaded bacterially-derived lipid composition can be evaluated to confirm the presence or level of the loaded agent using various methods, such as HPLC (e.g., to evaluate small molecules), immunoblotting (e.g., to evaluate proteins), and / or quantitative PCR (e.g., to evaluate nucleotides). However, it should be understood by those skilled in the art that loading of bacterially derived lipid compositions with heterologous functional agents of interest is not limited to the methods described above.

[0253] In some cases, heterologous functional agents may be conjugated to bacterially derived lipid compositions, in which the agent is indirectly or directly linked or bound to the bacterially derived lipid composition. For example, one or more agents can be chemically linked to the bacterially derived lipid composition so that the one or more agents are directly bound (e.g., by covalent or ionic bonds) to the lipid bilayer of the bacterially derived lipid composition. In some cases, conjugation of various agents to bacterially derived lipid compositions can be achieved by first mixing one or more agents with a suitable cross-linking agent (e.g., N-ethylcarbodiimide (EDC), which is generally used as a carboxyl-activating agent for amide bonds with primary amines and also reacts with phosphate groups) in a suitable solvent. After an incubation period sufficient to allow the drug to attach to the crosslinker, the crosslinker / drug mixture can then be combined with a bacterially-derived lipid composition and, after a further incubation period, subjected to a sucrose gradient (e.g., 8, 30, 45, and 60% sucrose gradient) to separate the free drug and free bacterially-derived lipid composition from the drug conjugated to the bacterially-derived lipid composition. As part of combining the mixture with the sucrose gradient and the accompanying centrifugation step, the bacterially-derived lipid composition conjugated to the drug is then seen as a band in the sucrose gradient, and the conjugated bacterially-derived lipid composition can then be collected, washed, and dissolved in a solution appropriate for use as described herein.

[0254] In some cases, the bacterial-derived lipid composition is stably associated with the heterologous functional agent before and after delivery of the bacterial-derived lipid composition, while in other cases, the bacterial-derived lipid composition is associated with the agent such that the agent dissociates from the bacterial-derived lipid composition after delivery of the bacterial-derived lipid composition.

[0255] Bacterial-derived lipid compositions may be loaded or formulated with various concentrations of heterologous functional agents depending on the particular agent or use. For example, in some cases, the bacterial-derived lipid compositions are loaded or formulated to contain about 0.001, 0.01, 0.1, 1.0, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 95 (or any range between about 0.001 and 95) or more weight percent agent. In some cases, the bacterially-derived lipid composition is loaded or formulated to contain about 95, 90, 80, 70, 60, 50, 40, 30, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1.0, 0.1, 0.01, 0.001 (or any range between about 95 and 0.001) or less by weight of the agent. For example, the bacterially-derived lipid composition can contain about 0.001 to about 0.01, about 0.01 to about 0.1, about 0.1 to about 1, about 1 to about 5, or about 5 to about 10, or about 10 to about 20% by weight of the agent. In some cases, the bacterial-derived lipid composition may be loaded or formulated with about 1, 5, 10, 50, 100, 200, or 500, 1,000, 2,000 (or any range from about 1 to 2,000) or more μg / ml of agent. The bacterial-derived lipid composition may be loaded or formulated with about 2,000, 1,000, 500, 200, 100, 50, 10, 5, 1 (or any range from about 2,000 to 1) or more μg / ml of agent.

[0256] In some cases, the bacterially-derived lipid composition is loaded, or the LPMP is formulated, to contain at least 0.001%, at least 0.01%, at least 0.1%, at least 1.0%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% by weight of the heterologous functional agent. In some cases, the bacterial-derived lipid composition may be loaded or formulated with at least 1 μg / ml, at least 5 μg / ml, at least 10 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 200 μg / ml, at least 500 μg / ml, at least 1,000 μg / ml, or at least 2,000 μg / ml of the agent.

[0257] In some cases, bacterially derived lipid compositions are formulated with heterologous functional agents, e.g., by suspending the bacterially derived lipid composition in a solution containing or consisting of the agent, e.g., by vigorous mixing. The agent (e.g., a cell-penetrating agent, e.g., a nucleic acid, an enzyme, a surfactant, an ion, a fluorescent agent, or a zwitterionic liquid, or an ionizable lipid) may comprise, for example, less than 1% or at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the solution.

[0258] formulation agricultural formulations The bacterially derived lipid compositions described herein can be formulated into agricultural compositions.

[0259] To facilitate application, handling, transportation, storage, and effective activity, bacterial lipid compositions can be formulated with other substances. For example, bacterial lipid compositions can be formulated into baits, concentrated emulsions, dusts, emulsifiable concentrates, smoke bombs, gels, granules, microencapsulated products, seed treatments, suspension concentrates, suspoemulsions, tablets, water-soluble liquids, water-dispersible granules or dry flowables, wettable powders, and ultra-low volume solutions. For details on formulation types, see "Catalogue of Pesticide Formulation Types and International Coding System" Technical Monograph no. 2, 5th Edition by CropLife International (2002).

[0260] Bacterial lipid compositions can be applied as aqueous suspensions or emulsions prepared from concentrated formulations of such agents. These water-soluble, water-suspendable, or emulsifiable formulations can be either solids, commonly known as wettable powders, or water-dispersible granules; liquids, commonly known as emulsifiable concentrates, or aqueous suspensions. Wettable powders, which can be compressed to form water-dispersible granules, contain an intimate mixture of the bacterial lipid composition, a carrier, and a surfactant. The carrier is typically selected from attapulgite clay, montmorillonite clay, diatomaceous earth, or purified silicates. Effective surfactants, comprising about 0.5% to about 10% of the wettable powder, are found in nonionic surfactants such as sulfonated lignin, condensed naphthalene sulfonates, naphthalene sulfonates, alkylbenzene sulfonates, alkyl sulfates, and ethylene oxide adducts of alkylphenols.

[0261] Emulsifiable concentrates can contain a suitable concentration of bacterial lipid composition, e.g., about 50 to about 500 grams per liter of liquid, dissolved in a carrier, either a water-miscible solvent or a mixture of a water-immiscible organic solvent and an emulsifier. Useful organic solvents include aromatics, particularly xylene, and petroleum fractions, especially the high-boiling naphthalene and olefinic portions of petroleum, such as heavy aromatic naphtha. Other organic solvents, such as terpene solvents including rosin derivatives, aliphatic ketones such as cyclohexanone, and complex alcohols such as 2-ethoxyethanol, can also be used. Suitable emulsifiers for emulsifiable concentrates can be selected from conventional anionic and nonionic surfactants.

[0262] The aqueous suspension comprises a suspension of the water-insoluble bacterial lipid composition dispersed in an aqueous carrier at a concentration ranging from about 5% to about 50% by weight. The suspension is prepared by finely grinding the composition and vigorously mixing it with a carrier consisting of water and a surfactant. Ingredients such as inorganic salts and synthetic or natural gums may be added to increase the density and viscosity of the aqueous carrier.

[0263] The bacterial-derived lipid composition may also be applied as a granular composition, particularly useful for application to soil. Granular compositions typically contain about 0.5% to about 10% by weight of the bacterial-derived lipid composition dispersed in a carrier comprising clay or similar material. Such compositions are typically prepared by dissolving the formulation in a suitable solvent and applying it to a granular carrier preformed to an appropriate particle size ranging from about 0.5 to about 3 mm. Such compositions may also be formulated by making a dough or paste of the carrier and compound, grinding, and drying to obtain the desired granular particle size.

[0264] Dusts containing the bacterially-derived lipid composition are prepared by intimately mixing the bacterially-derived lipid composition in powder form with a suitable dusty agricultural carrier, such as kaolin clay or crushed volcanic rock. Dusts may suitably comprise from about 1% to about 10% of the packet. They may be applied as a seed dressing or as a leaf application using a dust blower machine.

[0265] It is equally practical to apply the formulation in the form of a solution in a suitable organic solvent widely used in agricultural chemistry, usually petroleum such as spray oil.

[0266] The bacterial lipid composition can also be applied in the form of an aerosol composition. In such a composition, the packet is dissolved or dispersed in a carrier that is a pressure-generating propellant mixture. The aerosol composition is packaged in a container, through which the mixture is dispensed through an atomizing valve.

[0267] Another embodiment is an oil-in-water emulsion, the emulsion comprising oily globules each provided with a layer-structured liquid crystal coating, each oily globule comprising at least one agriculturally active compound and individually coated with a single or multiple layers comprising (1) at least one non-ionic lipophilic surfactant, (2) at least one non-ionic hydrophilic surfactant, and (3) at least one ionic surfactant, the globules having an average particle diameter of less than 800 nanometers. Further information regarding this embodiment is disclosed in U.S. Patent Publication No. 20070027034, published February 1, 2007. For ease of use, this embodiment is referred to as an "OIWE."

[0268] Furthermore, generally, when the molecules disclosed above are used in formulations, such formulations may also contain other components. These components include, but are not limited to, wetting agents, spreading agents, adhesives, penetrating agents, buffers, sequestering agents, drift reducing agents, compatibilizers, antifoaming agents, detergents, and emulsifiers (this is a non-exhaustive and non-mutually exclusive list). Some components are described below.

[0269] Wetting agents are substances that, when added to a liquid, increase the spreading or penetration of the liquid by reducing the interfacial tension between the liquid and the surface onto which the liquid spreads. Wetting agents are used for two main functions in pesticide formulations: during processing and manufacturing to increase the wetting rate of powders in water to create concentrates or suspension concentrates for soluble liquids, and during mixing of the product with water in the spray tank to reduce the wetting time of wettable powders and improve the penetration of water into water-dispersible granules. Examples of wetting agents used in wettable powder, suspension concentrate, and water-dispersible granule formulations are sodium lauryl sulfate, sodium dioctyl sulfosuccinate, alkylphenol ethoxylates, and fatty alcohol ethoxylates.

[0270] Dispersants are substances that adsorb onto particle surfaces, helping to preserve particle dispersion and prevent particle reagglomeration. Dispersants are added to pesticide formulations to facilitate dispersion and suspension during manufacturing and ensure particle redispersion in the water in the spray tank. They are widely used in wettable powders, suspension concentrates, and water-dispersible granules. Surfactants used as dispersants have the ability to strongly adsorb onto particle surfaces and provide a charge or steric barrier against particle reagglomeration. The most commonly used surfactants are anionic, nonionic, or a mixture of the two types. For wettable powder formulations, the most common dispersant is sodium lignosulfonate. For suspension concentrates, very good adsorption and stabilization are achieved using polyelectrolytes such as sodium naphthalene sulfonate formaldehyde condensate. Tristyrylphenol ethoxylate phosphate esters are also used. Nonionics such as alkylarylethylene oxide condensates and EO-PO block copolymers are sometimes combined with anionics as dispersants for suspension concentrates. In recent years, new types of ultra-high molecular weight polymer surfactants have been developed as dispersants. These have a very long hydrophobic "backbone" and numerous ethylene oxide chains that form the "teeth" of a "comb" surfactant. These high molecular weight polymers can provide excellent long-term stability to suspension concentrates because the hydrophobic backbone provides many anchoring points on the particle surface. Examples of dispersants used in pesticide formulations include sodium lignosulfonate, sodium naphthalene sulfonate formaldehyde condensate, tristyrylphenol ethoxylate phosphate ester, fatty alcohol ethoxylate, alkyl ethoxylate, EO-PO (ethylene oxide-propylene oxide) block copolymer, and graft copolymer.

[0271] An emulsifier is a substance that stabilizes the suspension of droplets of one liquid phase in another. Without an emulsifier, the two liquids would separate into two immiscible liquid phases. The most commonly used emulsifier blends contain an alkylphenol or aliphatic alcohol with 12 or more ethylene oxide units and an oil-soluble calcium salt of dodecylbenzenesulfonic acid. A hydrophile-lipophile balance ("HLB") value in the range of 8 to 18 usually provides a well-stable emulsion. Emulsion stability can be improved by adding a small amount of an EO-PO block copolymer surfactant.

[0272] Solubilizers are surfactants that form micelles in water at concentrations above the critical micelle concentration. The micelles can then dissolve or solubilize water-insoluble materials within the hydrophobic compartment of the micelle. The types of surfactants commonly used for solubilization are nonionic, sorbitan monooleate, sorbitan monooleate ethoxylate, and methyl oleate ester.

[0273] Surfactants may be used alone or with other additives, such as mineral or vegetable oils, as adjuvants in spray tank mixtures to improve the biological performance of bacterially derived lipid compositions on targets. The type of surfactant used for bioaugmentation generally depends on the nature and mode of action of the bacterially derived lipid composition. However, they are often non-ionic, such as alkyl ethoxylates, linear fatty alcohol ethoxylates, and fatty amine ethoxylates.

[0274] Carriers or diluents in agricultural formulations are materials that are added to bacterially derived lipid compositions to obtain the required strength of the product. Carriers are usually materials with high absorption capacity, while diluents are usually materials with low absorption capacity. Carriers and diluents are used in the formulation of dusts, wettable powders, granules, and water-dispersible granules.

[0275] Organic solvents are primarily used in the formulation of emulsifiable concentrates, oil-in-water emulsions, suspoemulsions, and ultra-low volume formulations, and, to a lesser extent, in granular formulations. Mixtures of solvents may also be used. The first major group of solvents are aliphatic paraffinic oils, such as kerosene or refined paraffin. The second major group (and most common) includes aromatic solvents, such as xylene, and the high molecular weight fractions of C9 and C10 aromatic solvents. Chlorinated hydrocarbons are useful as cosolvents to prevent crystallization of bacterial-derived lipid compositions when the formulation is emulsified in water. Alcohols are sometimes used as cosolvents to increase the solvent power. Other solvents include vegetable oils, seed oils, and esters of vegetable and seed oils.

[0276] Thickeners or gelling agents are primarily used in the formulation of suspension concentrates, emulsions, and suspoemulsions to modify the rheological or flow properties of liquids and prevent separation and settling of dispersed particles or droplets. Thickeners, gelling agents, and anti-settling agents generally fall into two categories: water-insoluble particles and water-soluble polymers. Clay and silica can be used to produce suspension concentrate formulations. Examples of these types of materials include, but are not limited to, montmorillonite, bentonite, magnesium aluminum silicate, and attapulgite. Water-soluble polysaccharides have been used as thickening and gelling agents for many years. The most commonly used types of polysaccharides are natural extracts of seeds and seaweed, or synthetic derivatives of cellulose. Examples of these types of materials include, but are not limited to, guar gum, locust bean gum, carrageenan, alginate, methylcellulose, sodium carboxymethylcellulose (SCMC), and hydroxyethylcellulose (HEC). Other types of anti-settling agents are based on modified starch, polyacrylates, polyvinyl alcohol, and polyethylene oxide. Another good anti-settling agent is xanthan gum.

[0277] Microorganisms can cause spoilage of formulated products. Therefore, preservatives are used to eliminate or reduce their effects. Examples of such agents include, but are not limited to, propionic acid and its sodium salt, sorbic acid and its sodium or potassium salt, benzoic acid and its sodium salt, p-hydroxybenzoic acid sodium salt, methyl p-hydroxybenzoate, and 1,2-benzisothiazolin-3-one (BIT).

[0278] The presence of surfactants often causes water-based formulations to foam during the mixing operation during production and application through a spray tank. To reduce the tendency to foam, antifoaming agents are often added during the production stage or before filling into bottles. Generally, there are two types: silicone and non-silicone. Silicone antifoams are usually aqueous emulsions of dimethylpolysiloxane, while non-silicone antifoams are water-insoluble oils such as octanol and nonanol, or silica. In both cases, the function of the antifoaming agent is to displace the surfactant from the gas-water interface.

[0279] "Green" agents (e.g., adjuvants, surfactants, solvents) can reduce the overall environmental footprint of crop protection formulations. Green agents are biodegradable and generally derived from natural and / or sustainable sources, such as plant and animal sources. Specific examples are vegetable oils, seed oils, and their esters, as well as alkoxylated alkyl polyglucosides.

[0280] In some cases, the bacterial-derived lipid composition can be a freeze-dried or lyophilized composition. See U.S. Patent No. 4,311,712. The bacterial-derived lipid composition can then be reconstituted when contacted with water or another liquid. Other components can be added to the freeze-dried or reconstituted bacterial-derived lipid composition, such as other heterologous functional agents, agriculturally acceptable carriers, or other materials according to the formulations described herein.

[0281] Other optional features of the composition include a carrier or delivery vehicle that protects the bacterial-derived lipid composition from UV and / or acidic conditions. In some cases, the delivery vehicle contains a pH buffer. In some cases, the composition is formulated to have a pH in the range of about 4.5 to about 9.0, including, for example, any one of the following pH ranges: about 5.0 to about 8.0, about 6.5 to about 7.5, or about 6.5 to about 7.0.

[0282] For more information on agricultural formulations, see "Chemistry and Technology of Agrochemical Formulations," edited by D.A. Knowles, copyright 1998 by Kluwer Academic Publishers. Also see "Insecticides in Agriculture and Environment—Retrospects and Prospects" by A.S. Perry, I. Yamamoto, I. Ishaaya, and R. Perry, copyright 1998 by Springer-Verlag.

[0283] Pharmaceutical preparations The bacterial-derived lipid composition is formulated into a pharmaceutical composition (i.e., bacterial-derived lipid composition) for administration to animals (e.g., humans). The pharmaceutical composition may be administered to animals (e.g., humans) together with a pharmaceutically acceptable diluent, carrier, and / or excipient. Depending on the mode of administration and dosage, the pharmaceutical composition of the methods described herein is formulated into a suitable pharmaceutical composition to allow easy delivery. A single dose may be in a unit dosage form, if necessary.

[0284] The bacterial-derived lipid composition may be formulated, for example, for oral, intranasal, intravenous (e.g., injection or infusion), intramuscular, or subcutaneous administration to an animal. For injectable formulations, various effective pharmaceutical carriers are known in the art (e.g., Remington: The Science and Practice of Pharmacy, 22 nd ed., (2012) and ASHP Handbook on Injectable Drugs, 18 th ed., (2014).

[0285] Suitable pharmaceutically acceptable carriers and excipients are non-toxic to recipients at the dosage and concentration employed.Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran and immunoglobulin, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine and lysine, and carbohydrates such as glucose, mannose, sucrose and sorbitol.Bacteria-derived lipid compositions may be formulated according to conventional pharmaceutical practice.The concentration of the compound in the formulation varies depending on many factors, including the dosage of the effective agent (e.g., bacteria-derived lipid composition and nucleic acid) to be administered and the route of administration.

[0286] For oral administration to animals, bacterial-derived lipid compositions can be prepared in the form of oral preparations. Preparations for oral use can include tablets, caplets, capsules, syrups, or oral liquid dosage forms containing active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugars, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate), granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid), binders (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol), and smoothing agents, glidants, and antiadherents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients may be colorants, flavoring agents, plasticizers, humectants, buffers, etc. Formulations for oral use may also be provided in unit dosage form as chewable tablets, non-chewable tablets, caplets, capsules (e.g., hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or soft gelatin capsules in which the active ingredient is mixed with water or an oil medium). The compositions disclosed herein may also further comprise immediate-release, extended-release, or sustained-release formulations.

[0287] For parenteral administration to animals, bacterial-derived lipid compositions may be formulated in the form of a liquid solution or suspension and administered by parenteral routes (e.g., subcutaneous, intravenous, or intramuscular). Pharmaceutical compositions may be formulated for injection or infusion. Pharmaceutical compositions for parenteral administration may be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, or cell culture media (e.g., Dulbecco's Modified Eagle's Medium (DMEM), alpha-Modified Eagle's Medium (α-MEM), and F-12 medium). Formulation methods are known in the art, see, for example, Gibson (ed.) Pharmaceutical Preformulation and Formulation (2nd ed.) Taylor & Francis Group, CRC Press (2009).

