WO2024076723A1 - Processing extracellular vesicles - Google Patents
Processing extracellular vesicles Download PDFInfo
- Publication number
- WO2024076723A1 WO2024076723A1 PCT/US2023/034623 US2023034623W WO2024076723A1 WO 2024076723 A1 WO2024076723 A1 WO 2024076723A1 US 2023034623 W US2023034623 W US 2023034623W WO 2024076723 A1 WO2024076723 A1 WO 2024076723A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- evs
- bacteria
- filter
- prevotella
- product
- Prior art date
Links
- 238000012545 processing Methods 0.000 title claims description 25
- 238000000034 method Methods 0.000 claims abstract description 220
- 238000003306 harvesting Methods 0.000 claims abstract description 106
- 230000001580 bacterial effect Effects 0.000 claims abstract description 87
- 241000894006 Bacteria Species 0.000 claims description 368
- 230000010412 perfusion Effects 0.000 claims description 87
- 239000001963 growth medium Substances 0.000 claims description 72
- 239000002609 medium Substances 0.000 claims description 61
- 238000004587 chromatography analysis Methods 0.000 claims description 50
- 238000004519 manufacturing process Methods 0.000 claims description 44
- 239000007788 liquid Substances 0.000 claims description 23
- 239000004695 Polyether sulfone Substances 0.000 claims description 22
- 229920006393 polyether sulfone Polymers 0.000 claims description 22
- 230000004907 flux Effects 0.000 claims description 21
- 238000009295 crossflow filtration Methods 0.000 claims description 18
- 238000001914 filtration Methods 0.000 claims description 12
- 239000000047 product Substances 0.000 description 155
- 210000004027 cell Anatomy 0.000 description 33
- 241000909283 Negativicutes Species 0.000 description 23
- 108091033409 CRISPR Proteins 0.000 description 21
- 239000011148 porous material Substances 0.000 description 21
- 238000010354 CRISPR gene editing Methods 0.000 description 20
- 239000002773 nucleotide Substances 0.000 description 20
- 125000003729 nucleotide group Chemical group 0.000 description 20
- 241001576959 Megasphaera sp. Species 0.000 description 18
- 230000008569 process Effects 0.000 description 18
- 241000894007 species Species 0.000 description 18
- 206010028980 Neoplasm Diseases 0.000 description 17
- 201000011510 cancer Diseases 0.000 description 17
- 239000002699 waste material Substances 0.000 description 17
- 239000002207 metabolite Substances 0.000 description 16
- 241000723109 Agathobaculum Species 0.000 description 15
- 241000352296 Megasphaera massiliensis Species 0.000 description 15
- 241000605036 Selenomonas Species 0.000 description 15
- 241001430183 Veillonellaceae Species 0.000 description 15
- 238000002360 preparation method Methods 0.000 description 15
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 15
- 241001584243 Fournierella massiliensis Species 0.000 description 13
- 241000752432 Harryflintia acetispora Species 0.000 description 13
- 241000605861 Prevotella Species 0.000 description 13
- 241000660744 Selenomonadaceae Species 0.000 description 13
- 241000660742 Sporomusaceae Species 0.000 description 13
- 241001112696 Clostridia Species 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 241000604449 Megasphaera Species 0.000 description 11
- 238000001035 drying Methods 0.000 description 10
- 238000003801 milling Methods 0.000 description 10
- 238000007873 sieving Methods 0.000 description 10
- 241001148134 Veillonella Species 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 239000008188 pellet Substances 0.000 description 9
- 241000909284 Acidaminococcaceae Species 0.000 description 8
- 241000604451 Acidaminococcus Species 0.000 description 8
- 241000605980 Faecalibacterium prausnitzii Species 0.000 description 8
- 241000186660 Lactobacillus Species 0.000 description 8
- 241000692844 Prevotellaceae Species 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 229940039696 lactobacillus Drugs 0.000 description 8
- 238000002703 mutagenesis Methods 0.000 description 8
- 231100000350 mutagenesis Toxicity 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 241001116699 Acidaminococcus intestini Species 0.000 description 7
- 241001607451 Oscillospiraceae Species 0.000 description 7
- 241001482483 Prevotella histicola Species 0.000 description 7
- 229930006000 Sucrose Natural products 0.000 description 7
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 7
- 241000868652 Tannerellaceae Species 0.000 description 7
- 241001464867 [Ruminococcus] gnavus Species 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 210000003097 mucus Anatomy 0.000 description 7
- 239000005720 sucrose Substances 0.000 description 7
- 238000005199 ultracentrifugation Methods 0.000 description 7
- 241000186000 Bifidobacterium Species 0.000 description 6
- 108010019670 Chimeric Antigen Receptors Proteins 0.000 description 6
- 241001608234 Faecalibacterium Species 0.000 description 6
- 241000192125 Firmicutes Species 0.000 description 6
- 241000282414 Homo sapiens Species 0.000 description 6
- 230000003115 biocidal effect Effects 0.000 description 6
- 239000012141 concentrate Substances 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 241001112693 Lachnospiraceae Species 0.000 description 5
- 241000194034 Lactococcus lactis subsp. cremoris Species 0.000 description 5
- 241000606210 Parabacteroides distasonis Species 0.000 description 5
- 241000736131 Sphingomonas Species 0.000 description 5
- 241001584876 Synergistaceae Species 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 230000005291 magnetic effect Effects 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 241001141113 Bacteroidia Species 0.000 description 4
- 241000946390 Catenibacterium Species 0.000 description 4
- 241001430149 Clostridiaceae Species 0.000 description 4
- 240000006024 Lactobacillus plantarum Species 0.000 description 4
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 4
- 241000192041 Micrococcus Species 0.000 description 4
- 241000588771 Morganella <proteobacterium> Species 0.000 description 4
- 241000588769 Proteus <enterobacteria> Species 0.000 description 4
- 241000589180 Rhizobium Species 0.000 description 4
- 241000692845 Rikenellaceae Species 0.000 description 4
- 235000014962 Streptococcus cremoris Nutrition 0.000 description 4
- 241001584890 Synergistia Species 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 229940072205 lactobacillus plantarum Drugs 0.000 description 4
- 238000011068 loading method Methods 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 230000005298 paramagnetic effect Effects 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000001694 spray drying Methods 0.000 description 4
- 241000186033 Alloiococcus Species 0.000 description 3
- 241000193830 Bacillus <bacterium> Species 0.000 description 3
- 241001202853 Blautia Species 0.000 description 3
- 241000186216 Corynebacterium Species 0.000 description 3
- 241001528480 Cupriavidus Species 0.000 description 3
- 241001552883 Enhydrobacter Species 0.000 description 3
- 241000186394 Eubacterium Species 0.000 description 3
- 241001468125 Exiguobacterium Species 0.000 description 3
- 241000368889 Fournierella Species 0.000 description 3
- 230000005526 G1 to G0 transition Effects 0.000 description 3
- 241000626621 Geobacillus Species 0.000 description 3
- 241000813462 Harryflintia Species 0.000 description 3
- 241000054885 Leuconostoc holzapfelii Species 0.000 description 3
- 241000589323 Methylobacterium Species 0.000 description 3
- 241000160321 Parabacteroides Species 0.000 description 3
- 108091008874 T cell receptors Proteins 0.000 description 3
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000010923 batch production Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000011210 chromatographic step Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 230000006870 function Effects 0.000 description 3
- 230000002519 immonomodulatory effect Effects 0.000 description 3
- 238000000099 in vitro assay Methods 0.000 description 3
- 239000000411 inducer Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 210000000813 small intestine Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000702460 Akkermansia Species 0.000 description 2
- 241001288806 Alloprevotella tannerae Species 0.000 description 2
- 241000193818 Atopobium Species 0.000 description 2
- 241001135237 Bacteroides heparinolyticus Species 0.000 description 2
- 241001135233 Bacteroides zoogleoformans Species 0.000 description 2
- 241000643891 Blautia massiliensis Species 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 2
- 241000755889 Christensenellaceae Species 0.000 description 2
- 241001112695 Clostridiales Species 0.000 description 2
- 241000928573 Cutibacterium Species 0.000 description 2
- 241001464974 Cutibacterium avidum Species 0.000 description 2
- 108010053770 Deoxyribonucleases Proteins 0.000 description 2
- 102000016911 Deoxyribonucleases Human genes 0.000 description 2
- 241000995910 Dielma fastidiosa Species 0.000 description 2
- 108010067770 Endopeptidase K Proteins 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 102100021455 Histone deacetylase 3 Human genes 0.000 description 2
- 101000731015 Homo sapiens Peptidoglycan recognition protein 1 Proteins 0.000 description 2
- 241001304190 Hungatella Species 0.000 description 2
- 241000588748 Klebsiella Species 0.000 description 2
- 241000588749 Klebsiella oxytoca Species 0.000 description 2
- 241000906776 Klebsiella quasipneumoniae Species 0.000 description 2
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical class C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 2
- 244000199866 Lactobacillus casei Species 0.000 description 2
- 235000013958 Lactobacillus casei Nutrition 0.000 description 2
- 241000186605 Lactobacillus paracasei Species 0.000 description 2
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 2
- 241000186612 Lactobacillus sakei Species 0.000 description 2
- 241000194036 Lactococcus Species 0.000 description 2
- 241000192132 Leuconostoc Species 0.000 description 2
