JPWO2020123569A5 - - Google Patents

Claims (29)

A method of delivering cargo to a target cell, comprising:
(a) providing a composition comprising a plurality of highly active achromosomal dynamic activity systems (ADAS), the ADAS having an initial ATP concentration of at least 1.25 mM, the ADAS comprising cargo; and (b) contacting a target cell with said composition of step (a) to deliver cargo to the target cell. 2. The method of claim 1, wherein the target cells are animal cells, plant cells, or insect cells. the cargo is a nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulator, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP) 2. The method of claim 1, wherein there is 2. The method of claim 1, wherein the cargo comprises a heterologous protein encoded by a plasmid in the ADAS. 2. The method of claim 1, wherein the cargo is encapsulated in ADAS. 2. The method of claim 1, wherein cargo is attached to the surface of the ADAS. ADAS are derived from a parent bacterium containing a cell division topology-specific factor, the parent bacterium being Escherichia, Acinetobacter, Agrobacterium, Anabaena, Aquifexus. Aquifex), Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Erwinia Genus Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum (Msoliborum), , Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Polaromonas, Pro Chlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella ( Shigella), Sinorhizobium, Synechococcus (Syn Echococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Vibrio Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exci Exiguobacterium, Geobacillus, Lactobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium, or Thermoanaerobacter, and the cell division topology-specific factor is the parent bacterium's endogenous minE or DivIVA. 2. The method of claim 1, wherein said providing comprises reconstituting lyophilized ADAS. 9. The method of claim 8, wherein the reconstituted ADAS has an ATP concentration that is at least 95% of the ATP concentration of the ADAS prior to lyophilization. Claim 1, wherein the ADAS has an initial ATP concentration of at least 1.3 nM, 1.4 mM, 1.5 mM, 1.6 mM, 2 mM, 2.5 mM, 3 mM, 4 mM, 5 mM, 10 mM, 20 mM, 30 mM, or 50 mM. The method described in . A method of treating an animal in need of treatment comprising:
(a) providing a composition comprising a plurality of highly active achromosomal dynamic activity systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM and the composition comprises viable bacterial cells and (b) contacting the animal with an effective amount of the composition of step (a) to treat the animal. 12. The method of claim 11, wherein the ADAS carries chemotherapy cargo or immunotherapy cargo. A method of treating a plant in need of treatment, comprising:
(a) providing a composition comprising a plurality of highly active achromosomal dynamic activity systems (ADAS), wherein the ADAS have an initial ATP concentration of at least 1.25 mM and the composition comprises viable bacterial cells and (b) a method comprising contacting the plant or pest thereof with an effective amount of the composition of step (a) to treat the plant. 14. The method of claim 13, wherein said providing comprises reconstituting lyophilized ADAS. 15. The method of claim 14, wherein the reconstituted ADAS has an ATP concentration that is at least 95% of the ATP concentration of the ADAS prior to lyophilization. 14. The method of Claim 13, wherein the ADAS comprises a cargo protein. 14. The method of claim 13, wherein the cargo protein is a growth factor, antibacterial, antifungal, or insecticide protein. 1. A method for producing a composition comprising a plurality of highly active achromosomal dynamic activity systems (ADAS), the composition being substantially free of viable bacterial cells, the method comprising:
(a) producing, providing, or obtaining a plurality of parent bacteria exhibiting reduced levels or activities of cell division topology-specific factors, said reduced levels of at least minE and minC or minD polypeptides; making, providing, or obtaining, including reducing the expression of one;
(b) exposing the parent bacteria to conditions permissive for the formation of minicells; and (c) separating the minicells from the parent bacteria, thereby substantially free of viable bacterial cells. A method, including making an object. 19. The method of claim 18, wherein the ATP concentration of ADAS increases after 12 hours of incubation at 37[deg.]C. 19. The method of claim 18, wherein the parent bacterium has a deletion of the minCDE operon. Parent bacteria include Escherichia, Acinetobacter, Agrobacterium, Anabaena, Aquifex, Azoarcus, Azotobacter, Bordetella Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocospheera Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum, Mesorhizobium, Methylococcus, Neisseria (, Nitrosomonas), Nostoc, Photobacterium, Photorhabdus, Polaromonas, Prochlorococcus, Pseudomonas, Cyclobacter ( Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella, Sinorhizobium, Synechococcus, Synechococcus (Synechocy stis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Wigglesworthia , Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exiguobacterium ), Geobacillus, Lactobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium, or Thermoanaerobacter ( Thermoanaerobacter). A composition comprising a plurality of highly active achromosomal dynamic activity systems (ADAS),
ADAS has an initial ATP concentration of at least 1 mM;
the composition is substantially free of viable bacterial cells;
The ADAS is derived from a parent bacterium that exhibits reduced expression of a minE polypeptide and at least one of a minC or minD polypeptide, and the ATP concentration of the ADAS is increased after 12 hours of incubation at 37°C.
Composition. ADAS are derived from a parent bacterium containing a cell division topology-specific factor, the parent bacterium being Escherichia, Acinetobacter, Agrobacterium, Anabaena, Aquifexus. Aquifex), Azoarcus, Azotobacter, Bordetella, Bradyrhizobium, Brucella, Buchnera, Burkholderia, Candidatus, Chromobacterium, Crocosphaera, Dechloromonas, Desulfitobacterium, Desulfotalea, Erwinia, Erwinia Genus Francisella, Fusobacterium, Gloeobacter, Gluconobacter, Helicobacter, Legionella, Magnetospirillum (Msoliborum), , Methylococcus, Neisseria, Nitrosomonas, Nostoc, Photobacterium, Photorhabdus, Polaromonas, Pro Chlorococcus, Pseudomonas, Psychrobacter, Ralstonia, Rubrivivax, Salmonella, Shewanella, Shigella ( Shigella), Sinorhizobium, Synechococcus (Syn Echococcus, Synechocystis, Thermosynechococcus, Thermotoga, Thermus, Thiobacillus, Trichodesmium, Vibrio, Vibrio Wigglesworthia, Wolinella, Xanthomonas, Xylella, Yersinia, Bacillus, Clostridium, Deinococcus, Exci Exiguobacterium, Geobacillus, Lactobacillus, Lactobacillus, Moorella, Oceanobacillus, Symbiobacterium, or Thermoanaerobacter and the cell division topology specificity factor is the parent bacterium's endogenous minE or DivIVA. 23. The composition of claim 22, wherein the ADAS comprises cargo. the cargo is a nucleic acid, plasmid, polypeptide, protein, enzyme, amino acid, small molecule, gene editing system, hormone, immunomodulator, carbohydrate, lipid, organic particle, inorganic particle, or ribonucleoprotein complex (RNP) 25. The composition of claim 24, comprising at least one of: 26. The composition of Claim 25, wherein the nucleic acid is DNA, RNA, or a plasmid. 25. The composition of claim 24, wherein cargo is expressed within ADAS. 25. The composition of claim 24, wherein the cargo is encapsulated in ADAS. 22. The composition of claim 21, wherein the parent bacterium has a deletion of the minCDE operon.