[0288] Heterofunctional drugs The bacterially-derived lipid composition may include a heterologous functional agent, such as a heterologous agricultural agent (e.g., an insecticide, fertilizer, herbicide, plant modifying agent) or a heterologous therapeutic agent (e.g., an antifungal, antibacterial, antiviral, insecticide, nematicide, antiparasitic, or insect repellent). For example, the bacterially-derived lipid composition may encapsulate the heterologous functional agent. Alternatively, the heterologous functional agent may be embedded or conjugated to the surface of the bacterially-derived lipid composition. In some cases, the bacterially-derived lipid composition includes two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different heterologous functional agents. The heterologous functional agents may be added at any step during the manufacturing process effective to introduce the agent into the bacterially-derived lipid composition.

[0289] In certain cases, a heterologous functional agent (e.g., a heterologous agricultural agent (e.g., an insecticide, fertilizer, herbicide, plant modifier, heterologous nucleic acid, heterologous polypeptide, or heterologous small molecule) or a heterologous therapeutic agent (e.g., an antifungal, antibacterial, antiviral, nematicide, antiparasitic, or insect repellent)) can be modified. For example, the modification can be a chemical modification, such as conjugation to a marker, e.g., a fluorescent or radioactive marker. In other examples, the modification can include conjugation or operational attachment to a moiety that enhances the stability, delivery, targeting, bioavailability, or half-life of the agent, e.g., a lipid, glycan, polymer (e.g., PEG), or cationic moiety.

[0290] Examples of heterofunctional agents are outlined below.

[0291] Heterogeneous agricultural formulations The bacterially derived lipid composition may include a heterologous agricultural agent such as an insecticide, herbicide, fertilizer, or plant modifier (e.g., an agent that affects plants or organisms associated with plants and can be loaded into the bacterially derived lipid composition).

[0292] For example, in some cases, the bacterial-derived lipid composition may contain an insecticide. The insecticide may be an antifungal agent, an antibacterial agent, an insecticide, a molluscicide, a nematicide, a viricide, or a combination thereof. The insecticide may be a chemical agent, such as those known in the art. Alternatively, or in addition, the insecticide may be a peptide, a polypeptide, a nucleic acid, a polynucleotide, or a small molecule. The insecticide may be an agent that can reduce the fitness of various plant pests, or may be an agent that targets one or more specific target plant pests (e.g., a specific species or genus of plant pests).

[0293] In some cases, the bacterial-derived lipid composition may contain one or more heterologous fertilization agents. Examples of heterologous fertilization agents include plant nutrients or plant growth regulators, such as those known in the art. Alternatively, or in addition, the fertilization agent may be a peptide, polypeptide, nucleic acid, or polynucleotide that can increase the fitness of the plant symbiont. The fertilization agent may be an agent that can increase the fitness of various plants or plant symbionts, or may be an agent that targets one or more specific target plants or plant symbionts (e.g., a specific species or genus of plant or plant symbiont).

[0294] In other cases, the bacterially-derived lipid composition may include one or more heterologous plant modifying agents. In some cases, the plant modifying agent may include a peptide or a nucleic acid.

[0295] antibacterial agents The bacterial-derived lipid compositions described herein may further comprise an antimicrobial agent. In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antimicrobial agents. For example, the antimicrobial agent can reduce the fitness (e.g., reduce growth or kill) of a bacterial plant pest (e.g., a bacterial plant pathogen). The bacterial-derived lipid composition containing an antibiotic can be contacted with a target pest or an infested plant in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of antibiotic concentration in or on the target pest, and (b) reduce the fitness of the target pest. The antimicrobial agent may be formulated in the bacterial-derived lipid composition for any of the methods described herein, or in certain cases, may be associated with the bacterial-derived lipid composition.

[0296] As used herein, the term "antimicrobial agent" refers to a substance that kills or inhibits the growth, proliferation, division, reproduction, or spread of bacteria, such as plant pathogenic bacteria, and includes a bactericide (e.g., a disinfectant compound, an antiseptic compound, or an antibiotic) or a bacteriostatic agent (e.g., a compound or antibiotic). Bactericidal antibiotics kill bacteria, while bacteriostatic antibiotics only slow the growth or reproduction of bacteria.

[0297] Disinfectants may include disinfectants, antiseptics, or antibiotics. The most used disinfectants are active chlorine (i.e., hypochlorites (e.g., sodium hypochlorite), chloramines, dichloroisocyanurate and trichloroisocyanurate, wet chlorine, chlorine dioxide, etc.), active oxygen (peroxides, e.g., peracetic acid, potassium persulfate, sodium perborate, sodium percarbonate, and urea perhydrate), iodine (iodine-iodine (povidone-iodine, Betadine), Lugol's solution, tincture of iodine, iodized nonionic surfactants), concentrated alcohols (mainly ethanol, 1-propanol, also called n-propanol, and 2-propanol, called isopropanol, and mixtures thereof, as well as 2-phenoxyethanol and 1- and 2-phenoxypropanol), phenolic substances (phenol (also called carbolic acid), cresols (combined with liquid potassium soap, called lysols), etc.). These agents may include halogenated (chlorinated, brominated) phenols such as hexachlorophene, triclosan, trichlorophenol, tribromophenol, pentachlorophenol, dibromophenol and their salts), cationic surfactants such as some quaternary ammonium cations (benzalkonium chloride, cetyltrimethylammonium bromide or chloride, didecyldimethylammonium chloride, cetylpyridinium chloride, benzethonium chloride), non-quaternary compounds such as chlorhexidine, glucoprotamine, octenidine dihydrochloride, strong oxidizing agents such as ozone and permanganate solutions, heavy metals and their salts such as colloidal silver, silver nitrate, mercury chloride, phenylmercury salts, copper sulfate, copper oxide-chloride, copper hydroxide, copper octoate, copper oxychloride sulfate, copper sulfate, copper sulfate pentahydrate, and the like. Heavy metals and their salts are the most toxic and environmentally harmful disinfectants, and therefore their use is strongly discouraged or even eliminated, in addition to suitably concentrated strong acids (phosphoric acid, nitric acid, sulfuric acid, amidosulfonic acid, toluenesulfonic acid) and alkalis (sodium, potassium, calcium hydroxide).

[0298] As antiseptics (i.e., disinfectants that can be used on the human or animal body, skin, mucous membranes, wounds, etc.), some of the disinfectants mentioned above can be used under appropriate conditions (mainly concentration, pH, temperature and toxicity to humans / animals). Among other things, the following are important: appropriately diluted chlorine preparations (i.e., Dacan solution, 0.5% sodium or potassium hypochlorite solution, pH adjusted to 7-8, or 0.5-1% sodium benzenesulfochloramide solution (chloramine B)); some iodine preparations, such as iodopovidone in various galenics (ointments, solutions, wound dressings), formerly in Lugol's solution; peroxide as urea perhydrate solution and pH-buffered 0.1-0.25% peracetic acid solution; alcohol with or without preservatives, primarily used for skin disinfection; weak organic acids, such as sorbic acid, benzoic acid, lactic acid, and salicylic acid; some phenolic compounds, such as hexachlorophene, triclosan, and dibromomethyl; and cationically active compounds, such as 0.05-0.5% benzalkonium, 0.5-4% chlorhexidine, and 0.1-2% octenidine solutions.

[0299] The bacterial-derived lipid composition may include an antibiotic. Any antibiotic known in the art may be used. Antibiotics are generally classified based on their mechanism of action, chemical structure, or spectrum of activity.

[0300] The antibiotics described herein may target any bacterial function or growth process and may be either bacteriostatic (e.g., slow or prevent bacterial growth) or bactericidal (e.g., kill bacteria). In some cases, the antibiotic is a bactericidal antibiotic. In some cases, the bactericidal antibiotic is an antibiotic that targets the bacterial cell wall (e.g., penicillin and cephalosporin), an antibiotic that targets the cell membrane (e.g., polymyxin), or an antibiotic that inhibits essential bacterial enzymes (e.g., rifamycin, lipiarmycin, quinolones, and sulfonamides). In some cases, the bactericidal antibiotic is an aminoglycoside (e.g., kasugamycin). In some cases, the antibiotic is a bactericidal antibiotic. In some cases, the bacteriostatic antibiotic targets protein synthesis (e.g., macrolides, lincosamides, and tetracyclines). Additional classes of antibiotics that can be used herein include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), or lipiarmycins (such as fidaxomicin). Examples of antibiotics include rifampicin, ciprofloxacin, doxycycline, ampicillin, and polymyxin B. The antibiotics described herein can have any level of target specificity (e.g., narrow spectrum or broad spectrum). In some cases, the antibiotic is a narrow-spectrum antibiotic, thus targeting a specific type of bacteria, such as gram-negative or gram-positive bacteria. Alternatively, the antibiotic may be a broad-spectrum antibiotic that targets a wide range of bacteria.

[0301] Other non-limiting examples of antibiotics can be found in Table 1 of WO 2021 / 041301, which is incorporated herein by reference in its entirety. One of skill in the art will understand that the appropriate concentration of each antibiotic in the composition will depend on factors such as antibiotic efficacy, stability, number of distinct antibiotics, formulation, and method of application of the composition.

[0302] antifungal agents The bacterial-derived lipid composition can further comprise an antifungal agent. In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antifungal agents. For example, the antifungal agent can reduce the fitness (e.g., reduce growth or kill) of a fungal plant pest (e.g., a bacterial plant pathogen). The bacterial-derived lipid composition containing the antifungal agent can be contacted with the target pest or an infested plant in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of antibiotic concentration in or on the target fungus, and (b) reduce the fitness of the target fungus. The antifungal agent may be formulated in the bacterial-derived lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterial-derived lipid composition.

[0303] As used herein, the terms "fungicide" or "antifungal agent" refer to a substance that kills or inhibits the growth, proliferation, division, reproduction, or spread of fungi, such as plant pathogenic fungi. Many different types of antifungal agents are commercially available. Non-limiting examples of antifungal agents include azoxystrobin, mancozeb, prothioconazole, folpet, tebuconazole, difenoconazole, captan, bupirimate, or fosetyl-Al. Additional exemplary fungicides include strobilurins, azoxystrobin, dimoxystrobin, enestrobrin, fluoxastrobin, kresoxim-methyl, metominostrobin, picoxystrobin, pyraclostrobin, trifloxystrobin, orysastrobin, carboxamide, carboxanilide, benalaxyl, benalaxyl-M, benodanil, carboxin, mebenil, mepronil, fenfuran, and fenhexamid. , flutolanil, furalaxyl, flucarbanil, furametpyr, metalaxyl, metalaxyl-M (mefenoxam), methfluoxam, metsulfovax, ofrace, oxadixyl, oxycarboxin, penthiopyrad, pyracarbollide, salicylanilide, tecloftalam, thifluzamide, tiadinil, N-biphenylamide, bixafen, boscalid, carboxylic acid morpholide, dimethomorph, flumorph, benzamide, Flumetover, fluopicolide (picobenzamide), zoxamide, carboxamide, carpropamid, diclocymet, mandipropamide, silthiofam, azoles, triazoles, bitertanol, bromuconazole, cyproconazole, difenoconazole, diniconazole, enilconazole, epoxiconazole, fenbuconazole, flusilazole, fluquinconazole, flutriafol, hexaconazole, imibenco nazole, ipconazole, metconazole, myclobutanil, penconazole, propiconazole, prothioconazole, simeconazole, tebuconazole, tetraconazole, triadimenol, triadimefon, triticonazole, imidazole, cyazofamid, imazalil, pefurazoate, prochloraz, triflumizole, benzimidazole, benomyl, carbendazim, fuberidazole, thiabendazole, ethaboxam,Etridiazole, hymexazole, nitrogen-containing heterocyclic compounds, pyridine, fadinam, pyrifenox, pyrimidine, bupirimate, cyprodinil, ferimzone, fenarimol, mepanipyrim, nuarimol, pyrimethanil, piperazine, triforine, pyrrole, fludioxonil, fenpiclonil, morpholine, aldimorph, dodemorph, fenpropimorph, tridemorph, dicarboximide, iprodione, procymidone, vinclozolin, acibenzolar-S-methyl, anilazine, captan, captafol, dazomet, diclomezine, fenoxanil, folpet, fenpropizin, famoxadone, fenamidone, octhilinone, probenazole, proquinazide , pyroquilon, quinoxyfen, tricyclazole, carbamates, dithiocarbamates, ferbam, mancozeb, maneb, metiram, metam, propineb, thiuram, zineb, ziram, diethofencarb, flubenthiavalicarb, iprovalicarb, propamocarb, guanidine, dodine, iminoctadine, guazatine, kasugamycin, polyoxins, streptomycin, validamycin A, organometallic compounds, fentin salts, sulfur-containing heterocyclic compounds, isoprothiolane, dithianon, organophosphate compounds, edifenphos, fosetyl, fosetyl-aluminum, iprobenfos, pyrazophos, tolclofos-methyl, organochlorines, thiophanate-methyl, chlorothalonil, dichlofluanid, tolilph fluanid, flusulfamide, phthalide, hexachlorobenzene, pencycuron, quintozene, nitrophenyl derivatives, binapacryl, dinocap, dinobuton, spiroxamine, cyflufenamid, cymoxanil, metrafenone, N-2-cyanophenyl-3,4-dichloroisothiazole-5-carboxamide (isothianil), N-(3',4',5'-trifluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazole-4-carboxamide, 3-[5-(4-chlorophenyl)-2,3-dimethylisoxazolidin-3-yl]-pyridine, N-(3',4'-dichloro-4-fluorobiphenyl-2-yl)-3-difluoromethyl-1-methylpyrazole-e-4-carboxamide,5-chloro-7-(4-methylpiperidin-1-yl)-6-(2,4,6-trifluorophenyl)-[1,2,4]tria-zolo[1,5-a]pyrimidine, 2-butoxy-6-iodo-3-propylchromen-4-one, N,N-dimethyl-3-(3-bromo-6-fluoro-2-methylindole-1-sulfonyl)-[1,2,4]triazole-1-sulfonamide, methyl-(2-chloro-5-[1-(3-methylbenzyloxyimino)-ethyl]benzyl)carbamate , methyl-(2-chloro-5-[1-(6-methylpyridin-2-ylmethoxy-imino)ethyl]benzyl)carbamate, methyl 3-(4-chlorophenyl)-3-(2-isopropoxycarbonylamino-3-methylbutyryl-amino)propionate, 4-fluorophenyl N-(1-(1-(4-cyanophenyl)ethanesulfonyl)but-2-yl)carbamate, N-(2-(4-[3-(4-chlorophenyl)prop-2-ynyloxy]-3-methoxyphenyl)ethyl) -2-meta-nesulfonylamino-3-methylbutyramide, N-(2-(4-[3-(4-chlorophenyl)prop-2-ynyloxy]-3-methoxyphenyl)ethyl)-2-ethane-esulfonylamino-3-methylbutyramide, N-(4'-bromobiphenyl-2-yl)-4-difluoromethyl-2-methylthiazole-5-carboxamide, N-(4'-trifluoromethylbiphenyl-2-yl)-4-difluoromethyl-2-methylthiazole-5-carboxamide, N Antifungal agents include, but are not limited to, 4'-chloro-3'-fluorobiphenyl-2-yl)-4-difluoromethyl-2-methyltrifluorobenzoate, 4'-chloro-3'-fluorobiphenyl-2-yl)-4-difluoromethyl-2-methyltrifluorobenzoate, or methyl 2-(ortho-((2,5-dimethylphenyloxy-methylene)phenyl)-3-methoxyacrylate. One of ordinary skill in the art will understand that the appropriate concentration of each antifungal agent in the composition will depend on factors such as antifungal efficacy, stability, number of distinct antifungal agents, formulation, and method of application of the composition.

[0304] insecticides The bacterial lipid composition may further comprise an insecticide. In some cases, the bacterial lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different insecticides. For example, the insecticide can reduce the fitness of insect plant pests (e.g., reduce growth or kill them). The bacterial lipid composition comprising an insecticide can be contacted with a target insect pest, or an infested plant, in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of insecticide concentration in or on the target insect, and (b) reduce the fitness of the target insect. The insecticide may be formulated in the bacterial lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterial lipid composition.

[0305] As used herein, the terms "insecticide" or "pesticide" refer to a substance that kills or inhibits the growth, growth, reproduction, or spread of insects, such as agricultural insect pests. Non-limiting examples of insecticides are set forth in Table 2 of WO 2021 / 041301, which is incorporated herein by reference in its entirety. Further non-limiting examples of suitable insecticides include biologicals, hormones, or pheromones, such as azadirachtin, Bacillus species, Beiberia species, Codormones, Metarhizium species, Paecilomyces species, thuringiensis, and Verticillium species, as well as active compounds with unknown or unspecified mechanisms of action, such as smoke inhibitors (such as aluminum phosphide, methyl bromide, and sulfuryl fluoride) and selective antifeedants (such as cryolite, flonicamid, and pymetrozine). Those skilled in the art will understand that the appropriate concentration of each pesticide in the composition will depend on factors such as pesticide efficacy, stability, number of separate pesticides, formulation, and method of application of the composition.

[0306] nematicides The bacterial-derived lipid composition may further comprise a nematicide. In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different nematicides. For example, the nematicide can reduce the fitness of nematode plant pests (e.g., reduce growth or kill). The bacterial-derived lipid composition comprising a nematicide can be contacted with the target nematode pest or the plant infested with it in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of nematicide concentration in or on the target nematode, and (b) reduce the fitness of the target nematode. The nematicide may be formulated in the bacterial-derived lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterial-derived lipid composition.

[0307] As used herein, the terms "nematicide" or "nematicide" refer to a substance that kills or inhibits the growth, growth, reproduction, or spread of nematodes, such as agricultural nematode pests. Non-limiting examples of nematicides are set forth in Table 3 of WO 2021 / 041301, which is incorporated herein by reference in its entirety. One of ordinary skill in the art will understand that the appropriate concentration of each nematicide in the composition will depend on factors such as nematicide efficacy, stability, number of separate nematicides, formulation, and method of application of the composition.

[0308] Molluscicide The bacterially derived lipid composition may further comprise a molluscicide. In some cases, the bacterially derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different molluscicides. For example, the molluscicide can reduce the fitness of a molluscan plant pest (e.g., reduce growth or kill). The bacterially derived lipid composition comprising a molluscicide can be contacted with a target mollusc pest or an infested plant in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of molluscicide concentration in or on the target mollusc, and (b) reduce the fitness of the target mollusc. The molluscicide may be formulated in the bacterially derived lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterially derived lipid composition.

[0309] As used herein, the term "molluscicide" or "molluscicide" refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of mollusks, such as agricultural mollusc pests. Many chemicals can be used as molluscicides, including metal salts such as iron(III) phosphate, aluminum sulfate, and sodium ferric EDTA,[3][4] metaldehyde, methiocarbamate, or acetylcholinesterase inhibitors. Those skilled in the art will understand that the appropriate concentration of each molluscicide in the composition will depend on factors such as the efficacy and stability of the molluscicide, the number of distinct molluscicides, the formulation, and the method of application of the composition.