- 241001140861 Longicatena caecimuris Species 0.000 description 2
- 241000590158 Paraclostridium benzoelyticum Species 0.000 description 2
- 102000052544 Peptidoglycan recognition protein Human genes 0.000 description 2
- 108010009051 Peptidoglycan recognition protein Proteins 0.000 description 2
- 102100032393 Peptidoglycan recognition protein 1 Human genes 0.000 description 2
- 241000191992 Peptostreptococcus Species 0.000 description 2
- 241001302521 Prevotella albensis Species 0.000 description 2
- 241001041813 Prevotella amnii Species 0.000 description 2
- 241000326476 Prevotella aurantiaca Species 0.000 description 2
- 241000938719 Prevotella baroniae Species 0.000 description 2
- 241000987248 Prevotella bergensis Species 0.000 description 2
- 241001135215 Prevotella bivia Species 0.000 description 2
- 241001646114 Prevotella brevis Species 0.000 description 2
- 241001299661 Prevotella bryantii Species 0.000 description 2
- 241001135217 Prevotella buccae Species 0.000 description 2
- 241001135206 Prevotella buccalis Species 0.000 description 2
- 241001147550 Prevotella colorans Species 0.000 description 2
- 241000385060 Prevotella copri Species 0.000 description 2
- 241001135208 Prevotella corporis Species 0.000 description 2
- 241000509620 Prevotella dentalis Species 0.000 description 2
- 241001678008 Prevotella dentasini Species 0.000 description 2
- 241001135209 Prevotella denticola Species 0.000 description 2
- 241001135219 Prevotella disiens Species 0.000 description 2
- 241001288803 Prevotella enoeca Species 0.000 description 2
- 241000714308 Prevotella falsenii Species 0.000 description 2
- 241001678472 Prevotella fusca Species 0.000 description 2
- 241001135221 Prevotella intermedia Species 0.000 description 2
- 241000495654 Prevotella jejuni Species 0.000 description 2
- 241000605951 Prevotella loescheii Species 0.000 description 2
- 241000124542 Prevotella maculosa Species 0.000 description 2
- 241001141018 Prevotella marshii Species 0.000 description 2
- 241001135223 Prevotella melaninogenica Species 0.000 description 2
- 241001141020 Prevotella micans Species 0.000 description 2
- 241000782070 Prevotella multiformis Species 0.000 description 2
- 241001221454 Prevotella multisaccharivorax Species 0.000 description 2
- 241001365165 Prevotella nanceiensis Species 0.000 description 2
- 241001135225 Prevotella nigrescens Species 0.000 description 2
- 241001135261 Prevotella oralis Species 0.000 description 2
- 241001135262 Prevotella oris Species 0.000 description 2
- 241001103687 Prevotella oryzae Species 0.000 description 2
- 241001135263 Prevotella oulorum Species 0.000 description 2
- 241000864367 Prevotella pallens Species 0.000 description 2
- 241001103688 Prevotella paludivivens Species 0.000 description 2
- 241000665168 Prevotella pleuritidis Species 0.000 description 2
- 241000605860 Prevotella ruminicola Species 0.000 description 2
- 241001116196 Prevotella saccharolytica Species 0.000 description 2
- 241000331195 Prevotella salivae Species 0.000 description 2
- 241001678470 Prevotella scopos Species 0.000 description 2
- 241000331194 Prevotella shahii Species 0.000 description 2
- 241001430102 Prevotella stercorea Species 0.000 description 2
- 241000530934 Prevotella timonensis Species 0.000 description 2
- 241001135264 Prevotella veroralis Species 0.000 description 2
- 241000186429 Propionibacterium Species 0.000 description 2
- 241000589516 Pseudomonas Species 0.000 description 2
- 108020004422 Riboswitch Proteins 0.000 description 2
- 241000831652 Salinivibrio sharmensis Species 0.000 description 2
- 241000607768 Shigella Species 0.000 description 2
- 241000191940 Staphylococcus Species 0.000 description 2
- 241001147795 Tyzzerella nexilis Species 0.000 description 2
- 241001592639 Veillonella tobetsuensis Species 0.000 description 2
- 241000975185 Weissella cibaria Species 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000011260 co-administration Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 108010074724 histone deacetylase 3 Proteins 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000000543 intermediate Substances 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 229940017800 lactobacillus casei Drugs 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000006241 metabolic reaction Methods 0.000 description 2
- -1 molecules Chemical class 0.000 description 2
- 210000000214 mouth Anatomy 0.000 description 2
- 239000012466 permeate Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 241000589291 Acinetobacter Species 0.000 description 1
- 241000588626 Acinetobacter baumannii Species 0.000 description 1
- 241000186046 Actinomyces Species 0.000 description 1
- 241000702462 Akkermansia muciniphila Species 0.000 description 1
- 241000186032 Alloiococcus otitis Species 0.000 description 1
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 1
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 241001633064 Atopobium vaginae Species 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 241000692822 Bacteroidales Species 0.000 description 1
- 241000606125 Bacteroides Species 0.000 description 1
- 241000985922 Bariatricus massiliensis Species 0.000 description 1
- 241001495171 Bilophila Species 0.000 description 1
- 241001453380 Burkholderia Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000186226 Corynebacterium glutamicum Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000252867 Cupriavidus metallidurans Species 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000192093 Deinococcus Species 0.000 description 1
- 241000192091 Deinococcus radiodurans Species 0.000 description 1
- 241001535083 Dialister Species 0.000 description 1
- 241001263137 Dielma Species 0.000 description 1
- 241001468127 Exiguobacterium aurantiacum Species 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000605909 Fusobacterium Species 0.000 description 1
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 1
- 241000589989 Helicobacter Species 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000186606 Lactobacillus gasseri Species 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 241001015936 Longicatena Species 0.000 description 1
- 241000481564 Methylobacterium jeotgali Species 0.000 description 1
- 241000191938 Micrococcus luteus Species 0.000 description 1
- 241000588772 Morganella morganii Species 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 241000927544 Olsenella Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 241000742045 Paraclostridium Species 0.000 description 1
- 241001112692 Peptostreptococcaceae Species 0.000 description 1
- 241000605894 Porphyromonas Species 0.000 description 1
- 241000588770 Proteus mirabilis Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241000589194 Rhizobium leguminosarum Species 0.000 description 1
- 241000316848 Rhodococcus <scale insect> Species 0.000 description 1
- 241000158504 Rhodococcus hoagii Species 0.000 description 1
- 241000605947 Roseburia Species 0.000 description 1
- 241000398180 Roseburia intestinalis Species 0.000 description 1
- 241001453443 Rothia <bacteria> Species 0.000 description 1
- 241000428493 Rothia amarae Species 0.000 description 1
- 241000192031 Ruminococcus Species 0.000 description 1
- 241000909295 Selenomonadales Species 0.000 description 1
- 241000736110 Sphingomonas paucimobilis Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 241000123710 Sutterella Species 0.000 description 1
- 241001584893 Synergistales Species 0.000 description 1
- 241001425419 Turicibacter Species 0.000 description 1
- 241000125947 Tyzzerella Species 0.000 description 1
- 241000660765 Veillonellales Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 241001148470 aerobic bacillus Species 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 210000003578 bacterial chromosome Anatomy 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 230000001851 biosynthetic effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 239000002962 chemical mutagen Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000010924 continuous production Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 238000011143 downstream manufacturing Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 238000010228 ex vivo assay Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000002175 goblet cell Anatomy 0.000 description 1
- 238000012789 harvest method Methods 0.000 description 1
- 229940037467 helicobacter pylori Drugs 0.000 description 1
- 239000012510 hollow fiber Substances 0.000 description 1
- 230000005745 host immune response Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000005934 immune activation Effects 0.000 description 1
- 230000008629 immune suppression Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 description 1
- 229960004359 iodixanol Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 210000003126 m-cell Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 230000007269 microbial metabolism Effects 0.000 description 1
- 229940076266 morganella morganii Drugs 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 210000001331 nose Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000000856 sucrose gradient centrifugation Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 230000009452 underexpressoin Effects 0.000 description 1
- 210000001215 vagina Anatomy 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D43/00—Separating particles from liquids, or liquids from solids, otherwise than by sedimentation or filtration
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
- B01D15/08—Selective adsorption, e.g. chromatography
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/10—Separation or concentration of fermentation products
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
Definitions
- Extracellular vesicles are natural lipoprotein nanoparticles produced by many species of bacteria. Their macromolecular content is a complex subset of proteins, glycans, lipids, and LPS. Bacteria can secrete extracellular vesicles into the culture medium.