[0310] antiviral agents The bacterial-derived lipid composition may further comprise an antiviral agent. In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different virucides. For example, the antivirucide can reduce (e.g., reduce or eliminate) the fitness of a viral plant pathogen. A bacterial-derived lipid composition comprising a virucide described herein can be contacted with a target virus or an infested plant in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of antivirucide concentration and (b) reduce or eliminate the target virus. The antiviral agent described herein may be formulated in the bacterial-derived lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterial-derived lipid composition.

[0311] As used herein, the term "antiviral agent" or "antiviral agent" refers to a substance that kills or inhibits the growth, proliferation, reproduction, development, or spread of viruses, such as agricultural viral pathogens. Many agents can be used as virucidal agents, including chemical agents or biological agents (e.g., nucleic acids, e.g., dsRNA). One skilled in the art will understand that the appropriate concentration of each antiviral agent in the composition will depend on factors such as the efficacy and stability of the antiviral agent, the number of distinct antiviral agents, the formulation, and the method of application of the composition.

[0312] herbicides The bacterially-derived lipid composition may further comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) herbicides. For example, the herbicides can reduce (e.g., reduce or eliminate) the fitness of the weed. The bacterially-derived lipid composition comprising the herbicide can be contacted with the target weed in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of herbicide concentration on the plant and (b) reduce the fitness of the weed. The herbicide may be formulated in the bacterially-derived lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterially-derived lipid composition.

[0313] As used herein, the term "herbicide" refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of weeds. Many chemicals can be used as herbicides, including glufosinate, propaquizafop, metamitron, metazachlor, pendimethalin, flufenacet, diflufenican, clomazone, nicosulfolone, mesotrione, pinoxaden, sulcotrione, prosulfocarb, sulfentrazone, bifenox, quinmerac, triallate, terbuthylazine, atrazine, oxyfluorfen, diuron, trifluralin, or chlorotoluron. Further examples of herbicides include benzoic acid herbicides such as dicamba esters, phenoxyalkanoic acid herbicides such as 2,4-D, MCPA and 2,4-DB esters, aryloxyphenoxypropionic acid herbicides such as clodinafop, cyhalofop, fenoxaprop, fluazifop, haloxyfop, and quizalofop esters, pyridine carboxylic acid herbicides such as aminopyralid, picloram, and clopyralid esters, pyrimidine carboxylic acid herbicides such as aminocyclopyrachlor esters, These include, but are not limited to, pyridyloxyalkanoic acid herbicides such as fluroxypyr and triclopyr esters, and hydroxybenzonitrile herbicides such as bromoxynil and ioxynil esters, esters of arylpyridinecarboxylic acid hydrazides, and arylpyrimidinecarboxylic acids of the general structure disclosed in U.S. Pat. No. 7,314,849, U.S. Pat. No. 7,300,907, and U.S. Pat. No. 7,642,220, each of which is incorporated herein by reference in its entirety.In certain embodiments, the herbicide is 2,4-D, 2,4-DB, acetochlor, acifluorfen, alachlor, ametryn, amitrole, asulam, atrazine, azafenidin, benefin, bensulfuron, bensulide, bentazon, bromacil, bromoxynil, butyrate, carfentrazone, chloramben, chlorimuron, chlorpropham, chlorsulfuron, clethodim, clomazone, clopyralid, chloransulam, cyanazine, cycloate, DCPA, desmedipham, dichlobenil, diclofenol, cyclohexan ... Hops, diclosulam, diethathyl, difenzoquat, diflufenzopyr, dimethenamid-p, diquat, diuron, DSMA, endothal, EPTC, ethalfluralin, ethametsurfuron, ethofumesate, fenoxaprop, fluazifop-P, flucarbazone, flufenacet, flumetsulam, flumiclorac, flumioxazin, fluometuron, fluroxypyr, fluthiacet, fomesafen, foramsulfuron, glufosinate, glyphosate, halosulfuron, haloxyfop, hexazinone, imaza Methabenz, imazamox, imazapic, imazaquin, imazethapyr, isoxaben, isoxaflutole, lactofen, linuron, MCPA, MCPB, mesotrione, methazole, metolachlor-s, metribuzin, metsulfuron, molinate, MSMA, napropamide, naptalam, nicosulfuron, norflurazon, oryzalin, oxadiazon, oxasulfuron, oxyfluorfen, paraquat, pebulate, pelargonic acid, pendimethalin, phenmedipham, picloram, primisulfuron, promethazine The compound may be selected from the group consisting of diamine, prometryn, pronamide, propachlor, propanil, prosulfuron, pyrazone, pyridate, pyrithiobac, quinclorac, quizalofop, rimsulfuron, sethoxydim, siduron, simazine, sulfentrazone, sulfometuron, sulfosulfuron, tebuthiuron, terbacil, thiazopyr, thifensulfuron, thiobencarb, tralkoxydim, triallate, triasulfuron, tribenuron, triclopyr, trifluralin, triflusulfuron, and benolate.Those skilled in the art will understand that the appropriate concentration of each herbicide in the composition will depend on factors such as herbicide efficacy, stability, the number of separate herbicides, the formulation, and the method of application of the composition.

[0314] Antidote The bacterial-derived lipid composition may further comprise a repellent. In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different repellents. For example, the repellent can repel any of the pests (e.g., insects, nematodes, or mollusks), microorganisms (e.g., plant pathogens or endophytes such as bacteria, fungi, or viruses), or weeds described herein. The bacterial-derived lipid composition containing the repellent can be contacted with the target plant or its infested plant in an amount and for a time sufficient to (a) achieve a target level of repellent concentration (e.g., a predetermined level or threshold level) and (b) reduce the level of pests on the plant compared to an untreated plant. The repellent may be formulated in the bacterial-derived lipid composition for any of the methods described herein, or in certain cases, may be associated with the bacterial-derived lipid composition.

[0315] In some cases, the repellent is an insect repellent. Some examples of well-known insect repellents include benzyl, benzyl benzoate, 2,3,4,5-bis(butyl-2-ene)tetrahydrofurfuran (MGK repellent 11), butoxypolypropylene glycol, N-butylacetanilide, normal-butyl-6,6-dimethyl-5,6-dihydro-1,4-pyrone-2-carboxylate (Indalone), dibutyl adipate, dibutyl phthalate, di-normal-butylsuccinate (Tabatrex), N,N-diethyl-meta-toluamide (DEET), dimethylcarbate (endo,endo)-dimethylbicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate), dimethyl phthalate, 2-ethyl-2-butyl-1,3-propanediol, 2-ethyl-1,3-hexanediol (Rutgers 612), di-normal-propyl isotinochromelonate (MGK repellent 326), 2-phenylcyclohexanol, p-methane-3,8-diol, and normal-propyl N,N-diethylsuccinamate. Other repellents include citronella oil, dimethyl phthalate, normal-butyl mesityl oxide oxalate, and 2-ethylhexanediol-1,3 (see Kirk-Othmer Encyclopedia of Chemical Technology, 2nd Ed., Vol. 11:724-728, and The Condensed Chemical Dictionary, 8th Ed., p 756).

[0316] The repellent may be a synthetic or non-synthetic insect repellent. Examples of synthetic insect repellents include methyl anthranilate and other anthranilic acid-based repellents, benzaldehyde, DEET (N,N-diethyl-m-toluamide), dimethylcarbate, dimethyl phthalate, icaridin (i.e., picaridin, Bayrepel, and KBR 3023), indalone (e.g., IR3535 (3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester), used in the "6-2-2" mixture (60% dimethyl phthalate, 20% indalone, 20% ethyl hexanediol)), metofluthrin, permethrin, SS220, or tricyclodecenyl allyl ether. Examples of natural insect repellents include beautyberry (Callicarpa) leaves, birch bark, port willow (Salix alba), catnip oil (e.g., nepetalactone), citronella oil, and essential oils of the Lemon eucalyptus (Corymbopsis citriola, e.g., p-menthane-3,8-diol (PMD)), neem oil, lemongrass, tea tree oil from Melaleuca alternifolia leaves, tobacco, or extracts thereof.

[0317] xenofertilization agents The bacterial-derived lipid composition can further comprise a heterologous fertilization agent. In some cases, the heterologous fertilization agent is associated with the bacterial-derived lipid composition. For example, the bacterial-derived lipid composition can encapsulate the heterologous fertilization agent. Additionally or alternatively, the heterologous fertilization agent can be embedded or conjugated to the surface of the bacterial-derived lipid composition.

[0318] Examples of heterologous fertilization agents include plant nutrients or plant growth regulators, such as those known in the art. Alternatively, or in addition, the fertilization agent may be a peptide, polypeptide, nucleic acid, or polynucleotide that can increase the fitness of the plant symbiont. The fertilization agent may be an agent that can increase the fitness of various plants or plant symbionts, or may be an agent that targets one or more specific target plants or plant symbionts (e.g., a specific species or genus of plant or plant symbiont).

[0319] In some cases, heterologous fertilization agents can be modified.For example, modification can be chemical modification, such as conjugation to a marker, for example, a fluorescent marker or a radioactive marker.In other examples, modification can include conjugation or operational binding to a moiety that enhances the stability, delivery, targeting, bioavailability, or half-life of a drug, for example, lipids, glycans, polymers (e.g., PEG), cationic moieties.

[0320] In some cases, heterofertilization agents include any material of natural or synthetic origin that is applied to soil or plant tissue to provide one or more plant nutrients essential for plant growth. Plant nutrients can include macronutrients, micronutrients, or a combination thereof. Plant macronutrients include nitrogen, phosphorus, potassium, calcium, magnesium, and / or sulfur. Plant micronutrients include copper, iron, manganese, molybdenum, zinc, boron, silicon, cobalt, and / or vanadium. Examples of plant nutrient fertilizers include nitrogen fertilizers, including, but not limited to, urea, ammonium nitrate, ammonium sulfate, non-pressurized nitrogen solution, aqueous ammonia, anhydrous ammonia, ammonium thiosulfate, sulfur-coated urea, urea-formaldehyde, IBDU, polymer-coated urea, calcium nitrate, ureaform, or methylene urea; phosphite fertilizers, such as diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, concentrated superphosphate, and triple superphosphate; or potassium fertilizers, such as potassium chloride, potassium sulfate, potassium-magnesium sulfate, and potassium nitrate. Such compositions may exist as free salts or ions within the composition. Fertilizers may be designated by the content of one or more of their components, such as nitrogen, phosphorous, or potassium. The content of these elements in a fertilizer may be indicated by the NPK value (N = nitrogen content by weight percent, P = phosphorous content by weight percent, and K = potassium content by weight percent).

[0321] On the other hand, inorganic fertilizers are made from non-biological materials and include, for example, ammonium nitrate, ammonium sulfate, urea, potassium chloride, potash, ammonium phosphate, anhydrous ammonia, and other phosphates. Inorganic fertilizers contain nutrients in soluble forms that are readily available and readily available to plants. Inorganic fertilizers are generally inexpensive and have a low unit cost for the desired elements. Those skilled in the art will understand that the exact amount of a given element in a fertilizer can be calculated and administered to plants or soil.

[0322] Fertilizers may be further classified as either organic or inorganic. Organic fertilizers include fertilizers with molecular frameworks that have a carbon skeleton, such as compositions derived from biological materials. Organic fertilizers are made from materials derived from living organisms. Animal manure, compost, bone meal, feather meal, and blood meal are common examples of organic fertilizers. On the other hand, organic fertilizers are typically not readily available to plants and require soil microorganisms to break down the fertilizer components into simpler structures before they can be used by plants. In addition, organic fertilizers not only induce plant growth responses observed with common inorganic fertilizers, but natural organic fertilizers can also stimulate the growth and activity of soil microbial populations. Increasing soil microbial populations (e.g., plant symbionts) has significant beneficial effects on the physical and chemical properties of the soil and increases disease and pest resistance.

[0323] In one aspect, a bacterially-derived lipid composition comprising a plant nutrient can be contacted with a plant in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of plant nutrient concentration in or on the plant, and (b) increase the fitness of the plant compared to an untreated plant.

[0324] In another embodiment, a bacterially-derived lipid composition comprising a plant nutrient can be contacted with a plant symbiont in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of plant nutrient concentration in or on the plant symbiont (e.g., a bacterial or fungal endosymbiont), and (b) increase the fitness of the plant symbiont compared to an untreated plant.

[0325] The heterofertilization agent may comprise a plant growth regulator. Exemplary plant growth regulators include auxins, cytokines, gibberellins, and abscisic acid. In some cases, the plant growth regulator is abscisic acid, amidochlor, ansimidol, 6-benzylaminopurine, brassinolide, butaline, chlormequat (chlormequat chloride), choline chloride, cyclanilide, daminozide, dikeglac, dimethipin, 2,6-dimethylprylidine, ethephon, flumetralin, fluprimidol, fluthiacet, forclofenuron, gibberellic acid, inabenfide, indole-3-acetic acid, maleic hydrazide, mefluidide, mepiquat (mepiquat chloride), naphthaleneacetic acid, N-6-benzyladenine, paclobutrazol, prohexadione (prohexadione-calcium), prohydrojasmone, thidiazuron, triapentenol, tributyl phosphorotrithioate, 2,3,5-tri-iodobenzoic acid, trinexapac-ethyl, and uniconazole. Other plant growth regulators that can be incorporated into the seed coating compositions are described in US2012 / 0108431, which is incorporated by reference in its entirety.

[0326] Plant Modifiers The bacterially derived lipid compositions described herein include one or more heterologous plant modifying agents. For example, the bacterially derived lipid composition may encapsulate the heterologous plant modifying agent. Alternatively, or in addition, the heterologous plant modifying agent may be embedded or conjugated to the surface of the bacterially derived lipid composition.

[0327] In some cases, the plant modifier may comprise a peptide or a nucleic acid. The plant modifier may be an agent capable of increasing the fitness of various plants or may be an agent that targets one or more specific plants (e.g., a specific species or genus of plants). Furthermore, in some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different plant modifiers.

[0328] Furthermore, in some cases, heterologous plant modifiers (e.g., agents containing nucleic acid molecules or peptides) can be modified. For example, the modification can be a chemical modification, such as conjugation to a marker, such as a fluorescent marker or a radioactive marker. In other examples, the modification can include conjugation or operational binding to a moiety that enhances the stability, delivery, targeting, bioavailability, or half-life of the agent, such as a lipid, glycan, polymer (e.g., PEG), or cationic moiety.

[0329] Polypeptides The bacterial-derived lipid composition may comprise a polypeptide. In some cases, the bacterial-derived lipid composition comprises a polypeptide or a functional fragment or derivative thereof.

[0330] Exemplary polypeptides can include enzymes (e.g., metabolic recombinases, helicases, integrases, RNAses, DNAses, or ubiquitinating proteins), pore-forming proteins, signaling ligands, cell-penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene-editing proteins (e.g., CRISPR-Cas systems, TALENs, or zinc fingers), riboproteins, protein aptamers, or chaperones.

[0331] The polypeptide may include a naturally occurring polypeptide or a recombinantly produced variant. In some cases, the polypeptide may be a functional fragment or variant thereof (e.g., an enzymatically active fragment or variant thereof). For example, the polypeptide may be a functionally active variant of any of the polypeptides described herein that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of a polypeptide described herein or a naturally occurring polypeptide, e.g., over a specific region or over the entire sequence. In some cases, a polypeptide may have at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identity to a protein of interest.

[0332] Polypeptides may be formulated in bacterial-derived lipid compositions. The compositions disclosed herein may contain any number or type (e.g., class) of polypeptides, such as at least about one of one polypeptide, two, three, four, five, ten, fifteen, twenty, or more polypeptides. The appropriate concentration of each polypeptide in the composition depends on factors such as efficacy, stability of the polypeptide, the number of distinct polypeptides in the composition, and the method of application of the formulation. In some cases, each polypeptide in a liquid composition is about 0.1 ng / mL to about 100 mg / mL. In some cases, each polypeptide in a solid composition is about 0.1 ng / g to about 100 mg / g.

[0333] Methods for producing polypeptides are routine in the art. See generally, Smales & James (Eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press (2005); and Crommelin, Sindelar & Meibohm (Eds.), Pharmaceutical Biotechnology: Fundamentals and Applications, Springer (2013).

[0334] Although methods for producing polypeptides involve expression in plant cells, recombinant proteins can also be produced using insect cells, yeast, bacteria, mammalian cells, or other cells under the control of an appropriate promoter. Mammalian expression vectors can include non-transcribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5' or 3' flanking non-transcribed sequences, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 viral genome, such as SV40 origin, early promoter, enhancer, splice, and polyadenylation sites, can be used to provide other genetic elements required for the expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green & Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (2012).

[0335] Various mammalian cell culture systems can be used to express and produce recombinant polypeptide agents. Examples of mammalian expression systems include CHO cells, COS cells, HeLA, and BHK cell lines. Host cell culture processes for the production of protein therapeutics are described, for example, in Zhou and Kantardjieff (Eds.), Mammalian Cell Cultures for Biologics Manufacturing (Advances in Biochemical Engineering / Biotechnology), Springer (2014). Protein purification is described in Franks, Protein Biotechnology: Isolation, Characterization, and Stabilization, Humana Press (2013); and in Cutler, Protein Purification Protocols (Methods in Molecular Biology), Humana Press (2010). Protein therapeutic formulation is described in Meyer (Ed.), Therapeutic Protein Drug Products: Practical Approaches to Formulation in the Laboratory, Manufacturing, and the Clinic, Woodhead Publishing Series (2012).

[0336] In some cases, the bacterial-derived lipid composition comprises an antibody or an antigen-binding fragment thereof. For example, the agent described herein may be an antibody that blocks or enhances the activity and / or function of a component. The antibody may act as an antagonist or agonist of a polypeptide (e.g., an enzyme or a cellular receptor). The production and use of antibodies against target antigens are known in the art. For antibody engineering, the use of degenerate oligonucleotides, recombinant antibody production methods including 5'-RACE, phage display, and mutagenesis, antibody testing and characterization, antibody pharmacokinetics and pharmacodynamics, antibody purification and storage, and screening and labeling techniques, see, for example, Zhiqiang An (Ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, 1st Edition, Wiley, 2009, and Greenfield (Ed.), Antibodies: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 2013.

[0337] nucleic acid In some cases, the bacterial-derived lipid composition comprises a nucleic acid (polynucleotide). Numerous nucleic acids are useful in the bacterial-derived lipid compositions and methods described herein. The bacterial-derived lipid composition may comprise any number or type (e.g., class) of heterologous nucleic acid (e.g., DNA molecules (e.g., plasmids) or RNA molecules, such as mRNA, guide RNA (gRNA), or inhibitory RNA molecules or precursors thereof (e.g., siRNA, shRNA, or miRNA, or precursors of any of these), or hybrid DNA-RNA molecules, for example, at least about one class or variant of nucleic acid, or 2, 3, 4, 5, 10, 15, 20 or more classes or variants of nucleic acid. The appropriate concentration of each nucleic acid in the composition depends on factors such as efficacy, stability of the nucleic acid, number of distinct nucleic acids, formulation, and method of application of the composition. Examples of nucleic acids useful herein include DNA molecules (e.g., plasmids), mRNA, siRNA, and the like. A, Dicer substrate small interfering RNA (dsiRNA), antisense RNA, small interfering RNA (siRNA) or siRNA precursor (e.g., one or more strands of RNA that hybridize intermolecularly or intramolecularly to form an at least partially double-stranded RNA having at least about 20 consecutive base pairs), small hairpin (shRNA), microRNA (miRNA) or miRNA precursor, asymmetric interfering RNA (aiRNA), peptide nucleic acid (PNA), morpholino, locked nucleic acid (LNA), piwi-interacting RNA (piRNA), ribozyme, deoxyribozyme (DNAzyme), aptamer (DNA, RNA), circular RNA (circRNA), guide RNA (gRNA), or a DNA molecule encoding any of these RNAs.

[0338] Nucleic acid encoding a peptide In some cases, the bacterial lipid composition includes a nucleic acid encoding a polypeptide, the nucleic acid encoding the polypeptide having a length (nt) of about 10 to about 50,000 nucleotides, such as about 25 to about 100 nt, about 50 to about 150 nt, about 100 to about 200 nt, about 150 to about 250 nt, about 200 to about 300 nt, about 250 to about 350 nt, about 300 to about 500 nt, about 10 to about 1000 nt, about 50 to about 1000 nt, about 100 to about 1000 nt, about 1000 to about 2000 nt, about 2000 to about 3000 nt, about 3000 to about 4000 nt, about 4000 to about 5000 nt, or about 5000 nt. It may have an amino acid sequence of from about 10,000 to about 6,000 nt, from about 6,000 to about 7,000 nt, from about 7,000 to about 8,000 nt, from about 8,000 to about 9,000 nt, from about 9,000 to about 10,000 nt, from about 10,000 to about 15,000 nt, from about 10,000 to about 20,000 nt, from about 10,000 to about 25,000 nt, from about 10,000 to about 30,000 nt, from about 10,000 to about 40,000 nt, from about 10,000 to about 45,000 nt, from about 10,000 to about 50,000 nt, or any range therebetween.

[0339] The bacterial lipid composition may also comprise an active variant of the nucleic acid sequence of interest. In some cases, the nucleic acid variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the nucleic acid sequence of interest, for example, across a specified region or across the entire sequence. In some cases, the bacterial lipid composition comprises an active polypeptide encoded by the nucleic acid variant. In some cases, an active polypeptide encoded by a nucleic acid variant has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a subject or naturally-occurring polypeptide sequence, e.g., over a specified region or over the entire amino acid sequence.

[0340] A specific method for expressing a nucleic acid encoding a protein may involve expression in cells, including insects, yeast, plants, bacteria, or other cells, under the control of an appropriate promoter. Expression vectors may include non-transcribed elements such as origins of replication, suitable promoters and enhancers, and other 5' or 3' adjacent non-transcribed sequences, as well as 5' or 3' non-translated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and termination sequences. DNA sequences derived from the SV40 virus genome, such as SV40 origin, early promoter, enhancer, splice, and polyadenylation sites, may be used to provide other genetic elements required for the expression of heterologous DNA sequences. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Green et al., Molecular Cloning: A Laboratory Manual, Fourth Edition, Cold Spring Harbor Laboratory Press, 2012.

[0341] Genetic modification using recombinant methods is generally known in the art.The nucleic acid sequence encoding desired gene can be obtained using recombinant methods known in the art, such as screening a library from cells that express the gene, extracting the gene from a vector that is known to contain the gene, or using standard techniques to directly isolate it from the cells and tissues that contain it.Alternatively, the gene of interest can be produced synthetically instead of being cloned.

[0342] The expression of natural or synthetic nucleic acids is typically achieved by operably linking the nucleic acid encoding the gene of interest to a promoter and incorporating the construct into an expression vector. The expression vector may be suitable for replication and expression in bacteria. The expression vector may also be suitable for replication and integration in eukaryotes. A typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters useful for expressing the desired nucleic acid sequence.

[0343] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 base pairs (bp) upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that promoter function is retained when elements are inverted or moved relative to one another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription.

[0344] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can confer high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is elongation growth factor 1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0345] Alternatively, the promoter may be an inducible promoter. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired, or can turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0346] The expression vector to be introduced can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells to be transfected or infected with the viral vector. In other embodiments, the selectable marker can be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0347] Reporter genes can be used to identify potentially transformed cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that encode polypeptides that are absent from or expressed by the recipient source, and whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., FEBS Letters 479:79-82, 2000). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. Generally, the construct with the smallest 5'-flanking region that exhibits the highest level of reporter gene expression is identified as the promoter. Such promoter regions may be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0348] In some cases, an organism may be genetically modified to alter the expression of one or more proteins. The expression of one or more proteins may be modified for a specific time, for example, during the developmental or differentiation state of the organism. In one example, the invention includes compositions for altering the expression of one or more proteins, for example, proteins that affect activity, structure, or function. The expression of one or more proteins may be restricted to a specific location(s) or may be widespread throughout the organism.

[0349] mRNA The bacterial-derived lipid composition may comprise mRNA molecules, for example, mRNA molecules that encode polypeptides.The mRNA molecules can be synthesized and modified, for example, chemically modified.The mRNA molecules can be chemically synthesized or transcribed in vitro.The mRNA molecules can be placed on a plasmid, for example, a viral vector, a bacterial vector, or a eukaryotic expression vector.In some examples, the mRNA molecules can be delivered to cells by transfection, electroporation, or transduction (for example, adenovirus or lentivirus transduction).

[0350] In some cases, the modified RNA agent described herein has modified nucleosides or nucleotides.Such modifications are known and are described, for example, in International Publication No. 2012 / 019168.Additional modifications are described, for example, in International Publication No. 2015 / 038892, International Publication No. 2015 / 038892, International Publication No. 2015 / 089511, International Publication No. 2015 / 196130, International Publication No. 2015 / 196118 and International Publication No. 2015 / 196128 A2, which are incorporated herein by reference in their entirety.

[0351] In some cases, the modified RNA encoding the polypeptide of interest has one or more terminal modifications, such as a 5' cap structure and / or a poly-A tail (e.g., 100-200 nucleotides in length). The 5' cap structure may be selected from the group consisting of CapO, Capl, ARCA, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some cases, the modified RNA also contains a 5' UTR and a 3' UTR that contain at least one Kozak sequence. Such modifications are known and are described, for example, in International Publication Nos. WO 2012 / 135805 and WO 2013 / 052523, which are incorporated herein by reference in their entireties. Additional termination modifications are described, for example, in WO 2014 / 164253 and WO 2016 / 011306, WO 2012 / 045075, and WO 2014 / 093924, which are incorporated by reference in their entireties. Chimeric enzymes for synthesizing capped RNA molecules (e.g., modified mRNAs) that may contain at least one chemical modification are described in WO 2014 / 028429, which is incorporated by reference in its entirety.

[0352] In some cases, the modified mRNA may be circularized or ligated to generate a translationally competent molecule that supports the interaction between polyA-binding protein and 5'-end binding protein. The mechanism of circularization or ligation can occur via at least three different routes: 1) chemical, 2) enzymatic, and 3) ribozyme-catalyzed. The newly formed 5'- / 3'-bond may be intramolecular or intermolecular. Such modifications are known and are described, for example, in WO 2013 / 151736.

[0353] The method of producing and purifying modified RNA is known and disclosed in the art.For example, modified RNA is produced only by in vitro transcription (IVT) enzyme synthesis.The method of producing IVT polynucleotide is known in the art and is described in International Publication No. WO2013 / 151666, WO2013 / 151668, WO2013 / 151663, WO2013 / 151669, WO2013 / 151670, WO2013 / 151664, WO2013 / 151665, WO2013 / 151671, WO2013 / 151672, WO2013 / 151667 and WO2013 / 151736. Purification methods include purifying RNA transcripts containing poly-A tails by contacting the sample with a surface linked to multiple thymidines or derivatives thereof and / or multiple uracils or derivatives thereof (polyT / U) under conditions such that the RNA transcripts bind to the surface, and eluting the purified RNA transcripts from the surface using ion (e.g., anion) exchange chromatography (WO 2014 / 152031), which allows for the separation of longer RNAs up to 10,000 nucleotides in length via a scalable method (WO 2014 / 144767), and subjecting the modified mRNA sample to DNAse treatment (WO 2014 / 152030).

[0354] Formulations of modified RNA are known and are described, for example, in International Publication No. 2013 / 090648. For example, the formulation may be, but is not limited to, nanoparticles, poly(lactic-co-glycolic acid) (PLGA) microparticles, lipidoids, lipoplexes, liposomes, polymers, carbohydrates (including simple sugars), cationic lipids, fibrin gels, fibrin hydrogels, fibrin glues, fibrin sealants, fibrinogen, thrombin, rapidly cleared lipid nanoparticles (reLNPs), and combinations thereof.

[0355] Modified RNAs encoding polypeptides in the fields of human disease, antibodies, viruses, and various in vivo settings are known and are disclosed, for example, in Table 6 of WO 2013 / 151666, WO 2013 / 151668, WO 2013 / 151663, WO 2013 / 151669, WO 2013 / 151670, WO 2013 / 151664, WO 2013 / 151665, WO 2013 / 151736, Tables 6 and 7 of WO 2013 / 151672, Tables 6, 178 and 179 of WO 2013 / 151671, and Tables 6, 185 and 186 of WO 2013 / 151667. Any of the foregoing may be synthesized as an IVT polynucleotide, a chimeric polynucleotide or a circular polynucleotide, each of which may contain one or more modified nucleotides or terminal modifications.

[0356] inhibitory RNA In some cases, the bacterial-derived lipid composition contains an inhibitory RNA molecule, e.g., an inhibitory RNA molecule that acts via the RNA interference (RNAi) pathway. In some cases, the inhibitory RNA molecule reduces the level of gene expression and / or reduces protein levels. In some cases, the inhibitory RNA molecule inhibits gene expression. For example, the inhibitory RNA molecule may include a small interfering RNA or its precursor, a small hairpin RNA, and / or a microRNA or its precursor that targets a gene. Certain RNA molecules can inhibit gene expression through the biological process of RNA interference (RNAi). RNAi molecules typically contain 15-50 base pairs (e.g., about 18-25 base pairs) and include RNA or RNA-like structures with nucleobase sequences identical (or complementary) or nearly identical (or substantially complementary) to the coding sequence of a target gene expressed in a cell. RNAi molecules include, but are not limited to, short interfering RNA (siRNA), double-stranded RNA (dsRNA), small hairpin RNA (shRNA), meroduplex, Dicer substrate, and multivalent RNA interference (U.S. Patent Nos. 8,084,599, 8,349,809, 8,513,207, and 9,200,276). shRNA is an RNA molecule containing a hairpin turn that reduces the expression of a target gene through RNAi. shRNA can be delivered to cells in the form of a plasmid, e.g., a viral vector, or a bacterial vector, for example, by transfection, electroporation, or transduction. MicroRNAs are non-coding RNA molecules typically about 21 or 22 nucleotides in length. MiRNAs bind to target sites on mRNA molecules and silence mRNA, for example, by causing mRNA cleavage, mRNA destabilization, or inhibition of mRNA translation. In some cases, inhibitory RNA molecules reduce the level and / or activity of negative functional regulators. In other cases, the inhibitory RNA molecule reduces the level and / or activity of a positive functional regulator. Inhibitory RNA molecules can be chemically synthesized or transcribed in vitro.

[0357] In some cases, the nucleic acid is DNA, RNA, or PNA. In some cases, the RNA is an inhibitory RNA. In some cases, the inhibitory RNA molecule inhibits gene expression. In some cases, the nucleic acid is an mRNA, modified mRNA, or DNA molecule that increases the expression of an enzyme (e.g., metabolic recombinase, helicase, integrase, RNAse, DNAse, or ubiquitinating protein), a pore-forming protein, a signaling ligand, a cell-penetrating peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene-editing protein (e.g., CRISPR-Cas system, TALEN, or zinc finger), a riboprotein, a protein aptamer, or a chaperone. In some cases, the nucleic acid is an mRNA, modified mRNA, or DNA molecule that increases expression of an enzyme (e.g., a metabolic enzyme, a recombinase enzyme, a helicase enzyme, an integrase enzyme, an RNAse enzyme, a DNAse enzyme, or a ubiquitinating protein), a pore-forming protein, a signaling ligand, a cell-penetrating peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene-editing protein (e.g., a CRISPR-Cas system, a TALEN, or a zinc finger), a riboprotein, a protein aptamer, or a chaperone. In some embodiments, the nucleic acid encodes an enzyme, a pore-forming protein, a signaling ligand, a cell-penetrating peptide, a transcription factor, a receptor, an antibody, a nanobody, a gene-editing protein, a riboprotein, a protein aptamer, or a chaperone. In some cases, the increase in expression is about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% increase in expression compared to a reference level (e.g., expression in an untreated subject). In some cases, the increase in expression is about 2-fold, about 4-fold, about 5-fold, about 10-fold, about 20-fold, about 25-fold, about 50-fold, about 75-fold, or about 100-fold or more increase in expression compared to a reference level (e.g., expression in an untreated subject).

[0358] In some cases, the nucleic acid is an antisense RNA, dsiRNA, siRNA, shRNA, miRNA, aiRNA, PNA, morpholino, LNA, piRNA, ribozyme, DNAzyme, aptamer (DNA, RNA), circRNA, gRNA, or DNA molecule (e.g., a plasmid) that acts to reduce expression of, for example, an enzyme (metabolic enzyme, recombinase enzyme, helicase enzyme, integrase enzyme, RNAse enzyme, DNAse enzyme, polymerase enzyme, ubiquitinating protein, superoxide management enzyme, or energy-producing enzyme), transcription factor, secreted protein, structural factor (actin, kinesin, or tubulin), riboprotein, protein aptamer, chaperone, receptor, signaling ligand, or transporter. In some cases, the decrease in expression is about a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than a 100% decrease in expression compared to a reference level (e.g., expression in an untreated subject). In some cases, the decrease in expression is about a 2-fold, 4-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, or 100-fold or more decrease in expression compared to a reference level (e.g., expression in an untreated subject).

[0359] RNAi molecules contain sequences that are substantially complementary or completely complementary to all or a fragment of a target gene. RNAi molecules can complement the sequence at the boundary between introns and exons, preventing the newly generated nuclear RNA transcripts of a specific gene from maturing into mRNA due to transcription. RNAi molecules that are complementary to a specific gene can hybridize with the mRNA of the target gene and prevent its translation. Antisense molecules can be DNA, RNA, or their derivatives or hybrids. Examples of such derivative molecules include, but are not limited to, peptide nucleic acid (PNA) and phosphorothioate-based molecules such as deoxyribonucleic acid guanidine (DNG) or ribonucleic acid guanidine (RNG).

[0360] RNAi molecules can be provided as ready-to-use RNA synthesized in vitro, or as the sense and antisense RNA sequences (or the DNA encoding the sense and antisense RNA sequences) transfected into cells, which produce RNAi molecules upon transcription.The hybridization of RNA molecules with, for example, target mRNA leads to the degradation of hybridized complexes by RNAse H and / or the inhibition of the formation of translation complexes.Neither of these can produce the product of the original gene.

[0361] The length of the RNAi molecule that hybridizes to the target transcript can be about 10 nucleotides, about 15 nucleotides, or 30 nucleotides, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or more. In embodiments, the RNAi molecule hybridizes to the target transcript to form a complete or nearly complete double-stranded region of at least about 17 base pairs, and in embodiments, the double-stranded region comprises at least about 10 consecutive base pairs. The degree of identity of the antisense sequence to the target transcript can be at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0362] RNAi molecules may also contain overhangs, i.e., typically unpaired overhanging nucleotides, which are not directly involved in the double helix structure normally formed by the core sequences of the paired sense and antisense strands defined herein. RNAi molecules may contain 3' and / or 5' overhangs of about 1 to 5 bases independently on each of the sense and antisense strands. In some cases, both the sense and antisense strands contain 3' and 5' overhangs. In some cases, one or more of the 3' overhanging nucleotides of one strand are base-paired with one or more of the 5' overhanging nucleotides of the other strand. In other cases, one or more of the 3' overhanging nucleotides of one strand are not base-paired with one or more of the 5' overhanging nucleotides of the other strand. The sense and antisense strands of an RNAi molecule may or may not contain the same number of nucleotide bases. The antisense and sense strands may form a duplex, with the 5' end having only blunt ends, the 3' end having only blunt ends, both the 5' and 3' ends being blunt, or neither the 5' nor the 3' end being blunt. In other cases, one or more of the nucleotides in the overhang contain thiophosphate, phosphorothioate, deoxynucleotide inverted (3' to 3' linked) nucleotides, or are modified ribonucleotides or deoxynucleotides.

[0363] Small interfering RNA (siRNA) molecules contain a nucleotide sequence identical to about 15 to about 25 consecutive nucleotides of a target mRNA. In some cases, the siRNA sequence begins with the dinucleotide AA, contains about 30-70% (about 30-60%, about 40-60%, or about 45-55%) GC content, and does not share a high percentage of identity with any nucleotide sequence other than the target in the genome to be introduced, as determined, for example, by a standard BLAST search.

[0364] siRNA and shRNA resemble intermediates in the processing pathway of endogenous microRNA (miRNA) genes (Bartel, Cell 116:281-297, 2004). In some cases, siRNA may function as miRNA, and vice versa (Zeng et al., Mol. Cell 9:1327-1333, 2002; Doench et al., Genes Dev. 17:438-442, 2003). Exogenous siRNA downregulates mRNA with seed complementarity to siRNA (Birmingham et al., Nat. Methods 3:199-204, 2006). Multiple target sites within the 3'UTR result in stronger downregulation (Doench et al., Genes Dev. 17:438-442, 2003).

[0365] Known effective siRNA sequences and cognate binding sites are also well represented in the relevant literature. RNAi molecules are readily designed and generated by techniques known in the art. Furthermore, computational tools exist that can improve the likelihood of finding effective and specific sequence motifs (Pei et al., Nat. Methods 3(9):670-676, 2006; Reynolds et al., Nat. Biotechnol. 22(3):326-330, 2004; Khvorova et al., Nat. Struct. Biol. 10(9):708-712, 2003; Schwarz et al., Cell 115(2):199-208, 2003; Ui-Tei et al., Nucleic Acids Res. 32(3):936-948, 2004; Heale et al., Nucleic Acids Res. 33(3):e30, 2005; Chalk et al. al.,Biochem.Biophys.Res.Commun.319(1):264-274,2004; and Amarzguioui et al.,Biochem.Biophys.Res.Commun.316(4):1050-1058,2004).

[0366] RNAi molecules regulate the expression of the RNA coded by genes.Since multiple genes can share a certain degree of sequence homology with each other, in some cases, RNAi molecules can be designed to target a class of genes that have sufficient sequence homology.In some cases, RNAi molecules can contain sequences that are shared between different gene targets or have complementary sequences that are unique to specific gene targets.In some cases, RNAi molecules can be designed to target the conserved region of RNA sequences that have homology between several genes, thereby targeting several genes in a gene family (for example, different gene isoforms, splice variants, mutant genes, etc.).In some cases, RNAi molecules can be designed to target sequences that are unique to the specific RNA sequence of a single gene.

[0367] Inhibitory RNA molecules can be modified to contain, for example, modified nucleotides, such as 2'-fluoro, 2'-o-methyl, 2'-deoxy, unlocked nucleic acid, 2'-hydroxy, phosphorothioate, 2'-thiouridine, 4'-thiouridine, 2'-deoxyuridine. Without being bound by theory, it is believed that such modifications can increase nuclease resistance and / or serum stability or reduce immunogenicity.

[0368] In some cases, RNAi molecule or its precursor is linked to delivery polymer via physiologically unstable bond or linker.Physiologically unstable linker is selected to undergo chemical transformation (for example, cleavage) when it exists in certain physiological conditions (for example, disulfide bond is cleaved in the reducing environment of cytoplasm).The release of molecule from polymer by cleavage of physiologically unstable bond promotes the molecule to interact with appropriate cellular components for activity.