- Methods for increasing the production of EVs by bacteria into the medium for a given culture include using perfusion culture systems instead of batch culture systems. Also, increasing the size of the cultures (such as to a commercial scale such as a 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater) (for batch or perfusion cultures) also increases yields.
- Methods are developed to harvest the increased yields of EVs from bacterial cultures. Such methods include filter systems (such as two-filter systems) and chromatography techniques (such as monoliths) to decrease volumes, increase concentrations, and/or increase purity of EVs after medium (that contains EVs) is removed from cultures.
- the disclosure provides a method of producing extracellular vesicles (EVs), the method comprising growing EV-producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
- a perfusion culture e.g., wherein the perfusion culture comprises culture media that comprises EVs
- the disclosure provides a method of producing extracellular vesicles (EVs), the method comprising growing EV-producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
- the perfusion culture increases EV yields by at least about 10- fold, e.g., by at least about 15-fold or by at least about 17-fold or by at least about 50-fold, as compared to a batch culture of the same bacteria.
- the perfusion culture increases EV yields after 24, 48, or 72 hours of culturing, as compared to a batch culture of the same bacteria.
- EV production of the bacteria is coupled to growth in batch culture.
- EV production of the bacteria is not coupled to growth in batch culture.
- the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
- a filter system (such as a one-filter system or a two-filter system) removes EVs, metabolites, and waste products of the culture media.
- the filter system is a two-filter system.
- the method further comprises filtering the culture media.
- the method further comprises performing chromatography on the culture media.
- the method further comprises performing tangential flow filtration on the culture media.
- the method further comprises drying the culture media.
- the culture media is dried after the growing step.
- the culture media is dried after the filtering step.
- the culture media is dried after the chromatography step.
- the culture media is dried after the tangential flow filtration step.
- the method further comprises milling the dried culture media.
- the disclosure provides a culture media produced by a perfusion culture method provided herein.
- the disclosure provides a culture media produced by a method of producing extracellular vesicles provided herein.
- the disclosure provides a method of processing bacterial culture media that comprises extracellular vesicles (EVs), the method comprising passing bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter (e.g., wherein the output of the product harvest filter comprises EVs) and the second filter is a medium exchange filter.
- the first filter is a product harvest filter (e.g., wherein the output of the product harvest filter comprises EVs)
- the second filter is a medium exchange filter.
- the bacterial culture is a perfusion culture.
- the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
- the product harvest filter and the medium exchange filter comprise the same material.
- the product harvest filter comprises PES (polyethersulfone).
- the medium exchange filter comprises PES (polyethersulfone).
- the product harvest filter and the medium exchange filter comprise PES (polyethersulfone).
- EVs, media, waste and metabolites pass through the product harvest filter.
- the pore size of the product harvest filter is about 0.5 micron.
- media, waste and metabolites pass through the medium exchange filter.
- the pore size of the medium exchange filter is less than about 0.5 micron.
- the pore size of the medium exchange filter is about 0.05 micron.
- the pore size of the medium exchange filter is about 0.02 micron.
- the pore size of the medium exchange filter is about 0.01 micron.
- the pore size of the medium exchange filter comprises a size cut off of 750kD (kilodalton).
- the pore size of the medium exchange filter comprises a size cut off of 500kD.
- the medium exchange filter runs at a higher flux than the product harvest filter.
- the flux ratio (medium exchange filterproduct harvest filter) is about 5: 1.
- the flux ratio (medium exchange filterproduct harvest filter) is about 9: 1.
- the flux ratio (medium exchange filter product harvest filter) is about 10: 1.
- the flux ratio reduces sieving of the product harvest filter (e.g., as compared to the amount of sieving if the flux ratio was 1 : 1 or if the product harvest filter was used alone).
- the volume of the output of the product harvest filter is about l/5x the volume than if a single-filter system was used.
- the volume of the output of the product harvest filter is about l/9x the volume than if a single-filter system was used. [41] In some embodiments, the volume of the output of the product harvest filter is about l/10x the volume than if a single-filter system was used.
- the output of the product harvest filter comprises a higher concentration of EVs than if a single-filter system was used.
- the output of the product harvest filter comprises a concentration of EVs that is at least about 5x higher than if a single-filter system was used.
- the output of the product harvest filter comprises a concentration of EVs that is at least about 9x higher than if a single-filter system was used.
- the output of the product harvest filter comprises a concentration of EVs that is at least about lOx higher than if a single-filter system was used.
- the method further comprises performing chromatography on the output of the product harvest filter.
- the method further comprises performing tangential flow filtration on the output of the product harvest filter.
- the method further comprises drying the output of the product harvest filter.
- the output is dried after the filtering step.
- the output is dried after the chromatography step.
- the output is dried after the tangential flow filtration step.
- the method further comprises milling the dried output of the product harvest filter.
- the disclosure provides an output of a product harvest filter produced by a method of processing bacterial culture media provided herein.
- the disclosure provides a method of processing a liquid that comprises extracellular vesicles (EVs) to prepare an EV eluate, the method comprising performing chromatography on the liquid.
- EVs extracellular vesicles
- the liquid comprises bacterial culture media.
- the bacterial culture media is from a perfusion culture.
- the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
- the liquid comprises a product-containing volume.
- the product-containing volume is output from a single-filter system.
- the product-containing volume is output from a two-filter system.
- the product-containing volume is output from a product harvest filter.
- the product harvest filter comprises PES (polyethersulfone).
- the pore size of the product harvest filter is about 0.5 micron.
- the chromatography comprises a chromatography column.
- the chromatography column comprises a monolith.
- the chromatography column comprises a monolith ion exchange column.
- the method comprises processing on one column.
- the liquid is loaded onto the column and then EV-containing eluate is eluted.
- the method comprises processing on two columns.
- the liquid is loaded onto a first column, then EV-containing eluate is eluted from the first column, and while EV-containing eluate is eluted from the first column, liquid is loaded onto the second column.
- EV-containing eluate is eluted from the second column, and while EV-containing eluate is eluted from the second column, liquid is loaded onto the first column.
- the loading and eluting steps on the first and second columns are alternated to process the liquid continuously.
- bacterial culture media is filtered prior to performing the chromatography.
- the chromatography enriches EV yield by greater than about 5-fold.
- the chromatography enriches EV yield by about 6-fold.
- the chromatography enriches EV yield by about 12-fold.
- the yield of EVs from the chromatography is greater than about 50%.
- the yield of EVs from the chromatography is greater than about 60%.
- the method further comprises performing tangential flow filtration on the EV eluate. [77] In some embodiments, the method further comprises drying the EV eluate. In some embodiments, the EV eluate is dried after the chromatography step. In some embodiments, the EV eluate is dried after the tangential flow filtration step.
- the method further comprises milling the dried EV eluate.
- the disclosure provides an EV eluate produced by a method of processing a liquid that comprises extracellular vesicles (EVs) provided herein.
- EVs extracellular vesicles
- the disclosure provides a method comprising:
- the disclosure provides an output of a product harvest filter produced by a method provided herein.
- the disclosure provides a method comprising:
- the disclosure provides an eluate produced by a method provided herein.
- the disclosure provides a method comprising:
- EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs (e.g., as described herein);
- the disclosure provides an eluate produced by a method provided herein.
- the method comprises EVs from a bacterial strain that is associated with mucus.
- the method comprises EVs from anaerobic bacteria.
- the anaerobic bacteria are obligate (e.g., strict) anaerobes.
- the anaerobic bacteria are facultative anaerobes.
- the anaerobic bacteria are aerotolerant anaerobes.
- the EVs are from monoderm bacteria.
- the EVs are from diderm bacteria.
- the EVs are from Gram negative bacteria.
- the EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae;
- the EVs are from Gram positive bacteria.