[0369] An RNAi molecule polymer conjugate can be formed by covalently linking a molecule to a polymer. The polymer is polymerized or modified to contain reactive group A. The RNAi molecule is also polymerized or modified to contain reactive group B. Reactive groups A and B are selected so that they can be linked via a reversible covalent bond using methods known in the art.

[0370] The conjugation of RNAi molecules to polymers can be carried out in the presence of excess polymer.RNAi molecules and polymers can be oppositely charged during conjugation, and the presence of excess polymer can reduce or eliminate the aggregation of conjugates.Alternatively, excess carrier polymers such as polycations can be used.Excess polymers can be removed from the conjugated polymer before the administration of conjugates.Alternatively, excess polymers can be administered simultaneously with conjugates.

[0371] The production and use of inhibitors based on non-coding RNAs, such as ribozymes, RNAse P, siRNA, and miRNA, are known in the art, as described, for example, in Sioud, RNA Therapeutics: Function, Design, and Delivery (Methods in Molecular Biology). Humana Press (2010).

[0372] Gene editing The bacterial lipid composition can contain components of a gene editing system. For example, the agent can introduce modifications (such as insertions, deletions (e.g., knockouts), translocations, inversions, point mutations, or other mutations) into genes. Exemplary gene editing systems include zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR) systems. ZFN, TALEN, and CRISPR-based methods are described, for example, in Gaj et al., Trends Biotechnol 31(7):397-405, 2013.

[0373] Further description of the components and processes of the gene editing system can be found in International Patent Application Publication No. WO 2021 / 041301, which is incorporated herein by reference in its entirety.

[0374] Xenotherapy The bacterially-derived lipid compositions can include therapeutic agents (e.g., agents that affect an animal (e.g., a mammal, e.g., a human), an animal pathogen, or its pathogen vector, such as a therapeutic peptide, a therapeutic nucleic acid (e.g., a therapeutic RNA), a therapeutic small molecule, or a pathogen control agent (e.g., an antifungal, antibacterial, virucide, antiviral, insecticide, nematicide, antiparasitic, or insect repellent). Such agent-loaded bacterially-derived lipid compositions can be formulated with a pharmaceutically acceptable carrier for delivery to the animal, animal pathogen, or its pathogen vector. antibacterial agents

[0375] The bacterial-derived lipid composition can further comprise an antimicrobial agent. For example, a bacterial-derived lipid composition comprising an antibiotic can be administered to an animal in an amount and for a time sufficient to achieve a target level (e.g., a predetermined level or threshold level) of antibiotic concentration in or on the animal and / or treat or prevent bacterial infection in the animal. The antimicrobial agent can be formulated in the bacterial-derived lipid composition for any of the methods described herein, and in certain cases, can be associated with the bacterial-derived lipid composition. In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antimicrobial agents.

[0376] As used herein, the term "antimicrobial agent" refers to a substance that kills or inhibits the growth, proliferation, division, reproduction, or spread of bacteria, such as plant pathogenic bacteria, and includes a bactericide (e.g., a disinfectant compound, an antiseptic compound, or an antibiotic) or a bacteriostatic agent (e.g., a compound or antibiotic). Bactericidal antibiotics kill bacteria, while bacteriostatic antibiotics only slow the growth or reproduction of bacteria.

[0377] Exemplary fungicides include: Examples include those already discussed above with regard to antimicrobial agents in the "Heterogeneous Agricultural Formulations" section.

[0378] As antiseptics (i.e., disinfectants that can be used on the human or animal body, skin, mucous membranes, wounds, etc.), some of the disinfectants mentioned above can be used under appropriate conditions (mainly concentration, pH, temperature and toxicity to humans / animals). Among other things, the following are important: appropriately diluted chlorine preparations (i.e., Dacan solution, 0.5% sodium or potassium hypochlorite solution, pH adjusted to 7-8, or 0.5-1% sodium benzenesulfochloramide solution (chloramine B)); some iodine preparations, such as iodopovidone in various galenics (ointments, solutions, wound dressings), formerly in Lugol's solution; peroxide as urea perhydrate solution and pH-buffered 0.1-0.25% peracetic acid solution; alcohol with or without preservatives, primarily used for skin disinfection; weak organic acids, such as sorbic acid, benzoic acid, lactic acid, and salicylic acid; some phenolic compounds, such as hexachlorophene, triclosan, and dibromomethyl; and cationically active compounds, such as 0.05-0.5% benzalkonium, 0.5-4% chlorhexidine, and 0.1-2% octenidine solutions.

[0379] The bacterial-derived lipid composition may include an antibiotic. Any antibiotic known in the art may be used. Antibiotics are generally classified based on their mechanism of action, chemical structure, or spectrum of activity.

[0380] The antibiotics described herein may target any bacterial function or growth process and may be either bacteriostatic (e.g., slow or prevent bacterial growth) or bactericidal (e.g., kill bacteria). In some cases, the antibiotic is a bactericidal antibiotic. In some cases, the bactericidal antibiotic is an antibiotic that targets the bacterial cell wall (e.g., penicillin and cephalosporin), an antibiotic that targets the cell membrane (e.g., polymyxin), or an antibiotic that inhibits essential bacterial enzymes (e.g., rifamycin, lipiarmycin, quinolones, and sulfonamides). In some cases, the bactericidal antibiotic is an aminoglycoside (e.g., kasugamycin). In some cases, the antibiotic is a bactericidal antibiotic. In some cases, the bacteriostatic antibiotic targets protein synthesis (e.g., macrolides, lincosamides, and tetracyclines). Additional classes of antibiotics that can be used herein include cyclic lipopeptides (such as daptomycin), glycylcyclines (such as tigecycline), oxazolidinones (such as linezolid), or lipiarmycins (such as fidaxomicin). Examples of antibiotics include rifampicin, ciprofloxacin, doxycycline, ampicillin, and polymyxin B. The antibiotics described herein can have any level of target specificity (e.g., narrow spectrum or broad spectrum). In some cases, the antibiotic is a narrow-spectrum antibiotic, thus targeting a specific type of bacteria, such as gram-negative or gram-positive bacteria. Alternatively, the antibiotic may be a broad-spectrum antibiotic that targets a wide range of bacteria.

[0381] Examples of antibacterial agents suitable for treating animals include penicillins (amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mesocricillin, nafcillin, oxacillin, penicillin G, Cristicillin 300 AS, Pentys, Permanent, Faizelpen, Faizelpen-AS, Wicillin, penicillin V, piperacillin, pivampicillin, pivomecillinam, ticarcillin), cephalosporins (cefastril (cefastril), cefadroxil (cefadroxil), cephalexin (cephalexin), cephaloglycin (cephaloglycin), cephalonium (cephalonium), cephaloridine (cephalorazine), cephalothin (cephalothin), cephapirin ( Cephapirin), cefatrizine, cefazaflour, cefazedone, cefazolin (cefazolin), cephradine (cephradine), cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil (cefproxil), cefuroxime, cefuzonam, cefcapene, cefdaroxime, cefdinir, cefeditoren, cefetamet, cefixime, cefmenoxime, cefozizime, cefotaxime , cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime, cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, ceftobiprole, ceftaroline, cefaclomezine, cephaloram, cefaparole, cefcanel, cefedrole, cefempidone, cefetrizole, cefibitril, cefmatilen, cefmepidium, Cefovecin, cefoxazole, cefrotil, cefsumide, cefuracetim, ceftioxid, combinations, ceftazidime / avibactam, ceftarozane / tazobactam), monobactams (aztreonam), carbapenems (imipenem, imipenem / cilastatin, doripenem, ertapenem, meropenem, meropenem / vaborbactam), macrolides (azithromycin, erythromycin, clarithromycin, dirithromycin, roxithromycin, telitromycin),Lincosamides (clindamycin, lincomycin), streptogramins (pristinamycin, quinupristin / dalfopristin), aminoglycosides (amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, tobramycin), quinolones (flumequine, nalidixic acid, oxyphosphate, piromidic acid, pipemidic acid, rosoxacin, second generation, ciprofloxacin) enoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, besifloxacin, delafloxacin, clinafloxacin, gemifloxacin, prulifloxacin, sitaf These include loxacin, trovafloxacin), sulfonamides (sulfamethizole, sulfamethoxazole, sulfisoxazole, trimethoprim-sulfamethoxazole), tetracyclines (demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, tigecycline), and others (lipopeptides, fluoroquinolones, lipoglycopeptides, cephalosporins, macrocyclics, chloramphenicol, metronidazole, tinidazole, nitrofurantoin, glycopeptides, vancomycin, teicoplanin, lipoglycopeptides, telavancin, oxazolidinone, linezolid, cycloserine 2, rifamycin, rifampin, rifabutin, rifapentine, rifalazil, polypeptides, bacitracin, polymyxin B, tuberactinomycin, viomycin, capreomycin).

[0382] Those skilled in the art will understand that the appropriate concentration of each antibiotic in the composition will depend on factors such as antibiotic efficacy, stability, number of distinct antibiotics, formulation, and method of application of the composition.

[0383] antifungal agents The bacterial-derived lipid composition described herein can further comprise an antifungal agent.For example, the bacterial-derived lipid composition comprising an antifungal agent can be administered to an animal in an amount and for a time sufficient to reach a target level (e.g., a predetermined level or threshold level) of the antifungal drug concentration in or on the animal and / or to treat or prevent fungal infection in the animal.The antifungal agent described herein can be formulated in the bacterial-derived lipid composition for any of the methods described herein, and in certain cases, can be associated with the bacterial-derived lipid composition.In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antifungal agents.

[0384] As used herein, the term "fungicide" or "antifungal agent" refers to a substance that kills or inhibits the growth, proliferation, division, reproduction, or spread of fungi, including fungi that are pathogenic to animals. Many different types of antifungal agents are commercially available. Non-limiting examples of antifungal agents include allylamines (amoroline, butenafine, naftifine, terbinafine), imidazoles (bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, ketoconazole, isoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, tioconazole, terconazole), triazoles (albaconazole, efinaconazole, fluconazole, isabconazole, itraconazole, posaconazole, laconazole, lanofungal ... Antifungal agents include buconazole, terconazole, voriconazole, thiazoles (abafungin), polyenes (amphotericin B, nystatin, natamycin, trichomycin), echinocandins (anidulafungin, caspofungin, micafungin), and others (tolnaftate, flucytosine, butenafine, griseofulvin, ciclopirox, selenium sulfide, tavaborole). One of ordinary skill in the art will recognize that the appropriate concentration of each antifungal agent in the composition will depend on factors such as the efficacy and stability of the antifungal agent, the number of distinct antifungal agents, the formulation, and the method of application of the composition.

[0385] insecticides The bacterial lipid composition may further comprise an insecticide. For example, the insecticide can reduce the fitness (e.g., reduce growth or kill) of an insect vector of an animal pathogen. The bacterial lipid composition comprising the insecticide can be contacted with an insect in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of insecticide concentration in or on the insect, and (b) reduce the fitness of the insect. In some cases, the insecticide can reduce the fitness (e.g., reduce growth or kill) of a parasitic insect. The bacterial lipid composition comprising the insecticide can be contacted with a parasitic insect, or an animal infected therewith, in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined level or threshold level) of insecticide concentration in or on the parasitic insect, and (b) reduce the fitness of the parasitic insect. The insecticides described herein may be formulated in bacterial lipid compositions for any of the methods described herein, and in certain cases, may be associated with bacterial lipid compositions. In some cases, the bacterially derived lipid composition includes two or more (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different pesticides.

[0386] As used herein, the term "insecticide" or "pesticide" refers to a substance that kills or inhibits the growth, growth, reproduction, or transmission of insects, such as insect vectors of animal pathogens or parasitic insects. Non-limiting examples of insecticides are set forth in Table 4 of WO 2021 / 041301, which is incorporated herein by reference in its entirety. Further non-limiting examples of suitable insecticides include those already discussed above in connection with insecticides in the "Heterogeneous Agricultural Formulations" section. One of ordinary skill in the art will understand that the appropriate concentration of each insecticide in the composition will depend on factors such as insecticide efficacy, stability, the number of separate insecticides, the formulation, and the method of application of the composition.

[0387] nematicides The bacterially derived lipid composition may further comprise a nematicide as a therapeutic agent. Non-limiting examples of nematicides used as therapeutic agents include those already discussed above in relation to nematicides in the "Heterologous Agricultural Formulations" section.

[0388] antiparasitic agents The bacterial lipid composition may further comprise an antiparasitic agent. For example, the antiparasitic agent can reduce the fitness of parasitic protozoa (e.g., reduce growth or kill). The bacterial lipid composition comprising the antiparasitic agent described herein can be contacted with a protozoa in an amount and for a time sufficient to (a) reach a target level (e.g., a predetermined level or threshold level) of the antiparasitic agent concentration in or on the protozoa or an animal infected with the protozoa, and (b) reduce the fitness of the protozoa. This can be useful for treating or preventing parasites in animals. For example, the bacterial lipid composition comprising the antiparasitic agent described herein can be administered to an animal in an amount and for a time sufficient to reach a target level (e.g., a predetermined level or threshold level) of the antiparasitic agent concentration in or on the animal, and / or treat or prevent parasitic (e.g., parasitic nematode, parasitic insect, or protozoa) infection in the animal. The antiparasitic agents described herein may be formulated in, and in certain cases associated with, a bacterially-derived lipid composition for any of the methods described herein. In some cases, the bacterially-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antiparasitic agents.

[0389] As used herein, the term "antiparasitic agent" or "anti-parasitic agent" refers to a substance that kills or inhibits the growth, proliferation, reproduction, or spread of parasites, such as parasitic protozoa, parasitic nematodes, or parasitic insects. Examples of antiparasitic agents include anthelmintics (bephenium, diethylcarbamazine, ivermectin, niclosamide, piperazine, praziquantel, pyrantel, pyrvinium, benzimidazole, albendazole, flubendazole, mebendazole, thiadiabendazole, levamisole, nitazoxanide, monopantel, emodepside, spiroindole), scabicides (benzyl benzoate, benzyl benzoate / disulfiram), and the like. Antiparasitic drugs include liceicides (piperonyl butoxide / pyrethrin, spinosad, moxidectin), scabicides (crotamiton), tapeworm insecticides (niclosamide, praziquantel, albendazole), amoebicides (rifampin, amphotericin B), or antiprotozoal drugs (melarsoprol, eflornithine, metronidazole, tinidazole, miltefosine, artemisinin). In certain cases, antiparasitic drugs such as levamisole, fenbendazole, oxfendazole, albendazole, moxidectin, eprinomectin, doramectin, ivermectin, or clorsulon may be used to treat or prevent infection in livestock animals. Those skilled in the art will understand that the appropriate concentration of each antiparasitic agent in the composition will depend on factors such as antiparasitic agent efficacy, stability, the number of distinct antiparasitic agents, the formulation, and the method of application of the composition.

[0390] antiviral agents The bacterial-derived lipid composition may further comprise an antiviral agent.The bacterial-derived lipid composition comprising an antiviral agent can be administered to an animal in an amount and for a time sufficient to reach a target level (e.g., a predetermined level or threshold level) of antiviral concentration in or on the animal and / or treat or prevent viral infection in the animal.The antiviral agent described herein may be formulated in the bacterial-derived lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterial-derived lipid composition.In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different antiviral agents.

[0391] As used herein, the term "antiviral agent" or "antiviral agent" refers to a substance that kills or inhibits the growth, proliferation, reproduction, development, or spread of viruses, such as viral pathogens that infect animals. Many agents can be used as antiviral agents, including chemical or biological agents (e.g., nucleic acids, e.g., dsRNA). Examples of antiviral agents useful herein include abacavir, acyclovir (acyclovir), adefovir, amantadine, amprenavir (agenase), ampligen, arbidol, atazanavir, atripla, baravir, cidofovir, combivir, dolutegravir, darunavir, delavirdine, dianosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir, ecolieber, famciclovir, fomivirsen, fosamprenavir, foscarnet, phosphonet, fusion inhibitors, ganciclovir, ibacitabine, immunovir, idoxuridine, imiquimod, indinavir, inosine, integration inhibitors, interferon type III, interferon type II, interferon type I, interferon, lamivudine , lopinavir, loviride, maraviroc, moroxydine, methisazone, nelfinavir, nevirapine, nexavir, nitazoxanide, nucleoside analogues, norvir, oseltamivir (Tamiflu), peginterferon alfa-2a, penciclovir, peramivir, pleconaril, podophyllotoxin, lutegravir, ribavirin, rimantadine, ritonavir, pyramidine, saquinavir , sofosbuvir, stavudine, synergistic enhancer (antiretroviral), telaprevir, tenofovir, tenofovir disoproxil, tipranavir, trifluridine, trizivir, tromantadine, Truvada, valacyclovir (Valtrex), valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine, zanamivir (Relenza), or zidovudine. One skilled in the art will understand that the appropriate concentration of each antiviral agent in the composition will depend on factors such as the efficacy and stability of the antiviral agents, the number of distinct antiviral agents, the formulation, and the method of application of the composition.

[0392] Antidote The bacterial-derived lipid composition may further comprise a repellent. For example, the repellent can repel vectors of animal pathogens, such as insects. The repellents described herein may be formulated in the bacterial-derived lipid composition for any of the methods described herein, and in certain cases, may be associated with the bacterial-derived lipid composition. In some cases, the bacterial-derived lipid composition comprises two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10) different repellents.

[0393] For example, a bacterially-derived lipid composition containing a repellent described herein can be contacted with an insect vector or vector habitat in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined or threshold level) of repellent concentration and / or (b) reduce the level of insects near or on a nearby animal compared to a control. Alternatively, a bacterially-derived lipid composition containing a repellent described herein can be contacted with an animal in an amount and for a time sufficient to (a) achieve a target level (e.g., a predetermined or threshold level) of repellent concentration and / or (b) reduce the level of insects near or on the animal compared to an untreated animal.

[0394] Some examples of well-known insect repellents include benzyl, benzyl benzoate, 2,3,4,5-bis(butyl-2-ene)tetrahydrofurfuran (MGK repellent 11), butoxypolypropylene glycol, N-butylacetanilide, normal-butyl-6,6-dimethyl-5,6-dihydro-1,4-pyrone-2-carboxylate (Indalone), dibutyl adipate, dibutyl phthalate, di-normal-butylsuccinate (Tabatrex), N,N-diethyl-meta-toluamide (DEET), dimethylcarbate (endo,endo)-dimethylbicyclo[2.2.1]hept-5-ene-2,3-dicarboxylate), dimethyl phthalate, 2-ethyl-2-butyl-1,3-propanediol, 2-ethyl-1,3-hexanediol (Rutgers 612), di-normal-propyl isotinochromelonate (MGK repellent 326), 2-phenylcyclohexanol, p-methane-3,8-diol, and normal-propyl N,N-diethylsuccinamate. Other repellents include citronella oil, dimethyl phthalate, normal-butyl mesityl oxide oxalate, and 2-ethylhexanediol-1,3 (see Kirk-Othmer Encyclopedia of Chemical Technology, 2nd Ed., Vol. 11:724-728, and The Condensed Chemical Dictionary, 8th Ed., p 756).

[0395] In some cases, the repellent is an insect repellent, including synthetic and non-synthetic insect repellents. Examples of synthetic insect repellents include methyl anthranilate and other anthranilic acid-based repellents, benzaldehyde, DEET (N,N-diethyl-m-toluamide), dimethylcarbate, dimethyl phthalate, icaridin (i.e., picaridin, Bayrepel, and KBR 3023), indalone (e.g., IR3535 (3-[N-butyl-N-acetyl]-aminopropionic acid, ethyl ester), used in the "6-2-2" mixture (60% dimethyl phthalate, 20% indalone, 20% ethyl hexanediol)), metofluthrin, permethrin, SS220, or tricyclodecenyl allyl ether. Examples of natural insect repellents include beautyberry (Callicarpa) leaves, birch bark, port willow (Salix alba), catnip oil (e.g., nepetalactone), citronella oil, and essential oils of the Lemon eucalyptus (Corymbopsis citriola, e.g., p-menthane-3,8-diol (PMD)), neem oil, lemongrass, tea tree oil from Melaleuca alternifolia leaves, tobacco, or extracts thereof.

[0396] Uses of bacterially derived lipid compositions The bacterially derived lipid compositions are useful in a variety of agricultural or therapeutic applications. Exemplary methods of using the bacterially derived lipid compositions are further described below.

[0397] Delivery to plants For example, provided herein are methods of delivering bacterially-derived lipid compositions to plants by contacting the plant, or a portion thereof (e.g., plant leaves, seeds, pollen, roots, fruits, shoots, flowers, cells, protoplasts, or tissues (e.g., meristem tissue)) with the bacterially-derived lipid composition. In some cases, plants may be treated with bacterially-derived lipid compositions that do not contain heterologous functional agents. In other cases, the bacterially-derived lipid composition contains heterologous functional agents, such as insecticides (e.g., antibacterial agents, antifungal agents, nematicides, molluscicides, antiviral agents, herbicides), parasite control agents (e.g., repellents), fertilizers, or plant-modifying agents.

[0398] In some embodiments, provided herein are methods of increasing plant fitness, the methods comprising delivering a bacterially-derived lipid composition to a plant by contacting the plant, or a portion thereof (e.g., a plant leaf, seed, pollen, root, fruit, shoot, flower, cell, protoplast, or tissue (e.g., meristem tissue)) with a bacterially-derived lipid composition described herein (e.g., in an effective amount and for a duration) to increase the fitness of the plant compared to an untreated plant (e.g., a plant that has not been delivered with the bacterially-derived lipid composition).

[0399] The increased fitness of plants as a result of the delivery of bacterially derived lipid compositions can manifest in many ways, for example, by improving the plant's production, for example, by improving yield, improving plant vigor (e.g., improving tolerance to abiotic or biotic stress or improving resistance to harmful organisms), or improving the quality of products harvested from the plant. Improved plant yield refers to an increase in the yield of a plant's product (e.g., measured by plant biomass, grain, seed or fruit yield, protein content, carbohydrate or oil content, or leaf area) by a measurable amount over the yield of the same product of a plant produced under the same conditions but without application of the composition or compared to application of a conventional agricultural formulation.

[0400] Increased plant fitness as a result of delivery of the bacterially-derived lipid composition can also be measured by other methods, such as vigor assessment, stand (number of plants per unit area), plant height, stem circumference, stem length, number of leaves, leaf size, plant canopy, visual appearance (such as greener leaf color), root assessment, heading, protein content, increased germination, larger leaves, more leaves, fewer dead basal leaves, stronger tillers, less fertilizer required, less seed required, more productive tillers, earlier flowering, earlier fruit or seed maturation, fewer plant nodes (perennial roots), increased shoot growth, earlier germination, or an increase or improvement in any combination of these factors that is measurable or significant compared to the same factors in plants produced under the same conditions but without administration of the composition or with application of conventional agricultural formulations.

[0401] Provided herein are methods for increasing or killing weed fitness when a herbicide is included in the bacterially-derived lipid composition. In such cases, the method can be effective for reducing the fitness of the weed by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, compared to untreated weeds (e.g., weeds to which the bacterially-derived lipid composition has not been administered). For example, the method can be effective for killing weeds, thereby reducing the weed population by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, compared to untreated weeds. In some cases, the method substantially eliminates weeds.

[0402] Plants that can be delivered (i.e., "treated") with bacterially derived lipid compositions according to the present methods include whole plants and parts thereof, including, but not limited to, shoot plant organs / structures (e.g., leaves, stems, and tubers), roots, flowers and floral organs / structures (e.g., bracts, sepals, petals, stamens, ovaries, anthers, and ovules), seeds (including embryos, endosperm, cotyledons, and seed coats) and fruits (mature ovules), plant tissues (e.g., meristem tissue, vascular tissue, aboveground tissue, etc.) and cells (e.g., guard cells, egg cells, etc.), and their progeny. Plant parts can also refer to plant parts such as shoots, roots, stems, seeds, thorns, leaves, petals, flowers, ovules, bracts, branches, petioles, internodes, bark, pubescence, rhizomes, leaves, pollen, stamens, etc.

[0403] Examples of types of plants and weeds that can be treated with the bacterially-derived lipid compositions may be found in WO 2021 / 041301, which is incorporated herein by reference in its entirety.

[0404] Delivery of plants to pests For example, provided herein is a method for delivering bacterial-derived lipid compositions to plant pests by contacting the plant pests with bacterial-derived lipid compositions.In some cases, plant pests may be treated with bacterial-derived lipid compositions that do not contain heterologous functional agents.In other cases, the bacterial-derived lipid compositions contain heterologous functional agents, such as insecticides (e.g., antibacterial agents, antifungal agents, nematicides, molluscicides, antiviral agents, or herbicides) or pest control agents (e.g., repellents).For example, the method may be useful for reducing the fitness of pests, for example, as a result of delivering bacterial-derived lipid compositions, to prevent or treat pest infestation.

[0405] In some embodiments, provided herein are methods of reducing the fitness of a pest, the methods comprising delivering to the pest a bacterially-derived lipid composition described herein (e.g., in an effective amount and for an effective duration) to reduce the fitness of the pest compared to an untreated pest (e.g., a pest that has not received the bacterially-derived lipid composition).

[0406] In some embodiments, provided herein are methods of reducing (e.g., treating) a fungal infection in a plant having a fungal infection, the methods comprising delivering a bacterial-derived lipid composition described herein to a pest of the plant. In some embodiments, provided herein are methods of reducing (e.g., treating) a fungal infection in a plant having a fungal infection, the methods comprising delivering a bacterial-derived lipid composition described herein to a pest of the plant, the bacterial-derived lipid composition comprising an antifungal agent.

[0407] In some embodiments, provided herein are methods for reducing (e.g., treating) a bacterial infection in a plant having a bacterial infection, the method comprising delivering a bacterial-derived lipid composition described herein to an insect pest of the plant.

[0408] In some embodiments, provided herein are methods for reducing (e.g., treating) a bacterial infection in a plant having a bacterial infection, the method comprising delivering a bacterial-derived lipid composition described herein to a pest of the plant, wherein the bacterial-derived lipid composition comprises an antimicrobial agent.

[0409] In some embodiments, provided herein are methods for reducing the fitness of an insect plant pest, the method comprising delivering a bacterially-derived lipid composition described herein to the insect plant pest of the plant.

[0410] In some embodiments, provided herein are methods for reducing the fitness of an insect plant pest, the method comprising delivering a bacterially-derived lipid composition described herein to an insect plant pest of the plant, wherein the bacterially-derived lipid composition comprises an insecticide.

[0411] In some embodiments, provided herein are methods for reducing the fitness of a nematode plant pest, the method comprising delivering a bacterial-derived lipid composition described herein to the nematode plant pest.

[0412] In some embodiments, provided herein are methods for reducing the fitness of a nematode plant pest, the method comprising delivering a bacterially-derived lipid composition described herein to the nematode plant pest, wherein the bacterially-derived lipid composition comprises a nematicide.

[0413] In some embodiments, provided herein are methods for reducing the fitness of weeds, the methods comprising delivering to the weeds a bacterial-derived lipid composition described herein.

[0414] In some embodiments, provided herein are methods of reducing the fitness of weeds, the methods comprising delivering to the weeds a bacterially-derived lipid composition described herein, wherein the bacterially-derived lipid composition comprises a herbicide (e.g., glufosinate).

[0415] Suitable antimicrobial agents, insecticides, nematicides and herbicides include those already mentioned above.

[0416] The reduction in the fitness of a pest as a result of the delivery of a bacterially-derived lipid composition can manifest in many ways. In some cases, the reduction in the fitness of a pest can manifest as a deterioration or decline in the physiological function of the pest (e.g., reduced health or survival) as a result of the delivery of a bacterially-derived lipid composition. In some cases, the fitness of an organism can be measured by one or more parameters, including, but not limited to, reproductive rate, fecundity, lifespan, survival rate, mobility, fecundity, pest development, body weight, metabolic rate or activity, or survival, compared to a pest that has not been administered a bacterially-derived lipid composition.

[0417] In some cases, a decrease in the fitness of the pest may manifest as a decrease in the production of one or more nutrients (e.g., vitamins, carbohydrates, amino acids, or polypeptides) in the pest compared to a pest that has not been administered the bacterially-derived lipid composition.

[0418] In some cases, a decrease in the fitness of the pest may manifest as an increase in the pest's susceptibility to pesticides or allelochemicals and / or a decrease in the pest's resistance to pesticides compared to pests that have not been administered the bacterially-derived lipid composition.

[0419] In some cases, the methods or compositions provided herein may be effective in reducing the resistance of a pest to a parasite or pathogen (e.g., a fungal, bacterial, or viral pathogen or parasite) compared to a pest that has not been administered the bacterial-derived lipid composition.

[0420] In some cases, the methods or compositions provided herein may be effective in reducing the ability of a pest to carry or transmit a plant pathogen (e.g., a plant virus (e.g., TYLCV) or a plant bacterium (e.g., an Agrobacterium species)) compared to a pest that has not been administered the bacterially-derived lipid composition.

[0421] Delivery to plant symbionts Provided herein are methods for delivering the bacterial-derived lipid compositions disclosed herein to plant symbionts. Included are methods for delivering bacterial-derived lipid compositions to symbionts (e.g., bacterial endosymbionts, fungal endosymbionts, or insects) by contacting the symbiont with the bacterial-derived lipid composition. The methods can be useful for increasing the fitness of plant symbionts, such as symbionts that are beneficial to the fitness of plants. In some cases, plant symbionts can be treated with bacterial-derived lipid compositions that do not contain heterologous functional agents. In other cases, the bacterial-derived lipid composition contains heterologous functional agents, such as fertilization agents.

[0422] Thus, the methods can be used to increase the fitness of a plant symbiont. In one aspect, provided herein is a method of increasing the fitness of a symbiont, the method comprising delivering to the symbiont (e.g., in an effective amount and for an effective duration) a bacterially-derived lipid composition described herein to increase the fitness of the symbiont compared to an untreated symbiont (e.g., a symbiont that has not been delivered a bacterially-derived lipid composition).

[0423] In some embodiments, provided herein are methods for increasing the fitness of a fungus (e.g., a fungal endosymbiont of a plant), the methods comprising delivering a bacterial-derived lipid composition described herein to the endosymbiont.

[0424] In some embodiments, provided herein are methods for increasing the fitness of bacteria (e.g., bacterial endosymbionts of plants), the methods comprising delivering a bacterial-derived lipid composition described herein to the bacteria.

[0425] In some embodiments, provided herein are methods for increasing the fitness of an insect (e.g., an insect symbiont of a plant), the methods comprising delivering to the insect a bacterial-derived lipid composition described herein.

[0426] In some cases, increased fitness of a symbiont may manifest as improved physiology (e.g., improved health or survival) of the symbiont as a result of administration of a bacterially-derived lipid composition. In some cases, the fitness of an organism may be measured by one or more parameters, including, but not limited to, reproductive rate, lifespan, mobility, fecundity, body weight, metabolic rate or activity, or survival, compared to a symbiont to which the bacterially-derived lipid composition has not been delivered. For example, a method or composition provided herein may be effective in improving the overall health of a symbiont or improving the overall survival of a symbiont, compared to a symbiont not administered a bacterially-derived lipid composition.

[0427] In some cases, increased fitness of the symbiont may manifest as an increased frequency or effectiveness of a desired activity performed by the symbiont (e.g., pollination, predation of pests, seed dispersal, or decomposition of waste or organic matter) compared to a symbiont that has not been administered the bacterial-derived lipid composition.

[0428] In some cases, increased fitness of the symbiont may be manifested as increased production of one or more nutrients (e.g., vitamins, carbohydrates, amino acids, or polypeptides) in the symbiont compared to a symbiont that has not been administered the bacterial-derived lipid composition.

[0429] In some cases, increased fitness of the symbiont may manifest as decreased susceptibility of the symbiont to insecticides and / or increased resistance of the symbiont to insecticides compared to symbionts not administered the bacterial-derived lipid composition.

[0430] In some cases, the increased fitness of the symbiont may manifest as a decreased sensitivity of the symbiont to the allelochemical agent and / or an increased resistance of the symbiont to the allelochemical agent compared to a symbiont not administered the bacterial-derived lipid composition. In some cases, the allelochemical agent is caffeine, soybean statin N, a monoterpene, a diterpene acid, or a phenolic compound. In some cases, the methods or compositions provided herein may decrease the sensitivity of the symbiont to the allelochemical agent by increasing the symbiont's ability to metabolize or degrade the allelochemical agent into usable substrates.

[0431] In some cases, the methods or compositions provided herein may be effective in increasing the resistance of a symbiont to a parasite or pathogen (e.g., a fungal, bacterial, or viral pathogen, or a parasitic mite (e.g., Varroa destructor in honeybees)) compared to a symbiont that has not been administered the bacterial-derived lipid composition.

[0432] In some cases, increased fitness of the symbiont may manifest as other fitness advantages, such as improved tolerance to certain environmental factors (e.g., high or low temperature tolerance), improved ability to survive in certain habitats, or improved ability to sustain certain diets (e.g., improved ability to metabolize soybeans versus corn), compared to symbionts not administered the bacterial-derived lipid composition.

[0433] The fitness of the symbiont may be assessed using any standard method in the art. In some cases, the fitness of the symbiont may be assessed by assessing individual symbionts. Alternatively, the fitness of the symbiont may be assessed by assessing the symbiont population. For example, an increase in the fitness of the symbiont may manifest as increased competitive success against other insects, thereby resulting in an increase in the size of the symbiont population.

[0434] Delivery to animal pathogens Provided herein is a method for delivering the bacterial lipid composition described herein to an animal (e.g., human) pathogen by contacting the pathogen with the bacterial lipid composition. As used herein, the term "pathogen" refers to an organism, such as a microorganism or invertebrate, that causes disease or disease symptoms in an animal, for example, by (i) directly infecting an animal, (ii) producing an agent (e.g., a bacterium that produces a pathogenic toxin, etc.) that causes disease or disease symptoms in an animal, and / or (iii) inducing an immune (e.g., inflammatory response) in an animal (e.g., a biting insect, e.g., a bedbug). As used herein, a pathogen includes, but is not limited to, bacteria, protozoa, parasites, fungi, nematodes, insects, viroids and viruses, or any combination thereof, and each pathogen can cause disease or symptoms in an animal, such as a human, by contacting itself or another pathogen.

[0435] In some cases, animal (e.g., human) pathogens may be treated with bacterially derived lipid compositions that do not contain heterologous functional agents. In other cases, the bacterially derived lipid compositions contain heterologous functional agents, such as heterologous therapeutic agents (e.g., antibacterial agents, antifungal agents, insecticides, nematicides, antiparasitic agents, antivirals, or repellents). The methods may be useful, for example, to reduce the fitness of animal pathogens, to prevent or treat pathogen infection or to control pathogen spread, as a result of delivery of the bacterially derived lipid compositions.

[0436] Examples of pathogens that can be targeted according to the methods described herein include bacteria (e.g., Streptococcus spp., Pneumococcus spp., Pseudomonas spp., Shigella spp., Salmonella spp., Campylobacter spp., or Escherichia spp.), fungi (Saccharomyces spp. or Candida spp.), parasitic insects (e.g., Cimex spp.), parasitic nematodes (e.g., Heligmosomoides spp.), or parasitic protozoa (e.g., Trichomonas spp.).

[0437] For example, provided herein are methods for reducing the fitness of a pathogen, the methods comprising delivering a bacterial lipid composition described herein to the pathogen, wherein the method reduces the fitness of the pathogen compared to an untreated pathogen. In some embodiments, the methods comprise delivering the composition to at least one habitat where the pathogen grows, survives, reproduces, feeds, or invades. In some of the methods described herein, the composition is delivered as a pathogen-soluble composition for ingestion by the pathogen. In some of the methods described herein, the composition is delivered (e.g., to the pathogen) as a liquid, solid, aerosol, paste, gel, or gas.

[0438] Also provided herein is a method for reducing the fitness of a parasitic insect, the method comprising delivering a bacterially derived lipid composition to the parasitic insect. In some cases, the method comprises delivering a bacterially derived lipid composition described herein to the parasitic insect, wherein the bacterially derived lipid composition comprises an insecticide. For example, the parasitic insect may be a bedbug. Other non-limiting examples of parasitic insects are provided herein. In some cases, the method reduces the fitness of the parasitic insect compared to an untreated parasitic insect.

[0439] Further provided herein are methods for reducing the fitness of parasitic nematodes, the methods comprising delivering a bacterially derived lipid composition described herein to the parasitic nematode. In some cases, the methods comprise delivering a bacterially derived lipid composition described herein to the parasitic nematode, wherein the bacterially derived lipid composition comprises a nematicide. For example, the parasitic nematode is Heligmosomoides polygyrus. Other non-limiting examples of parasitic nematodes are provided herein. In some cases, the methods reduce the fitness of the parasitic nematode compared to an untreated parasitic nematode.

[0440] Further provided herein are methods for reducing the fitness of parasitic protozoa, the methods comprising delivering a bacterially derived lipid composition described herein to the parasitic protozoa. In some cases, the methods comprise delivering a bacterially derived lipid composition described herein to the parasitic protozoa, wherein the bacterially derived lipid composition comprises an antiparasitic agent. For example, the parasitic protozoa may be Trichomonas vaginalis. Other non-limiting examples of parasitic protozoa are provided herein. In some cases, the methods reduce the fitness of the parasitic protozoa compared to untreated parasitic protozoa.

[0441] The reduction in the fitness of a pathogen as a result of delivery of a bacterially-derived lipid composition can manifest in many ways. In some cases, the reduction in the fitness of a pathogen can manifest as a deterioration or decline in the physiological function of the pathogen (e.g., reduced health or survival) as a result of delivery of a bacterially-derived lipid composition. In some cases, the fitness of an organism can be measured by one or more parameters, including, but not limited to, reproductive rate, fecundity, lifespan, survival rate, mobility, fecundity, pathogen development, body weight, metabolic rate or activity, or survival, compared to a pathogen not administered with a bacterially-derived lipid composition. For example, the methods or compositions provided herein can be effective in reducing the overall health of a pathogen or reducing the overall survival of a pathogen. In some cases, the increase in pathogen survival is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% above a reference level (e.g., a level observed in a pathogen not receiving a bacterially-derived lipid composition). In some cases, the methods and compositions are effective in reducing the reproduction (e.g., reproduction rate, fecundity) of a pathogen compared to a pathogen that has not been administered the bacterial-derived lipid composition. In some cases, the methods and compositions are effective in reducing other physiological parameters, such as mobility, weight, lifespan, fecundity, or metabolic rate, by greater than about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to a reference level (e.g., a level seen in a pathogen that has not received the bacterial-derived lipid composition).