- the EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
- the EVs are from bacteria of the genus Prevotella.
- the EVs are from bacteria of the genus Veillonella.
- the EVs are from bacteria of the genus Parabacteroides.
- the EVs are from bacteria of the Oscillospiraceae family. [101] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Tannerellaceae family.
- the EVs are from bacteria of the Prevotellaceae family.
- the EVs are from bacteria of the Veillonellaceae family.
- the EVs are from bacteria of class, order, family, genus, species and/or strain of bacteria provided in Table 1, Table 2, Table 3, and/or Table 4.
- the disclosure provides a product produced by a method provided herein.
- Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity.
- Figures 2A and 2B are graphs showing comparisons of EV yields (EV product batches (-fold)) from batch culture versus perfusion culture yields over time (hours).
- Figure 2A shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is coupled with growth.
- Figure 2B shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is not coupled with growth.
- the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).
- FIG. 3 is a schematic showing a set up for a two-filter system.
- Harvest volume transfers from the fermenter to the two filters: product harvest filter and medium exchange filter.
- Captured product from the product harvest filter transfers to a product reservoir and can be further processed, such as through a capture step(s). Metabolites and waste products that passed through the medium exchange filter transfer to a waste reservoir.
- Figure 4 is a graph showing a theoretical result of using a two-filter (dual membrane perfusion) system. Shifting flux from the “Product Harvest” filter to the “Medium Exchange” filter increases the Flux Ratio, and results in product concentration (-fold; upward sloping line) increasing and permeate volume (downward sloping line; volume (vol)/day) decreasing with increasing ratio. DETAILED DESCRIPTION
- the disclosure provides methods developed to harvest EVs (such as increased yields of EVs) from bacterial cultures. Yields can be increased, for example, by using perfusion culture systems instead of batch culture systems and/or by increasing the size of the cultures (such as to a commercial scale such as a 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater) (for batch or perfusion cultures).
- Such harvest methods include filter systems (such as two-filter systems) and chromatography techniques (such as monoliths) to decrease volumes, increase concentrations, and/or increase purity of EVs after medium (that contains EVs) is removed from cultures.
- perfusion culture can increase EV yields by at least 10-fold, e.g., by at least 15-fold or 17-fold or 50-fold, after 72 hours of culturing.
- a filter system removes metabolites, and waste products of the culture, yet does not remove the bacterial cells.
- the filter system also removes product (EVs).
- the volumes of media from a perfusion culture are greater than for a batch process (for example, up to ten times greater per day).
- filter area and flow rates need to be managed to ensure sufficient removal of product, metabolites, and waste products, and to avoid the need for expensive enlarged filter areas.
- flow rates and volumes need to be managed to minimize filter sieving.
- a two-filter system can address one or more of these considerations.
- Additional further processing of the output of the perfusion culture can be performed.
- additional further processing can include filtration (such as with a two-filter system), chromatography, tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
- a two-filter system can be used to process the culture media.
- the two-filter system can be used as part of a continuous process (e.g., as opposed to a batch or intermittent process).
- the two filters can be run at the same time, e.g., but at different flow rates.
- One filter functions to collect product (e.g., EVs) (the product harvest filter).
- the pore size of the product harvest filter is selected to allow product (e.g., EVs) to pass through, such as a 0.5 micron pore size.
- Product e.g., EVs
- the second filter functions to collect media, metabolites, and waste products (the medium exchange filter), yet product (e.g., EVs) does not pass through.
- the pore size of the medium exchange filter is selected to allow media, metabolites, and waste products to pass through but to not allow product to pass through, such as a 0.05 micron (or smaller, such as 0.02 micron or 0.01 micron) pore size.
- the medium exchange filter can be selected based on size cut-off: such as a 750kD or 500kD size limits for what can pass through.
- size cut-off such as a 750kD or 500kD size limits for what can pass through.
- Both the product harvest filter and the medium exchange filter can be made of the same material, such as PES (polyethersulfone).
- the medium exchange filter runs at a higher flux than the product harvest filter.
- the flux ratio (medium exchange filterproduct harvest filter) can be 5: 1, or 9: 1, or 10: 1.
- this allows the product harvest filter to collect higher concentration product (EVs) in a smaller volume, such as 1/5, 1/9 or 1/10 the volume than if a single-filter system was used.
- EVs concentration product
- a smaller volume such as 12,000 liters is further processed.
- This reduced volume provides advantages for further processing of the product-containing volume (e.g., the output of the product harvest filter when a two-filter system is used).
- a two-filter (dual-membrane) perfusion system in place of a single-filter perfusion system reduces downstream volume and increases product concentration.
- Additional further processing of the output of the two-filter system can be performed.
- Such additional further processing can include chromatography, tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
- chromatography Downstream of growing a bacterial culture for EV production (such as by perfusion culture (particularly when perfusion culturing is performed at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater))), chromatography can be used to process culture media or a product-containing volume.
- Chromatography can be used to process a product-containing volume, such as after culture media containing product (e.g., EVs) is filtered, such as through a single-filter system or a two-filter system.
- a monolith can be used (e.g., such as a monolith supplied by Sartorius).
- a monolith is cast as a single block and is inserted into a chromatographic housing. The monolith is characterized by a highly interconnected network of channels.
- Considerations include the feasibility and specificity of chromatography to capture the product (e.g., EVs); the large size of the chromatography matrices (such as monoliths); binding capacity; process volumes (including load, buffers and waste); and pool volume management.
- One or two columns can be used to process culture media (e.g., from a perfusion culture) or a product-containing volume, such as after culture media containing product (e.g., EVs) is filtered, such as through a single-filter system or a two-filter system.
- culture media or product-containing volume is loaded onto the column and then the product is eluted.
- culture media or product-containing volume is loaded onto a first column. While product is being eluted from the first column, culture media or product-containing volume is loaded onto a second column.
- culture media or product-containing volume is loaded onto the first column while product is being eluted from the second column.
- the loading and eluting steps on the first and second columns can continue to be alternated to process culture media or product-containing volume continuously. This allows for continuous capture, such as to achieve pure and highly concentrated product (this may be considered a process intermediate if additional further processing is performed).
- pH can affect column loading capacity, such as by three-fold. Enrichment factors can be greater than about 5-fold, e.g., 6- or 12-fold. Yields from the chromatography can be, for example, greater than about 50%, e.g., greater than about 60%.
- Additional further processing of the output of the chromatography can be performed.
- Such additional further processing can include tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
- EVs Extracellular vesicles
- EVs may be naturally-produced vesicles derived from bacteria. EVs are comprised of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate moieties, and are isolated from culture supernatant. The natural production of these vesicles can be artificially enhanced (for example, increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (for example, by media or temperature alterations).
- EV compositions may be modified to reduce, increase, add, or remove bacterial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (for example, lymph node), absorption (for example, gastrointestinal), and/or yield (for example, thereby altering the efficacy).
- purified EV composition or “EV composition” refers to a preparation of EVs that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components.
- Extracellular vesicles may also be obtained from mammalian cells and from can be obtained from microbes such as archaea, fungi, microscopic algae, protozoans, and parasites.
- “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J.
- isolated or “enriched” encompasses a microbe, an EV (such as a bacterial EV) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man.
- Isolated bacteria or EVs may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated.
- isolated bacteria or EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure, for example, substantially free of other components.
- Metal refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or bacterial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or bacterial metabolic reaction.
- a substance is “pure” if it is substantially free of other components.
- the terms “purify,” “purifying,” and “purified” refer to an EV (such as an EV from bacteria) preparation or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (for example, whether in nature or in an experimental setting), or during any time after its initial production.
- An EV preparation or compositions may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, and a purified microbe or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.”
- purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
- EV compositions (or preparations) are, for example, purified from residual habitat products.
- the term “purified EV composition” or “EV composition” refers to a preparation that includes EVs from bacteria that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments, the EVs are concentrated by 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or more than 10,000-fold.
- Strain refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species.
- the genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof.
- strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome.
- strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
- Bacteria propagated as sources of EVs can be selected based on assays in the art that identify bacteria with properties of interest. For example, in some embodiments, bacteria are selected for the ability to modulate host immune response and/or affect cytokine levels.
- EVs are selected from a bacterial strain that is associated with mucus.
- the mucus is associated with the gut lumen.
- the mucus is associated with the small intestine.
- the mucus is associated with the respiratory tract.
- EVs are selected from a bacterial strain that is associated with an epithelial tissue, such as oral cavity, lung, nose, or vagina.
- the EVs are from bacteria that are human commensals.
- the EVs are from human commensal bacteria that originate from the human small intestine.
- the EVs are from human commensal bacteria that originate from the human small intestine and are associated there with the outer mucus layer.
- taxonomic groups such as class, order, family, genus, species and/or strain
- Examples of taxonomic groups (such as class, order, family, genus, species and/or strain) of bacteria that can be used as a source of EVs described herein are provided in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere throughout the specification.
- the bacterial strain is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification).
- the EVs are from an oncotrophic bacteria.
- the EVs are from an immunostimulatory bacteria.
- the EVs are from an immunosuppressive bacteria.
- the EVs are from an immunomodulatory bacteria. In certain embodiments, EVs are generated from a combination of bacterial strains provided herein. In some embodiments, the combination is a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 bacterial strains.
- the combination includes EVs from bacterial strains provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification and/or bacterial strains having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification.
- bacteria from a taxonomic group for example, class, order, family, genus, species or strain
- Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification can be used as a source of EVs.
- the EVs are obtained from Gram negative bacteria.
- the Gram negative bacteria belong to the class Negativicutes.
- the Negativicutes represent a unique class of microorganisms as they are the only diderm members of the Firmicutes phylum. These anaerobic organisms can be found in the environment and are normal commensals of the oral cavity and GI tract of humans. Because these organisms have an outer membrane, the yields of EVs from this class were investigated. It was found that on a per cell basis these bacteria produce a high number of vesicles (10-150 EVs/cell). The EVs from these organisms are broadly stimulatory and highly potent in in vitro assays. Investigations into their therapeutic applications in several oncology and inflammation in vivo models have shown their therapeutic potential.
- the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae. and Sporomusaceae .
- the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus.
- Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestine, and Propionospora sp.
- the EVs are obtained from Gram positive bacteria.
- the EVs are from aerotol erant bacteria.
- the EVs are from monoderm bacteria.
- the EVs are from diderm bacteria.
- the EVs are from bacteria of the family: Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; or Akkermaniaceae .
- the EVs are from bacteria of the family Oscillospiraceae ; Clostridiaceae; Lachnospiraceae; or Christensenellaceae .
- the EVs are from bacteria of the genus Prevotella.
- the EVs are from bacteria of the genus Veillonella. [152] In some embodiments, the EVs are from bacteria of the mis Parabacteroides.
- the EVs are from a bacterial strain of the Oscillospiraceae family.
- the EVs are from a bacterial strain of the Tannerellaceae family.
- the EVs are from a bacterial strain of the Prevotellaceae family.
- the EVs are from a bacterial strain of the Veillonellaceae family.
- the EVs are obtained from aerobic bacteria.
- the EVs are obtained from anaerobic bacteria.
- the anaerobic bacteria comprise obligate anaerobes.
- the anaerobic bacteria comprise facultative anaerobes.
- the EVs are obtained from acidophile bacteria.
- the EVs are obtained from alkaliphile bacteria.
- the EVs are obtained from neutral ophile bacteria.
- the EVs are obtained from fastidious bacteria.
- the EVs are obtained from nonfasti di ous bacteria.
- bacteria from which EVs are obtained are lyophilized.
- bacteria from which EVs are obtained are gamma irradiated (for example, at 17.5 or 25 kGy).
- bacteria from which EVs are obtained are UV irradiated.
- bacteria from which EVs are obtained are heat inactivated
- bacteria from which EVs are obtained are acid treated.
- bacteria from which EVs are obtained are oxygen sparged
- the EVs are lyophilized.
- the EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
- the EVs are UV irradiated.
- the EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
- the EVs are acid treated.
- the EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
- the phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria.
- EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
- EVs can be isolated from a batch culture of bacteria.
- EVs can be isolated from a perfusion culture of bacteria.
- the EVs described herein are obtained from obligate anaerobic bacteria.
- obligate anaerobic bacteria include gram-negative rods (including the genera of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterella sppf, gram -positive cocci (primarily Peptostreptococcus sppf, gram -positive spore-forming (Clostridium sppf, non-spore-forming bacilli (Actinomyces,
- the obligate anaerobic bacteria are of a genus selected from the group consisting of Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter, and Tyzzerella.
- the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
- the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, mA Acidaminococcus.
- Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
- the EVs are from bacteria of the Negativicutes class.
- the EVs are from bacteria of the Veillonellaceae family.
- the EVs are from bacteria of the Selenomonadaceae family.
- the EVs are from bacteria of the Acidaminococcaceae family.
- the EVs are from bacteria of the Sporomusaceae family.
- the EVs are from bacteria of the Megasphaera genus.
- the EVs are from bacteria of the Selenomonas genus.
- the EVs are from bacteria of the Propionospora genus.
- the EVs are from bacteria of the Acidaminococcus genus.
- the EVs are from Megasphaera sp. bacteria.
- the EVs are from Selenomonas felix bacteria. [192] In some embodiments, the EVs are from Acidaminococcus intestini bacteria.
- the EVs are from Propionospora sp. bacteria.
- the EVs are from bacteria of the Clostridia class.
- the EVs are from bacteria of the Oscillospriraceae family.
- the EVs are from bacteria of the Faecalibacterium genus.
- the EVs are from bacteria of the Fournierella genus.
- the EVs are from bacteria of the Harryflintia genus.
- the EVs are from bacteria of the Agathobaculum genus.
- the EVs are from Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
- the EVs are from Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
- the EVs are from Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
- the EVs are from Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
- the EVs described herein are obtained from bacterium of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.
- the EVs described herein are obtained from a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Lactococcus lactis cremoris, Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxytoca, and Veillonella tobetsuensis.
- the EVs described herein are obtained from a Prevotella bacteria.
- the EVs described herein are obtained from a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella bacteria.
- the EVs described herein are obtained from a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
- sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
- the EVs described herein are obtained from a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
- sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
- the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
- the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, mA Acidaminococcus.
- Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
- the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
- the Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus.
- Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
- the bacteria from which the EVs are obtained are of the Negativicutes class.
- the bacteria from which the EVs are obtained are of the Veillonellaceae family. [213] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonadaceae family.
- the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.
- the bacteria from which the EVs are obtained are of the Sporomusaceae family.
- the bacteria from which the EVs are obtained are of the Megasphaera genus.
- the bacteria from which the EVs are obtained are of the Selenomonas genus.
- the bacteria from which the EVs are obtained are of the Propionospora genus.
- the bacteria from which the EVs are obtained are of the Acidaminococcus genus.
- the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
- the bacteria from which the EVs are obtained are Selenomonas felix bacteria.
- the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.
- the bacteria from which the EVs are obtained are Propionospora sp. bacteria.
- the bacteria from which the EVs are obtained are of the Clostridia class.
- the bacteria from which the EVs are obtained are of the Oscillospriraceae family.
- the bacteria from which the EVs are obtained are of the Faecalibacterium genus.
- the bacteria from which the EVs are obtained are of the Fournierella genus.
- the bacteria from which the EVs are obtained are of the Harryflintia genus.
- the bacteria from which the EVs are obtained are of the Agathobaculum genus.
- the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
- the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
- the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
- the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
- the bacteria from which the EVs are obtained are bacteria of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.
- the bacteria from which the EVs are obtained are a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Lactococcus lactis cremoris, Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxytoca, and Veillonella tobetsuensis.
- the bacteria from which the EVs are obtained are a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella t
- the bacteria from which the EVs are obtained are a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
- sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
- the bacteria from which the EVs are obtained are a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
- sequence identity for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
- the Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae .
- the Negativicutes class includes the genera Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
- the bacteria from which the EVs are obtained are of the Negativicutes class.
- the bacteria from which the EVs are obtained are of the Veillonellaceae family.
- the bacteria from which the EVs are obtained are of the Selenomonadaceae family.
- the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.
- the bacteria from which the EVs are obtained are of the Sporomusaceae family.
- the bacteria from which the EVs are obtained are of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Christensenellaceae; or Akkermaniaceae family.
- the bacteria from which the EVs are obtained are of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
- the bacteria from which the EVs are obtained are of the Megasphaera genus.
- the bacteria from which the EVs are obtained are of the Selenomonas genus.
- the bacteria from which the EVs are obtained are of the Propionospora genus.
- the bacteria from which the EVs are obtained are of the Acidaminococcus genus.
- the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
- the bacteria from which the EVs are obtained are Selenomonas felix bacteria.
- the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.
- the bacteria from which the EVs are obtained are Propionospora sp. bacteria.
- the bacteria from which the EVs are obtained are of the Clostridia class.