[0442] In some cases, the reduction in fitness of the pest may manifest as increased susceptibility of the pathogen to the anti-pathogenic agent and / or decreased resistance of the pathogen to the anti-pathogenic agent compared to a pathogen to which the bacterial-derived lipid composition has not been delivered.

[0443] In some cases, the reduced fitness of the pathogen may manifest as other fitness advantages, such as reduced tolerance to certain environmental factors (e.g., high or low temperature tolerance), reduced ability to survive in certain habitats, or reduced ability to sustain certain diets, compared to pathogens to which the bacterial-derived lipid composition has not been delivered.

[0444] The fitness of a pathogen can be evaluated using any standard method in the art. In some cases, the fitness during parasitism can be evaluated by evaluating individual pathogens. Alternatively, the fitness of a parasite can be evaluated by evaluating the pathogen population. For example, a decrease in the fitness of a pathogen can be manifested as a decrease in the success of competition with other pathogens, thereby resulting in a decrease in the size of the pathogen population.

[0445] The bacterial-derived lipid compositions and related methods described herein are useful for reducing the fitness of animal pathogens, thereby treating or preventing infection in animals.

[0446] Delivery to pathogen vectors Provided herein is a method for delivering the bacterial lipid composition described herein to a pathogen vector, such as the pathogen vector described herein, by contacting the pathogen vector with the bacterial lipid composition.As used herein, the term "vector" refers to an insect that can carry or transmit animal pathogens from reservoirs to animals.Exemplary vectors include insects such as those with piercing and sucking mouthparts, found in Hemiptera and some Hymenoptera, such as mosquitoes, bees, wasps, small insects, ticks, tsetse flies, flies, fleas and ants.

[0447] In some cases, animal (e.g., human) pathogen vectors may be treated with bacterial-derived lipid compositions that do not contain heterologous functional agents. In other cases, the bacterial-derived lipid compositions contain heterologous functional agents, such as heterologous therapeutic agents (e.g., antibacterial agents, antifungal agents, insecticides, nematicides, antiparasitic agents, antiviral agents, or repellents). The method may be useful, for example, to reduce the fitness of pathogen vectors as a result of delivery of the bacterial-derived lipid composition, in order to control the spread of pathogens. Examples of pathogen vectors that can be targeted according to the present method include insects, such as those described herein.

[0448] For example, provided herein are methods for reducing the fitness of an animal pathogen vector, the methods comprising delivering an effective amount of a bacterial-derived lipid composition described herein to the vector, wherein the method reduces the fitness of the vector compared to an untreated vector. In some cases, the methods comprise delivering the composition to at least one habitat in which the vector grows, lives, reproduces, feeds, or invades. In some cases, the composition is delivered as a soluble composition for ingestion by the vector. In some cases, the vector is an insect. In some cases, the insect is a mosquito, tick, mite, or louse. In some cases, the composition is delivered (e.g., to the pathogen vector) as a liquid, solid, aerosol, paste, gel, or gas.

[0449] For example, provided herein is a method for reducing the fitness of an insect vector of an animal pathogen, the method comprising delivering a bacterial-derived lipid composition described herein to the vector. In some cases, the method comprises delivering a bacterial-derived lipid composition to the vector, wherein the bacterial-derived lipid composition comprises an insecticide. For example, the insect vector may be a mosquito, tick, mite, or louse. Other non-limiting examples of pathogen vectors are provided herein. In some cases, the method reduces the fitness of the vector compared to an untreated vector.

[0450] In some cases, a decrease in vector fitness can manifest as a deterioration or decline in the physiological function of the vector (e.g., decreased health or survival) as a result of administration of the composition. In some cases, the fitness of the organism can be measured by one or more parameters, including, but not limited to, reproductive rate, lifespan, mobility, fecundity, body weight, metabolic rate or activity, or survival, compared to a vector organism to which the composition has not been delivered. For example, a method or composition provided herein can be effective in reducing the overall health of the vector or reducing the overall survival of the vector. In some cases, the reduction in vector survival is about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% compared to a reference level (e.g., a level observed in a vector that has not received the composition). In some cases, the methods and compositions are effective for reducing vector reproduction (e.g., reproductive rate) compared to a vector organism to which the composition has not been delivered. In some cases, the methods and compositions are effective to reduce other physiological parameters, such as mobility, weight, lifespan, fecundity, or metabolic rate, by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to a reference level (e.g., a level seen in a vector not delivering the composition).

[0451] In some cases, the reduced fitness of the vector may manifest as increased susceptibility of the vector to an insecticide and / or decreased resistance of the vector to an insecticide compared to a vector organism that has not been administered the composition. In some cases, the methods or compositions provided herein may increase the susceptibility of the vector to an insecticide by decreasing the vector's ability to metabolize or break down the insecticide into usable substances compared to a vector that has not been delivered the composition.

[0452] In some cases, reduced vector fitness may manifest as other fitness advantages, such as reduced tolerance to certain environmental factors (e.g., high or low temperature tolerance), reduced ability to survive in certain habitats, or reduced ability to sustain certain diets, compared to vector organisms to which the composition has not been delivered. In some cases, a method or composition provided herein may be effective to reduce vector fitness in any of multiple ways described herein. Furthermore, a composition may reduce vector fitness in any number of vector classes, orders, families, genera, or species (e.g., one vector species, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 200, 250, 500, or more vector species). In some cases, a composition acts on a single vector class, order, family, genus, or species.

[0453] The fitness of a vector may be assessed using any standard method in the art. In some cases, the fitness of a vector may be assessed by assessing an individual vector. Alternatively, the fitness of a vector may be assessed by assessing a vector population. For example, a decrease in the fitness of a vector may manifest as a decrease in the success of competition with other vectors, thereby resulting in a decrease in the size of the vector population.

[0454] By reducing the fitness of vectors carrying animal pathogens, the compositions provided herein are effective for reducing the transmission of vector-borne diseases. The compositions can be delivered to insects using any of the formulations and delivery methods described herein in an amount and for a duration effective to reduce disease transmission, e.g., reduce vertical or horizontal transmission between vectors and / or reduce transmission to animals. For example, the compositions described herein can reduce vertical or horizontal transmission of vector-borne pathogens by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, compared to vector organisms to which the compositions are not delivered. As another example, the compositions described herein can reduce the vector competence of insect vectors by about 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more, compared to vector organisms to which the compositions are not delivered. Delivery to animals

[0455] For example, provided herein are methods for delivering the bacterial-derived lipid compositions described herein to animal cells, tissues, or subjects (e.g., mammals, e.g., humans) by contacting the animal cells, tissues, subjects, or portions thereof with the bacterial-derived lipid composition. In some cases, animals may be treated with bacterial-derived lipid compositions that do not contain heterologous functional agents. In other cases, the bacterial-derived lipid composition contains heterologous functional agents, such as heterologous therapeutic agents (e.g., therapeutic proteins or peptide nucleic acids, or small molecules, antibacterial agents, antifungal agents, insecticides, nematicides, antiparasitic agents, antiviral agents, or repellents).

[0456] In one aspect, provided herein is a method of increasing the fitness of an animal (e.g., a human), the method comprising delivering to the animal a bacterially-derived lipid composition described herein (e.g., in an effective amount and for an effective duration) to increase the fitness of the animal compared to an untreated animal (e.g., an animal that has not received the bacterially-derived lipid composition).

[0457] Increased animal fitness as a result of delivery of the bacterially-derived lipid composition can be determined by any method that assesses animal fitness (e.g., mammalian fitness, e.g., human fitness (e.g., health)).

[0458] Provided herein are methods for modifying or increasing the fitness of an animal (e.g., a human), comprising delivering an effective amount of a bacterial lipid composition described herein to the animal, wherein the method modifies the animal, thereby introducing or increasing a beneficial trait in the animal compared to an untreated animal (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%). In particular, the method can increase the fitness of an animal, e.g., a mammal, e.g., a human, compared to an untreated animal (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%).

[0459] In a further aspect, provided herein is a method of increasing the fitness of an animal (e.g., a human), the method comprising contacting cells of the animal with an effective amount of a bacterially derived lipid composition described herein, wherein the method increases the fitness of the animal, e.g., the animal, e.g., the human, compared to an untreated animal (e.g., by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%).

[0460] In certain cases, the animal is a mammal, e.g., a human. In certain cases, the animal is a livestock animal or a veterinary animal. In certain cases, the animal is a mouse.

[0461] How to apply The plants described herein can be exposed to the bacterial-derived lipid compositions described herein by any suitable method that allows the composition to be delivered or administered to the plant.The bacterial-derived lipid composition can be delivered alone or in combination with other active substances (e.g., fertilizers) or inactive substances, and can be applied by spraying, injection (e.g., microinjection), pouring, or immersion through the plant in the form of a concentrated liquid, gel, solution, suspension, spray, powder, pellet, briquette, brick, etc., formulated to deliver an effective concentration of bacterial-derived lipid composition.The amount and location for application of the compositions described herein are generally determined by the plant's habitat, the stage of the plant's life cycle that can be targeted by the bacterial-derived lipid composition, the site to be applied, and the physical and functional characteristics of the bacterial-derived lipid composition.

[0462] In some cases, the composition is directly sprayed onto plants, for example, crops, for example, by backpack spraying, aerial spraying, crop spraying / dusting, etc. When bacterially derived lipid compositions are delivered to plants, the plants that receive bacterially derived lipid compositions can be at any stage of plant growth.For example, formulated bacterially derived lipid compositions can be applied as seed coating or root treatment in the early stage of plant growth, or as total plant treatment in the later stage of crop cycle.In some cases, bacterially derived lipid compositions can be applied to plants as topical agents.

[0463] Additionally, bacterially-derived lipid compositions may be applied as systemic formulations (e.g., in the soil in which the plant grows or in the water used to water the plant) that are absorbed and distributed throughout the plant's tissues. In some cases, the plant or food organism may be genetically modified to express the bacterially-derived lipid composition.

[0464] Delayed or continuous release can also be achieved by coating the bacterial-derived lipid composition or bacterial-derived lipid composition with a dissolvable or bioerodible coating layer such as gelatin, or dispersing the agent in a dissolvable or erodible matrix, where the coating dissolves or erodes in the environment of use, thereby making the bacterial-derived lipid composition available. Such continuous release and / or dispensing devices can be advantageously used to consistently maintain effective concentrations of one or more of the bacterial-derived lipid compositions described herein.

[0465] In some cases, the bacterial-derived lipid composition is delivered to a part of a plant, such as a leaf, seed, pollen, root, fruit, shoot, or flower, or its tissue, cell, or protoplast. In some cases, the bacterial-derived lipid composition is delivered to a plant cell. In some cases, the bacterial-derived lipid composition is delivered to a plant protoplast. In some cases, the bacterial-derived lipid composition is delivered to a plant tissue. For example, the composition may be delivered to a plant meristem tissue (e.g., an apical meristem, a lateral meristem, or an interstitial meristem). In some cases, the composition is delivered to a plant permanent tissue (e.g., a simple tissue (e.g., a parenchyma, a sclerenchyma, or a sclerenchyma) or a complex permanent tissue (e.g., a xylem or a phloem)). In some cases, the composition is delivered to a plant embryo.

[0466] In some cases, the bacterially-derived lipid composition may be recommended for field application as the amount of bacterially-derived lipid composition per hectare (g / ha or kg / ha), or the amount of active ingredient per hectare (e.g., bacterially-derived lipid composition with or without a heterologous functional agent), or acid equivalent (kg ai / ha or g ai / ha). In some cases, a smaller amount of the heterologous functional agent in the composition may need to be applied to the soil, plant medium, seed plant tissue, or plant to achieve the same results as when the heterologous functional agent is applied in a composition lacking the bacterially-derived lipid composition. For example, the amount of heterologous functional agent can be applied at a level less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 50, or 100 times (or any range from about 2 to about 100 times, e.g., about 2 to 10 times, about 5 to 15 times, about 10 to 20 times, about 10 to 50 times) than the same heterologous functional agent applied in a non-bacterial-derived lipid composition, e.g., direct application of the same heterologous functional agent without the bacterial-derived lipid composition(s). The bacterial-derived lipid composition can be applied in various amounts per hectare, e.g., about 0.0001, 0.001, 0.005, 0.01, 0.1, 1, 2, 10, 100, 1,000, 2,000, 5,000 (or any range from about 0.0001 to 5,000) kg / ha. For example, about 0.0001 to about 0.01, about 0.01 to about 10, about 10 to about 1,000, or about 1,000 to about 5,000 kg / ha.

[0467] Processing method The bacterial-derived lipid compositions described herein can also be useful in various treatment methods. For example, the methods and compositions can be used to prevent or treat pathogen infections in animals (e.g., humans). As used herein, the term "treatment" refers to administering a pharmaceutical composition to an animal for prophylactic and / or therapeutic purposes. Preventing "infection" refers to the prophylactic treatment of an animal that is not yet sick, but is susceptible to or otherwise at risk of a particular disease. Treating infectious diseases refers to administering treatment to an animal that already has a disease to improve or stabilize the animal's condition. The method includes delivering the bacterial-derived lipid composition described herein to an animal, such as a human.

[0468] For example, provided herein are methods for treating an animal having a fungal infection, the methods comprising administering to the animal an effective amount of a bacterial-derived lipid composition. In some cases, the methods comprise administering to the animal an effective amount of a bacterial-derived lipid composition described herein, wherein the bacterial-derived lipid composition comprises an antifungal agent. In some cases, the antifungal agent is a nucleic acid that inhibits the expression of a gene in a fungus that causes the fungal infection.

[0469] In another aspect, provided herein are methods for treating an animal having a bacterial infection, the methods comprising administering to the animal an effective amount of a bacterial-derived lipid composition. In some cases, the methods comprise administering to the animal an effective amount of a bacterial-derived lipid composition described herein, wherein the bacterial-derived lipid composition comprises an antimicrobial agent. In some cases, the methods reduce or substantially eliminate the bacterial infection. In some cases, the animal is a human, a veterinary animal, or a livestock animal.

[0470] The present method is useful for treating infections in animals (e.g., caused by animal pathogens), which refers to treating an animal that is already suffering from a disease to improve or stabilize the animal's condition. This can involve reducing the colonization of one or more pathogens in, on, or around the animal compared to the starting amount (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) and / or providing a benefit to the individual (e.g., reducing colonization by an amount sufficient to resolve symptoms). In such cases, the treated infection can be manifested as a reduction in symptoms (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In some cases, the treated infection is effective to increase the survival chance of an individual (e.g., increase the chance of survival by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) or increase the overall survival of a population (e.g., increase the chance of survival by about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, the compositions and methods can be effective to "substantially eliminate" the infection, which refers to a reduction in the infection by an amount sufficient to provide a sustained resolution of symptoms in the animal (e.g., for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

[0471] The methods are useful for preventing infection (e.g., caused by animal pathogens) by preventing an increase in colonization in, on, or around an animal by one or more pathogens (e.g., about 1%, 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or greater than 100% compared to untreated animals) in an amount sufficient to maintain a starting pathogen population (e.g., approximately that found in healthy individuals), prevent the onset of infection, and / or prevent symptoms or conditions associated with infection. For example, prophylactic treatment may be used to prevent fungal infection in individuals preparing for invasive medical procedures (e.g., transplants, stem cell therapy, grafts, prosthetic devices, undergoing long-term or frequent intravenous catheterization, or undergoing intensive care unit treatment), immunocompromised individuals (e.g., those with cancer, HIV / AIDS, or taking immunosuppressants), or individuals undergoing long-term antibiotic therapy.

[0472] The bacterial-derived lipid composition can be formulated for administration or administered by any suitable method, including, for example, intravenous, intramuscular, subcutaneous, intradermal, transdermal, intra-arterial, intraperitoneal, intracerebral, intracranial, intra-articular, intraprostatic, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, topical, intratumoral, peritoneal, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, intraocular, intraorbital, oral, topical, transdermal, intravitreal (e.g., via intravitreal injection), eye drop, inhalation, injection, implant, infusion, continuous infusion, localized perfusion directly bathing target cells, catheter, lavage, cream, or lipid composition. The compositions used in the methods described herein can also be administered systemically or locally. The method of administration can vary depending on various factors (e.g., the compound or composition being administered and the severity of the condition, disease, or disorder being treated). In some cases, the bacterial-derived lipid composition is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, inhalation, intrathecally, intraventricularly, or intranasally. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic. Various administration schedules are contemplated herein, including, but not limited to, single or multiple administrations over various time periods, bolus administration, and pulse infusion.

[0473] The use of the bacterially-derived lipid composition for the prevention or treatment of infections described herein (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the severity and course of the disease, whether it is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history, and the patient's response to the bacterially-derived lipid composition. The bacterially-derived lipid composition can be administered to the patient, for example, once or over a series of treatments. For repeated administration over several days or longer, depending on the condition, treatment will generally be sustained until a desired suppression of disease symptoms occurs or the infection is no longer detectable. Such doses may be administered intermittently, for example, weekly or biweekly (e.g., so that the patient receives, for example, about two to about twenty doses of the bacterially-derived lipid composition). An initial higher loading dose, followed by one or more lower doses, may also be administered. However, other dosing regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

[0474] In some cases, the amount of bacterially derived lipid composition administered to an individual (e.g., a human) may be in the range of about 0.01 mg / kg to about 5 g / kg of the individual's body weight (e.g., about 0.01 mg / kg to 0.1 mg / kg, about 0.1 mg / kg to 1 mg / kg, about 1 mg / kg to 10 mg / kg, about 10 mg / kg to 100 mg / kg, about 100 mg / kg to 1 g / kg, or about 1 g / kg to 5 g / kg). In some cases, the amount of bacterially derived lipid composition administered to an individual (e.g., a human) is at least 0.01 mg / kg of the individual's body weight (e.g., at least 0.01 mg / kg, at least 0.1 mg / kg, at least 1 mg / kg, at least 10 mg / kg, at least 100 mg / kg, at least 1 g / kg, or at least 5 g / kg). The dosage can be administered as a single dose or as multiple doses (for example, 2, 3, 4, 5, 6, 7, or more than 7 doses).In some cases, the bacterial lipid composition administered to animals can be administered alone or in combination with additional therapeutic agents.The dosage of antibody administered in combination therapy can be reduced compared with single treatment.The progress of this therapy can be easily monitored by conventional techniques.

[0475] kit The present invention also provides a kit comprising a container with the bacterial-derived lipid composition described herein. The kit may further comprise instruction materials for applying or delivering the bacterial-derived lipid composition to plants according to the methods of the present invention. Those skilled in the art will understand that the instructions for applying the bacterial-derived lipid composition in the methods of the present invention can be in any form. Such instructions include, but are not limited to, written instructions (such as labels, booklets, pamphlets), oral instructions (such as audio cassettes or CDs), or video instructions (such as videotapes or DVDs).

[0476] Nucleic Acid Vaccines for Viral Infections Bacteria-derived lipid compositions can be used as nucleic acid vaccine compositions when the compositions contain antigenic polypeptides that combat various viral infections.

[0477] In some embodiments, the bacterial-derived lipid composition / nucleic acid vaccine formulation (i.e., BacLC / nucleic acid vaccine) comprises one or more polynucleotides (e.g., mRNA) encoding one or more antigenic polypeptides to combat various viral infections. The one or more polynucleotides (e.g., mRNA) encode one or more antigenic polypeptides derived from an infectious agent that causes an infectious disease, disorder, or condition, e.g., a viral infection caused by an RNA virus.

[0478] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes one or more wild-type or engineered antigens (or antibodies to antigens) of various types and strains of viruses described below.