- the bacteria from which the EVs are obtained are of the Oscillospriraceae family.
- the bacteria from which the EVs are obtained are of the Faecalibacterium genus.
- the bacteria from which the EVs are obtained are of the Fournierella genus.
- the bacteria from which the EVs are obtained are of the Harryflintia genus.
- the bacteria from which the EVs are obtained are of the Agathobaculum genus.
- the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
- the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
- the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
- the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
- the bacteria from which the EVs are obtained are a strain of Agathobaculum sp.
- the Agathobaculum sp. strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, CRISPR sequence) of the Agathobaculum sp.
- Strain A ATCC Deposit Number PTA-125892
- the Agathobaculum sp. strain is the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892).
- the bacteria from which the EVs are obtained are of the class Bacteroidia [phylum Bacteroidota ⁇ . In some embodiments, the bacteria from which the EVs are obtained are bacteria of order Bacteroidales. In some embodiments, the bacteria from which the EVs are obtained are of the family Porphyromonoadaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Prevotellaceae . In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia wherein the bacteria is diderm and the bacteria stain Gram negative.
- the bacteria from which the EVs are obtained are bacteria of the class Clostridia [phylum Firmicutes ⁇ . In some embodiments, the bacteria from which the EVs are obtained are of the order Eubacteriales. In some embodiments, the bacteria from which the EVs are obtained are of the family Oscillispiraceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Lachnospiraceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Peptostreptococcaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Clostridiales family XIII/ Incertae sedis 41.
- the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia that stain Gram positive. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram positive.
- the bacteria from which the EVs are obtained are of the class Negativicutes [phylum Firmicutes ⁇ . In some embodiments, the bacteria from which the EVs are obtained are of the order Veillonellales. In some embodiments, the bacteria from which the EVs are obtained are of the family Veillonelloceae. In some embodiments, the bacteria from which the EVs are obtained are of the order Selenomonadales. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the family Selenomonadaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Sporomusaceae .
- t the bacteria from which the EVs are obtained are of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are of the bacteria from which the EVs are obtained are the EVs are from bacteria of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
- the bacteria from which the EVs are obtained are of the class Synergistia [phylum Synergistota ⁇ . In some embodiments, the bacteria from which the EVs are obtained are of the order Synergistales. In some embodiments, the bacteria from which the EVs are obtained are of the family Synergistaceae . In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
- the bacteria from which the EVs are obtained are from one strain of bacteria, for example, a strain provided herein.
- the bacteria from which the EVs are obtained are from one strain of bacteria (for example, a strain provided herein) or from more than one strain provided herein.
- the bacteria from which the EVs are obtained are Lactococcus lactis cremoris bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
- the bacteria from which the EVs are obtained are Lactococcus bacteria, for example, Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
- the bacteria from which the EVs are obtained are of the Prevotella genus. In some embodiments, the bacteria from which the EVs are obtained are Prevotella histicola bacteria.
- the bacteria from which the EVs are obtained are Prevotella bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329).
- the bacteria from which the EVs are obtained are Prevotella bacteria, for example, Prevotella Strain B 50329 (NRRL accession number B 50329).
- the bacteria from which the EVs are obtained are Prevotella histicola bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella histicola Strain C deposited as ATCC designation number PTA-126140.
- the bacteria from which the EVs are obtained are Prevotella histicola bacteria, for example Prevotella histicola Strain C deposited as ATCC designation number PTA-126140).
- the bacteria from which the EVs are obtained are Bifidobacterium bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
- the bacteria from which the EVs are obtained are Bifidobacterium bacteria, for example, Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
- the bacteria from which the EVs are obtained are Veillonella bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Veillonella bacteria deposited as ATCC designation number PTA-125691.
- the bacteria from which the EVs are obtained are Veillonella bacteria, for example, Veillonella bacteria deposited as ATCC designation number PTA-125691.
- the bacteria from which the EVs are obtained are Ruminococcus gnavus bacteria.
- the Ruminococcus gnavus bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
- the Ruminococcus gnavus bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
- the Ruminococcus gnavus bacteria are Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
- the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
- the Megasphaera sp. bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the Megasphaera sp. bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera .s/ bacteria deposited as ATCC designation number PTA- 126770.
- the Megasphaera sp. bacteria are Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
- the bacteria from which the EVs are obtained are Fournierella massiliensis bacteria.
- the Fournierella massiliensis bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the Fournierella massiliensis bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the Fournierella massiliensis bacteria are Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
- the bacteria from which the EVs are obtained are Harryflintia acetispora bacteria.
- the Harryflintia acetispora bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the Harryflintia acetispora bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the Harryflintia acetispora bacteria are Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
- the bacteria from which the EVs are obtained are bacteria that produce metabolites, for example, the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.
- the bacteria from which the EVs are obtained are bacteria that produce butyrate. In some embodiments, the bacteria are from the genus Blautia;
- the bacteria from which the EVs are obtained are bacteria that produce iosine.
- the bacteria are from the genus Bifidobacterium; Lactobacillus; or Olsenella.
- the bacteria from which the EVs are obtained are bacteria that produce proprionate.
- the bacteria are from the genus Akkermansia; Bacteriodes; Dialister; Eubacterium; Megasphaera; Parabacteriodes;
- the bacteria from which the EVs are obtained are bacteria that produce tryptophan metabolites.
- the bacteria are from the genus Lactobacillus or Peptostreptococcus.
- the bacteria from which the EVs are obtained are bacteria that produce inhibitors of histone deacetylase 3 (HDAC3).
- the bacteria are from the species Bariatricus massiliensis, Faecalibacterium prausnitzii, Megasphaera massiliensis or Roseburia intestinalis.
- the bacteria are from the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus; Enhydrobacter; Exiguobacterium; Faecalibacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus;
- the bacteria are from the genus Cutibacterium. In some embodiments, the bacteria are from the species Cutibacterium avidum. In some embodiments, the bacteria are from the genus Lactobacillus. In some embodiments, the bacteria are from the species Lactobacillus gasseri. In some embodiments, the bacteria are from the genus Dysosmobacter . In some embodiments, the bacteria are from the species Dysosmobacter welbionis.
- the bacteria from which the EVs are obtained are of the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus;
- the bacteria from which the EVs are obtained are of the Cutibacterium genus. In some embodiments, the bacteria from which the EVs are obtained are Cutibacterium avidum bacteria.
- the bacteria from which the EVs are obtained are of the genus Leuconostoc.
- the bacteria from which the EVs are obtained are of the genus Lactobacillus.
- the bacteria from which the EVs are obtained are of the genus Akkermansia; Bacillus; Blautia; Cupriavidus; Enhydrobacter; Faecalibacterium; Lactobacillus; Lactococcus; Micrococcus; Morganella; Propionibacterium; Proteus; Rhizobium; or Streptococcus.
- the bacteria from which the EVs are obtained are Leuconostoc holzapfelii bacteria.
- the bacteria from which the EVs are obtained are Akkermansia muciniphila; Cupriavidus metallidurans; Faecalibacterium prausnitzii; Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; Lactobacillus sakei; or Streptococcus pyogenes bacteria.
- the bacteria from which the EVs are obtained are Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; or Lactobacillus sakei bacteria.
- the EVs described herein are obtained from a genus selected from the group consisting of Acinetobacter; Deinococcus; Helicobacter; Rhodococcus;
- the EVs described herein are obtained from a species selected from the group consisting of Acinetobacter baumanii; Deinococcus radiodurans; Helicobacter pylori; Rhodococcus equi; Weissella cibaria; Alloiococcus otitis; Atopobium vaginae; Catenibacterium mituokai; Corynebacterium glutamicum; Exiguobacterium aurantiacum; Geobacillus stearothermophilus; Methylobacterium jeotgali; Micrococcus luteus; Morganella morganii; Proteus mirabilis; Rhizobium leguminosarum; Rothia amarae; Sphingomonas paucimobilis; and Sphingomonas koreens.
- the EVs are from Leuconostoc holzapfelii bacteria. In some embodiments, the EVs are from Leuconostoc holzapfelii Ceb-kc-003 (KCCM11830P) bacteria.
- the bacteria from which the EVs are obtained are Megasphaera sp. bacteria (for example, from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387).
- the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number NCIMB 42787, NCIMB 43388 or NCIMB 43389).
- the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number DSM 26228).
- the bacteria from which the EVs are obtained are Parabacteroides distasonis bacteria (for example, from the strain with accession number NCIMB 42382).
- the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number NCIMB 43388 or NCIMB 43389), or a derivative thereof. See, for example, WO 2020/120714.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria from the strain with accession number NCIMB 43388 or NCIMB 43389.