[0479] Viral infection BacLC / nucleic acid vaccines are effective against acute febrile pharyngitis, pharyngoconjunctival fever, epidermolytic keratoconjunctivitis, infantile gastroenteritis, Coxsackie infections, infectious mononucleosis, Burkitt's lymphoma, acute hepatitis, chronic hepatitis, liver cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (e.g., gingivitis in children, tonsillitis and pharyngitis in adults, keratoconjunctivitis), latent HSV-1 infection (e.g., herpes labialis and herpes labialis), primary HSV-2 infection, latent HSV-2 infection, aseptic meningitis, infectious mononucleosis, cytomegalic inclusion disease, Kaposi's sarcoma, multicentric Castleman's disease, primary effusion lymphoma, and They may be suitable for combating infectious diseases, disorders, or conditions associated with viral infections, including, but not limited to, AIDS, influenza, Reye's syndrome, measles, post-infectious encephalomyelitis, mumps, hyperplastic epithelial lesions (e.g., common flat, plantar, and anogenital warts, pharyngeal papillomas, epidermodysplasia verruciformis), cervical cancer, squamous cell carcinoma, croup, pneumonia, bronchitis, the common cold, polymyelitis, rabies, bronchitis, pneumonia, influenza-like syndrome, severe bronchitis with pneumonia, German measles, congenital rubella, chickenpox, and shingles.

[0480] Exemplary viral infectious agents include adenovirus, herpes simplex type 1, herpes simplex type 2, encephalitis virus, papillomavirus, varicella-zoster virus, human cytomegalovirus, human herpesvirus type 8, human papillomavirus, BK virus, JC virus, smallpox, poliovirus, hepatitis B virus, human bocavirus, parvovirus B19, human astrovirus, Norwalk virus, coxsackievirus, hepatitis A virus, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C virus, yellow fever virus, dengue virus, West Nile virus, rubella virus, and hepatitis E virus. , human immunodeficiency virus (HIV), influenza A or B, Guanarito virus, Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo hemorrhagic fever virus, Marburg virus, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus, Nipah virus, rabies virus, hepatitis D, rotavirus, orbivirus, coltivirus, hantavirus, Middle East respiratory coronavirus, chikungunya virus, or banna virus.

[0481] The infectious agent may be a strain of a virus selected from the group consisting of the viruses in the table below. JPEG2024542547000072.jpg240137 JPEG2024542547000073.jpg99135

[0482] Other suitable viral infections and viral infectious agents are described in U.S. Patent Application Publication No. 2019 / 0015501 and U.S. Patent No. 11,007,260, both of which are incorporated by reference in their entireties.

[0483] Mosquito-borne viruses Dengue fever. In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a strain of dengue virus (flavivirus). In some embodiments, the polynucleotide (e.g., mRNA) encodes E protein domain III (DENV1-4 tandem mRNA), E protein domain I / II hinge region (DENV1-4 individual mRNA), prM protein (DENV1-4 tandem or individual mRNA), and C protein (DENV1-4 tandem or single mRNA).

[0484] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a strain of Chikungunya virus. In some embodiments, the antigenic polypeptide encodes a Chikungunya envelope and / or capsid antigenic polypeptide selected from the group consisting of C, E1, E2, E3, 6K, and C-E3-E2-6K-E1.

[0485] In some embodiments, the polynucleotides (e.g., mRNA) in the BacLC / nucleic acid vaccine are selected from the following strains and isolates: TA53, SA76, UG82, 37997, IND-06, Ross, S27, M-713424, E1-A226V, E1-T98, IND-63-WB1, Gibbs 63-263, TH35, 1-634029, AF15561, IND-73-MH5, 653496, C0392-95, P0731460, MY0211MR / 06 / BP, SV0444-95, K0146-95, TS I-GSD-218-VR1, TSI-GSD-218, M127, M125, 6441-88, MY003IMR / 06 / BP, MY0021MR / 06 / BP, TR206 / H804187, MY / 06 / 37348, M Y / 06 / 37350, NC / 2011-568, 1455-75, RSU1, 0706aTw, InDRE51CHIK, PR-S4, AMA2798 / H804298, Hu / 85 / NR / 001, PhH15483, 0 706aTw, 0802aTw, MY019IMR / 06 / BP, PR-S6, PER160 / H803609, 99659, JKT23574, 0811aTw, CHIK / SBY6 / 10, 2001908323-BDG E1, 2001907981-BDG E1, 2004904899-BDG E1, 2004904879-BDG E1, 2003902452-BDG E1, DH 130003, 0804aTw, 2002918310-BDG E1, JC2012, chik-sy, 3807, 3462, Yap 13-2148, PR-S5, 0802aTw, MY019IMR / 06 / Bp, 0706aTw, PhH15483, Hu / 85 / NR / 001, CHIKV-13-112A, InDRE 4CHIK, 0806aTw, 0712aTw, 3412-78, Yap 13-2039, LEIV-CHIKV / Moscow / 1, DH130003, and 20039.

[0486] Zika virus. In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a strain of Zika virus (a flavivirus). In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a ZIKV polypeptide from a ZIKV serotype selected from the group consisting of MR766, SPH2015, and ACD75819.

[0487] Venezuelan Equine Encephalitis (VEE) Virus In some embodiments, the polynucleotide (eg, mRNA) in the BacLC / nucleic acid vaccine encodes a strain of VEE virus.

[0488] The polynucleotides (e.g., mRNA) in the BacLC / nucleic acid vaccines may encode additional types and strains of viruses and mosquito-borne viruses, or fragments thereof, as described in U.S. Patent No. 11,007,260, the entire contents of which are incorporated herein by reference.

[0489] influenza In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a strain of influenza virus. In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes an influenza A or influenza B strain, or a combination thereof. In some embodiments, the influenza A or B strain is associated with an avian, swine, equine, canine, human, or non-human primate.

[0490] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a hemagglutinin protein or a fragment thereof. In some embodiments, the hemagglutinin protein is H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof. In some embodiments, the hemagglutinin protein does not include a head domain (HA1). In some embodiments, the hemagglutinin protein does not include a portion of the head domain (HA1). In some embodiments, the hemagglutinin protein does not include a cytoplasmic domain. In some embodiments, the hemagglutinin protein does not include a portion of the cytoplasmic domain.

[0491] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a truncated hemagglutinin protein. In some embodiments, the truncated hemagglutinin protein does not include a portion of the transmembrane domain. In some embodiments, the virus is selected from the group consisting of H1N1, H3N2, H5N1, H7N9, and H10N8.

[0492] The polynucleotides (e.g., mRNA) in the BacLC / nucleic acid vaccines may encode additional types and strains of influenza viruses, or fragments thereof, as described in U.S. Patent No. 2019 / 0015501, which is incorporated by reference herein in its entirety.

[0493] coronavirus Betacoronavirus. In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a peptide / protein comprising the spike protein (S) of a betacoronavirus (BetaCoV), or a fragment or subunit thereof.

[0494] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine comprises an open reading frame encoding a peptide / protein comprising the spike protein (S) of a betacoronavirus (BetaCoV), or a fragment or subunit thereof.

[0495] The polynucleotides (eg, mRNA) in the BacLC / nucleic acid vaccines can encode different types of viruses, or fragments thereof, as described in US Pat. No. 10,933,127, which is incorporated herein by reference in its entirety.

[0496] Middle East Respiratory Syndrome Coronavirus. In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a peptide / protein comprising the spike protein (S), spike S1 fragment (S1), envelope protein (E), membrane protein (M), and / or nucleocapsid protein (N) of MERS coronavirus, or a fragment or variant of any one of these proteins.

[0497] The polynucleotides (e.g., mRNA) in the BacLC / nucleic acid vaccines may encode different strains of MERS coronavirus, or fragments thereof, as described in U.S. Patent No. 2019 / 0351048, which is incorporated by reference in its entirety.

[0498] SARS-CoV-2 In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes one or more wild-type or engineered antigens (or antibodies to antigens) of SARS-CoV-2.

[0499] In some embodiments, the open reading frame of the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes one or more wild-type or engineered antigens (or antibodies to antigens) of SARS-CoV-2. In some embodiments, the open reading frame is codon-optimized.

[0500] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a peptide / protein comprising the spike protein (S), membrane (M) protein, envelope (E) protein, and / or nucleocapsid (NC) protein of the SARS-CoV-2 virus, or a fragment or variant thereof.

[0501] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes a peptide / protein comprising the spike protein (S) of the SARS-CoV-2 virus, or a fragment or variant thereof. In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine encodes at least one or two domains of the spike protein (S) of the SARS-CoV-2 virus, and less than the full-length spike protein.

[0502] In some embodiments, the polynucleotide (e.g., mRNA) in the BacLC / nucleic acid vaccine comprises an open reading frame (ORF) encoding a SARS-CoV-2 spike (S) protein with a double proline stabilizing mutation.

[0503] In some embodiments, the polynucleotide (e.g., mRNA) (or open reading frame thereof) in the BacLC / nucleic acid vaccine is Alpha (lineage B.1.1.7, Q.1-Q.8), Beta (lineages B.1.351, B.1.351.2, B.1.351.3), delta Gamma (lineages P.1, P.1.1, P.1.2) Epsilon (lineages B.1.427, B.1.429) Eta (lineage B.1.525) Iota (lineage B.1.526) Kappa (lineage B.1.617.1) B.1.617.3 Lambda (lineage C.37) Mu (lineages B.1.621, B.1.621.1) Zeta (lineage P.2). Omicron

[0504] In some embodiments, the polynucleotide (e.g., mRNA) (or open reading frame thereof) in the BacLC / nucleic acid vaccine encodes the SARS-CoV-2 spike (S) protein and / or RBD, or a fragment thereof. In some embodiments, the S antigen and / or RBD antigen fragment thereof comprises one or more mutations in the RBD selected from the group consisting of K417N or K417T, N439N, N440K, G446V, L452R, Y453F, S477G or S477N, E484Q or E484K, F490S, N501S or N501Y, D614G, Q677P or Q677H, P681H or P681R.

[0505] In some embodiments, the polynucleotide (e.g., mRNA) (or open reading frame thereof) in the BacLC / nucleic acid vaccine encodes the SARS-CoV-2 spike (S) protein and / or RBD, or fragments thereof. In some embodiments, the S antigen contains spike trimer-stabilizing mutations, including, for example, K986P and V987P mutations (S-2P variants) and other proline substitutions, particularly F817P, A892P, A899P, and A942P, which can be combined together to obtain multiple proline variants, particularly hexaproline variants (HexaPro).

[0506] In some embodiments, the polynucleotide (e.g., mRNA) (or open reading frame thereof) in the BacLC / nucleic acid vaccine encodes the SARS-CoV-2 spike (S) protein and / or RBD, or a fragment thereof. In some embodiments, the S antigen comprises one or more mutations selected from the group consisting of substitutions L18F, T20N, P26S, D80A, D138Y, R190S, D215G, A570D, D614G, H655Y, P681H, A701V, T716I, S982A, T1027I, D1118H, and V1176F, and deletions delta69-70, delta144, delta242-244, and delta246-252.

[0507] In some embodiments, the polynucleotide (e.g., mRNA) (or open reading frame thereof) in the BacLC / nucleic acid vaccine encodes the SARS-CoV-2 spike (S) protein and / or RBD, or a fragment thereof. In some embodiments, the S antigen or RBD antigen fragment thereof comprises the following mutations: N501Y, E484K and N501Y, K417T or K417N, E484K and N501Y, K417N, N439N, Y453F, S477N, E484K, F490S, and N501Y, K417N, N439N, L452R, S477N, E484K, F490S, and N501Y.

[0508] Additional mutations can be found in WO 2021 / 154763A1, which is incorporated by reference in its entirety.

[0509] Nucleic acid sequence In some embodiments, the antigenic polypeptide encoded by the polynucleotide is a coronavirus, or a fragment or subunit thereof, hi some embodiments, the antigenic polypeptide is the spike protein (S) of the MERS virus (MERS-CoV), the SARS virus (SARS-CoV), or a fragment or subunit thereof.

[0510] In some embodiments, the antigenic polypeptide is a SARS virus, or a fragment or subunit thereof. The antigenic polypeptide may be a SARS-CoV-2 spike protein or a SARS-CoV-2 spike glycoprotein.

[0511] In some embodiments, the polynucleotide may be an mRNA, an siRNA or siRNA precursor, a microRNA (miRNA) or miRNA precursor, a plasmid, a Dicer substrate small interfering RNA (dsiRNA), a small hairpin RNA (shRNA), an asymmetric interfering RNA (aiRNA), a peptide nucleic acid (PNA), a morpholino, a locked nucleic acid (LNA), a piRNA (piRNA), a ribozyme, a deoxyribozyme (DNAzyme), an aptamer, a circular RNA (circRNA), a guide RNA (gRNA), or a DNA molecule encoding any of these RNAs. In one embodiment, the polynucleotide is an mRNA.

[0512] In some embodiments, the polynucleotide encodes a coronavirus antigen variant (e.g., a variant trimeric spike protein, such as a stabilized pre-fusion spike protein). An antigen variant or other polypeptide variant refers to a molecule whose amino acid sequence differs from a wild-type, native, or reference sequence. An antigen / polypeptide variant may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Typically, a variant has at least 50% identity with the wild-type, native, or reference sequence. In some embodiments, a variant shares at least 80% or at least 90% identity with the wild-type, native, or reference sequence.

[0513] Variant antigens / polypeptides encoded by the nucleic acids of the present disclosure may contain amino acid changes that confer any of a number of desirable properties, for example, enhancing their immunogenicity, enhancing their expression, and / or improving their stability or PK / PD properties in a subject. Variant antigens / polypeptides can be generated using routine mutagenesis techniques and, if necessary, assayed to determine whether they possess the desired properties. Assays for determining expression levels and immunogenicity are well known in the art, and examples of such assays are described in the Examples section. Similarly, the PK / PD properties of protein variants can be measured using art-recognized techniques, for example, by determining antigen expression in vaccinated subjects over time and / or by examining the durability of the induced immune response. The stability of the protein(s) encoded by the variant nucleic acids may be measured by assaying thermal stability or stability upon urea denaturation, or may be measured using in silico predictions. Methods for such experiments and in silico determinations are known in the art.

[0514] The term "identity" refers to the relationship between two or more polypeptide (e.g., antigen) or polynucleotide (nucleic acid) sequences, as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences, as determined by the number of matches between a series of two or more amino acid or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any) specified by a particular mathematical model or computer program (e.g., "algorithm"). The identity of related antigens or nucleic acids can be readily calculated by known methods. "Percent identity" when applied to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid or nucleic acid residues) of a candidate amino acid or nucleic acid sequence that are identical to the residues of the amino acid or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well known in the art. It is understood that identity depends on the calculation of percent identity, but the value may vary depending on gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide (e.g., antigen) have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such alignment tools include those in the BL...

Claims

1. 1. A bacterially derived lipid composition comprising: (a) a bacterial component comprising one or more lipids extracted from a bacterial source; and (b) A bacterially derived lipid composition comprising an ionizable lipid.

2. The bacterial-derived lipid composition of claim 1 , wherein the bacterial component comprises isolated bacterial extracellular vesicles.

3. 2. The bacterial-derived lipid composition of claim 1, wherein the bacterial components are modified by reconstituting membranes containing the bacterial components in the presence of the ionizable lipids.

4. The ionizable lipid is (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least six carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and heteroorganic group separated by a chain of at least two atoms; and (v) an N:P ratio of at least 10.

5. 2. The bacterially-derived lipid composition of claim 1, further comprising a sterol and a polyethylene glycol (PEG)-lipid conjugate.

6. The bacterial-derived lipid composition comprises: about 20 mol % to about 50 mol % of said ionizable lipid; about 20 mole % to about 60 mole % of said bacterial component; about 7 mol % to about 45 mol % of said sterol, and 6. The bacterial-derived lipid composition of claim 5, comprising about 0.5 mol% to about 3 mol% of said polyethylene glycol (PEG)-lipid conjugate.

7. 6. The bacterial-derived lipid composition of claim 5, wherein the bacterial-derived lipid composition comprises a molar ratio of ionizable lipid:bacterial lipid:sterol:PEG-lipid of about 35:50:12.5:2.5 or about 35:20:42.5:2.

5.

8. 2. The bacterial-derived lipid composition of claim 1, wherein the ionizable lipid is selected from the group consisting of 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200), MD1 (cKK-E12), OF2, EPC, ZA3-Ep10, TT3, LP01, 5A2-SC8, Lipid 5, SM-102 (Lipid H), and ALC-315.

9. The ionizable lipid is 【Chemistry 1】 2. The bacterial lipid composition of claim 1, wherein R is independently a C8-C14 alkyl group.

10. The bacterial source is selected from the group consisting of Escherichia coli, Acinetobacter, Agrobacterium, Anabaena, Aquifex, Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Polaromonas, Prochloromonas, 2. The bacterial lipid composition of claim 1, wherein the bacterial lipid composition is selected from the genera Coccus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia, Wallinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exiguobacterium, Geobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium, and Thermoanaerobacterium.

11. 11. The bacterial-derived lipid composition of claim 10, wherein the bacterial source is Escherichia coli or Salmonella typhimurium.

12. 2. The bacterially derived lipid composition of claim 1, wherein the bacterially derived lipid composition is a lipophilic moiety selected from the group consisting of lipoplexes, liposomes, lipid nanoparticles, polymeric carriers, exosomes, lamellar bodies, micelles, and emulsions.

13. The bacterial lipid composition of claim 1, which is a lipid nanoparticle having a size of less than about 200 nm.

14. The bacterial-derived lipid composition of claim 13, wherein the lipid nanoparticles have an average polydispersity index (PDI) in the range of about 0.1 to about 0.

4.

15. The bacterial-derived lipid composition of any one of claims 1 to 14, further comprising one or more heterologous functional agents, wherein the heterologous functional agents may be encapsulated by, embedded in, or conjugated to the surface of the bacterial-derived lipid composition.

16. The bacterial-derived lipid composition of claim 15 , wherein the heterologous functional agent comprises a polynucleotide.

17. The bacterial lipid composition of claim 16, wherein the polynucleotide is mRNA.

18. 16. The bacterial-derived lipid composition of claim 15, wherein the bacterial-derived lipid nanoparticles have a total lipid:heterologous functional agent weight ratio of about 50:1 to about 10:

1.

19. 15. The bacterial-derived lipid composition of any one of claims 1 to 14, further comprising a HEPES or TRIS buffer at a pH of about 7.0 to about 8.

5.

20. 15. The bacterial-derived lipid composition of any one of claims 1 to 14, further comprising one or more cryoprotectants.

21. The bacterial-derived lipid composition according to any one of claims 1 to 14, wherein the bacterial-derived lipid composition is a freeze-dried composition.

22. 22. The bacterially-derived lipid composition of claim 21, wherein the bacterially-derived lipid composition comprises one or more lyoprotectants.

23. 1. A method of making a bacterially derived lipid composition, comprising: (b) reconstituting the bacterial components comprising one or more lipids extracted from (a) the bacterial source in the presence of an ionizable lipid to produce a bacterial-derived lipid composition, wherein the ionizable lipid has the following characteristics: (i) at least two ionizable amines; (ii) at least three lipid tails, each of said lipid tails being at least six carbon atoms in length; (iii) a pKa of about 4.5 to about 7.5; (iv) an ionizable amine and heteroorganic group separated by a chain of at least two atoms; and (v) an N:P ratio of at least 10; and loading said bacterially derived lipid composition with one or more heterologous functional agents.

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