- the Megasphaera massiliensis bacteria is the strain with accession number NCIMB 43388 or NCIMB 43389.
- the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787, or a derivative thereof. See, for example, WO 2018/229216.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787.
- the Megasphaera massiliensis bacteria is the strain deposited under accession number NCIMB 42787.
- the bacteria from which the EVs are obtained are Megasphaera spp. bacteria from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387, or a derivative thereof. See, for example, WO 2020/120714. In some embodiments, the Megasphaera sp.
- bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera sp. from a strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
- the Megasphaera sp. bacteria is the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
- the bacteria from which the EVs are obtained are Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382, or a derivative thereof. See, for example, WO 2018/229216.
- the Parabacteroides distasonis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382.
- the Parabacteroides distasonis bacteria is the strain deposited under accession number NCIMB 42382.
- the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria deposited under accession number DSM 26228, or a derivative thereof. See, for example, WO 2018/229216.
- the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria deposited under accession number DSM 26228.
- the Megasphaera massiliensis bacteria is the strain deposited under accession number DSM 26228.
- the bacteria from which the EVs are obtained are modified (for example, engineered) to reduce toxicity or other adverse effects, to enhance delivery) (for example, oral delivery) of the EVs (for example, by improving acid resistance, muco- adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (for example, M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the EVs (for example, either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the EVs (for example, through modified production of polysaccharides, pili, fimbriae, adhesins).
- the engineered bacteria described herein are modified to improve EV manufacturing (for example, higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times).
- the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may results in the overexpression and/or underexpression of one or more genes.
- the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
- the EVs described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
- the therapeutic moiety is a cancer-specific moiety.
- the cancer-specific moiety has binding specificity for a cancer cell (for example, has binding specificity for a cancer-specific antigen).
- the cancer-specific moiety comprises an antibody or antigen binding fragment thereof.
- the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
- the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof.
- the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the bacterium and a second part that is capable of binding to a cancer cell (for example, by having binding specificity for a cancer-specific antigen).
- the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
- the first part has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen).
- the first and/or second part comprises an antibody or antigen binding fragment thereof.
- the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptorbinding fragment thereof. In certain embodiments, co-administration of the cancer-specific moiety with the EVs (either in combination or in separate administrations) increases the targeting of the EVs to the cancer cells.
- the EVs described herein are engineered such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (for example, a magnetic bead).
- the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria. In some embodiments, the magnetic and/or paramagnetic moiety is linked to and/or a part of an EV-binding moiety that that binds to the EV. In some embodiments, the EV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the EV-binding moiety has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen). In some embodiments, the EV-binding moiety comprises an antibody or antigen binding fragment thereof.
- the EV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the EV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the magnetic and/or paramagnetic moiety with the EVs (either together or in separate administrations) can be used to increase the targeting of the EVs (for example, to cancer cells and/or a part of a subject where cancer cells are present.
- the EVs from bacteria described herein are prepared using any method known in the art.
- the EVs are prepared without an EV purification step.
- bacteria described herein are killed using a method that leaves the EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein.
- the bacteria are killed using an antibiotic (for example, using an antibiotic described herein).
- the bacteria are killed using UV irradiation.
- the bacteria are heat- killed.
- the EVs described herein are purified from one or more other bacterial components.
- Methods for purifying EVs from bacteria are known in the art.
- EVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): eO 134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety.
- the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (for example, at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
- the culture supernatants are then passed through filters to exclude intact bacterial cells (for example, a 0.22 pm filter).
- the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS.
- filtered supernatants are centrifuged to pellet bacterial EVs (for example, at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C).
- the EVs are further purified by resuspending the resulting EV pellets (for example, in PBS), and applying the resuspended EVs to an Optiprep (iodixanol) gradient or gradient (for example, a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (for example, at 200,000 x g for 4-20 hours at 4°C).
- EV bands can be collected, diluted with PBS, and centrifuged to pellet the EVs (for example, at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
- the purified EVs can be stored, for example, at -80°C or -20°C until use.
- the EVs are further purified by treatment with DNase and/or proteinase K.
- cultures of bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria.
- Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells.
- Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration.
- ammonium sulfate precipitation 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C.
- Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C.
- the resulting pellets contain bacteria EVs and other debris.
- filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C.
- the pellet of this centrifugation contains bacterial EVs and other debris such as large protein complexes.
- supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
- EVs can be obtained from bacteria cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (for example, XCell ATF from Repligen).
- ATF alternating tangential flow
- the ATF system retains intact cells (>0.22 pm) in the bioreactor, and allows smaller components (for example, EVs, free proteins) to pass through a filter for collection.
- the system may be configured so that the ⁇ 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as EVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor.
- the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture.
- EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
- EVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column.
- Samples are applied to a 35- 60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, for example, 4-24 hours at 4°C.
- EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.
- EVs used for in vivo injections purified EVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
- EVs in PBS are sterile- filtered to ⁇ 0.22 pm.
- samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (for example, Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
- filtration for example, Amicon Ultra columns
- dialysis for example, dialysis
- ultracentrifugation 200,000 x g, > 3 hours, 4°C
- the sterility of the EV preparations can be confirmed by plating a portion of the EVs onto agar medium used for standard culture of the bacteria used in the generation of the EVs and incubating using standard conditions.
- select EVs are isolated and enriched by chromatography and binding surface moieties on EVs.
- select EVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
- EVs are analyzed, for example, as described in Jeppesen, et al. Cell 177:428 (2019).
- EVs are lyophilized.
- EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
- EVs are UV irradiated.
- EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
- EVs are acid treated.
- EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
- the phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria.
- EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
- the growth environment can affect the amount of EVs produced by bacteria.
- the yield of EVs can be increased by an EV inducer, as provided in Table 5.
- Table 5 Culture Techniques to Increase EV Production
- the method can optionally include exposing a culture of bacteria to an EV inducer prior to isolating EVs from the bacterial culture.
- the culture of bacteria can be exposed to an EV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
- engineered bacteria for the production of the EVs described herein.
- the engineered bacteria are modified to enhance certain desirable properties.
- the engineered bacteria are modified to enhance the immunomodulatory and/or therapeutic effect of the EVs (for example, either alone or in combination with another therapeutic agent), to reduce toxicity and/or to improve bacterial and/or EV manufacturing (for example, higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
- the engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR/Cas9, or any combination thereof.
- the bacterium is modified by directed evolution.
- the directed evolution comprises exposure of the bacterium to an environmental condition and selection of bacterium with improved survival and/or growth under the environmental condition.
- the method comprises a screen of mutagenized bacteria using an assay that identifies enhanced bacterium.
- the method further comprises mutagenizing the bacteria (for example, by exposure to chemical mutagens and/or UV radiation) or exposing them to a therapeutic agent (for example, antibiotic) followed by an assay to detect bacteria having the desired phenotype (for example, an in vivo assay, an ex vivo assay, or an in vitro assay).
- a method of producing extracellular vesicles comprising growing EV- producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
- a filter system removes EVs, metabolites, and waste products of (e.g., from) the culture media.
- the first filter is a product harvest filter (e.g., and passing the bacterial culture media that comprises EVs through the product harvest filter produces an output of the product harvest filter) and the second filter is a medium exchange filter.
- the bacterial culture media is from a perfusion culture.
- the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter scale or 20,000 liter scale or greater).
- the medium exchange filter comprises PES (polyethersulfone).
- volume of the output of the product harvest filter is about l/5x the volume than if a single-filter system was used (e.g., the volume of the output of the product harvest filter is about l/5x the volume as compared to the volume that would result from a single-filter system).
- a method comprising:
- a method comprising: (i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs; and
- a method comprising:
- EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Phrislensenellaceae or Akkermaniaceae family.
- EVs are from Gram positive bacteria.
- EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
- Example 1 EV manufacturing platform
- Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity.
- a system provides culture intensification and clarification, using a high density perfusion process at 20,000L-scale process, and could increase manufacturing plant output >50-fold and includes using a two-filter system (such as hollow fiber product separation).
- purification and concentration provide continuous capture to achieve pure and highly concentrated process intermediates that contain EVs, and can include continuous chromatography capture and tangential flow filtration.
- the output can be further processed by drying (such as spray drying or lyophilization) of EVs, and can undergo post-processing, such as milling.
- Example 2 Perfusion culture yields
- Figures 2A and 2B are graphs showing comparisons of EV yields (EV product batches (-fold)) from batch culture versus perfusion culture yields over time (hours).
- Figure 2A shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is coupled with growth.
- Figure 2B shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is not coupled with growth.
- the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).
- FIG. 3 is a schematic showing a set up for a two-filter system.
- Harvest volume transfers from the fermenter to the two filters: product harvest filter and medium exchange filter.
- Captured product from the product harvest filter transfers to a product reservoir and can be further processed, such as through a capture step(s). Metabolites, and waste products that passed through the medium exchange filter transfer to a waste reservoir.
- This two-filter system can extend operating time. In addition to reducing sieving of the product harvest filter, this system allows the product harvest filter to collect higher concentration product (EVs) in a smaller volume than if a single filter system was used.
- EVs concentration product
- Figure 4 is a graph showing a theoretical result of using a two-filter (dual membrane perfusion) system. Overall perfusion rate remains constant at 6 volumes/day.
- Shifting flux from the “Product Harvest” filter to the “Medium Exchange” filter increases the Flux Ratio, and results in product concentration (-fold; upward sloping line) increasing and permeate volume (downward sloping line; volume (vol)/day) decreasing with increasing ratio.
- pH also affects and can improve capacity (column volume), enrichment, and yield. See Table 6 where the performance of two pH conditions (pH A and pH B) were evaluated.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Virology (AREA)
- Sustainable Development (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Provided herein are methods to harvest EVs (such as increased yields of EVs) from bacterial cultures.
Description
PROCESSING EXTRACELLULAR VESICLES
CROSS-REFERENCE TO RELATED APPLICATIONS
[1] This application claims the benefit of U.S. Provisional Application No. 63/414,284, filed on October 7, 2022, the content of which is hereby incorporated by reference in its entirety.
SUMMARY
[2] Extracellular vesicles (EVs) are natural lipoprotein nanoparticles produced by many species of bacteria. Their macromolecular content is a complex subset of proteins, glycans, lipids, and LPS. Bacteria can secrete extracellular vesicles into the culture medium. Methods for increasing the production of EVs by bacteria into the medium for a given culture (that is, the yields) include using perfusion culture systems instead of batch culture systems. Also, increasing the size of the cultures (such as to a commercial scale such as a 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater) (for batch or perfusion cultures) also increases yields. Methods are developed to harvest the increased yields of EVs from bacterial cultures. Such methods include filter systems (such as two-filter systems) and chromatography techniques (such as monoliths) to decrease volumes, increase concentrations, and/or increase purity of EVs after medium (that contains EVs) is removed from cultures.
[3] In some aspects, the disclosure provides a method of producing extracellular vesicles (EVs), the method comprising growing EV-producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
[4] In some embodiments, the perfusion culture increases EV yields by at least about 10- fold, e.g., by at least about 15-fold or by at least about 17-fold or by at least about 50-fold, as compared to a batch culture of the same bacteria.
[5] In some embodiments, the perfusion culture increases EV yields after 24, 48, or 72 hours of culturing, as compared to a batch culture of the same bacteria.
[6] In some embodiments, EV production of the bacteria is coupled to growth in batch culture.
[7] In some embodiments, EV production of the bacteria is not coupled to growth in batch culture.
[8] In some embodiments, the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
[9] In some embodiments, a filter system (such as a one-filter system or a two-filter system) removes EVs, metabolites, and waste products of the culture media.
[10] In some embodiments, the filter system is a two-filter system.
[11] In some embodiments, the method further comprises filtering the culture media.
[12] In some embodiments, the method further comprises performing chromatography on the culture media.
[13] In some embodiments, the method further comprises performing tangential flow filtration on the culture media.
[14] In some embodiments, the method further comprises drying the culture media. In some embodiments, the culture media is dried after the growing step. In some embodiments, the culture media is dried after the filtering step. In some embodiments, the culture media is dried after the chromatography step. In some embodiments, the culture media is dried after the tangential flow filtration step.
[15] In some embodiments, the method further comprises milling the dried culture media.
[16] In some aspects, the disclosure provides a culture media produced by a perfusion culture method provided herein.
[17] In some aspects, the disclosure provides a culture media produced by a method of producing extracellular vesicles provided herein.
[18] In some aspects, the disclosure provides a method of processing bacterial culture media that comprises extracellular vesicles (EVs), the method comprising passing bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter (e.g., wherein the output of the product harvest filter comprises EVs) and the second filter is a medium exchange filter.
[19] In some embodiments, the bacterial culture is a perfusion culture.
[20] In some embodiments, the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
[21] In some embodiments, the product harvest filter and the medium exchange filter comprise the same material.
[22] In some embodiments, the product harvest filter comprises PES (polyethersulfone).
[23] In some embodiments, the medium exchange filter comprises PES (polyethersulfone).
[24] In some embodiments, the product harvest filter and the medium exchange filter comprise PES (polyethersulfone).
[25] In some embodiments, EVs, media, waste and metabolites pass through the product harvest filter.
[26] In some embodiments, the pore size of the product harvest filter is about 0.5 micron.
[27] In some embodiments, media, waste and metabolites pass through the medium exchange filter.
[28] In some embodiments, the pore size of the medium exchange filter is less than about 0.5 micron.
[29] In some embodiments, the pore size of the medium exchange filter is about 0.05 micron.
[30] In some embodiments, the pore size of the medium exchange filter is about 0.02 micron.
[31] In some embodiments, the pore size of the medium exchange filter is about 0.01 micron.
[32] In some embodiments, the pore size of the medium exchange filter comprises a size cut off of 750kD (kilodalton).
[33] In some embodiments, the pore size of the medium exchange filter comprises a size cut off of 500kD.
[34] In some embodiments, the medium exchange filter runs at a higher flux than the product harvest filter.
[35] In some embodiments, the flux ratio (medium exchange filterproduct harvest filter) is about 5: 1.
[36] In some embodiments, the flux ratio (medium exchange filterproduct harvest filter) is about 9: 1.
[37] In some embodiments, the flux ratio (medium exchange filter product harvest filter) is about 10: 1.
[38] In some embodiments, the flux ratio (medium exchange filter product harvest filter) reduces sieving of the product harvest filter (e.g., as compared to the amount of sieving if the flux ratio was 1 : 1 or if the product harvest filter was used alone).
[39] In some embodiments, the volume of the output of the product harvest filter is about l/5x the volume than if a single-filter system was used.
[40] In some embodiments, the volume of the output of the product harvest filter is about l/9x the volume than if a single-filter system was used.
[41] In some embodiments, the volume of the output of the product harvest filter is about l/10x the volume than if a single-filter system was used.
[42] In some embodiments, the output of the product harvest filter comprises a higher concentration of EVs than if a single-filter system was used.
[43] In some embodiments, the output of the product harvest filter comprises a concentration of EVs that is at least about 5x higher than if a single-filter system was used.
[44] In some embodiments, the output of the product harvest filter comprises a concentration of EVs that is at least about 9x higher than if a single-filter system was used.
[45] In some embodiments, the output of the product harvest filter comprises a concentration of EVs that is at least about lOx higher than if a single-filter system was used.
[46] In some embodiments, the method further comprises performing chromatography on the output of the product harvest filter.
[47] In some embodiments, the method further comprises performing tangential flow filtration on the output of the product harvest filter.
[48] In some embodiments, the method further comprises drying the output of the product harvest filter. In some embodiments, the output is dried after the filtering step. In some embodiments, the output is dried after the chromatography step. In some embodiments, the output is dried after the tangential flow filtration step.
[49] In some embodiments, the method further comprises milling the dried output of the product harvest filter.
[50] In some aspects, the disclosure provides an output of a product harvest filter produced by a method of processing bacterial culture media provided herein.
[51] In some aspects, the disclosure provides a method of processing a liquid that comprises extracellular vesicles (EVs) to prepare an EV eluate, the method comprising performing chromatography on the liquid.
[52] In some embodiments, the liquid comprises bacterial culture media.
[53] In some embodiments, the bacterial culture media is from a perfusion culture.
[54] In some embodiments, the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
[55] In some embodiments, the liquid comprises a product-containing volume.
[56] In some embodiments, the product-containing volume is output from a single-filter system.
[57] In some embodiments, the product-containing volume is output from a two-filter system.
[58] In some embodiments, the product-containing volume is output from a product harvest filter.
[59] In some embodiments, the product harvest filter comprises PES (polyethersulfone).
[60] In some embodiments, the pore size of the product harvest filter is about 0.5 micron.
[61] In some embodiments, the chromatography comprises a chromatography column.
[62] In some embodiments, the chromatography column comprises a monolith.
[63] In some embodiments, the chromatography column comprises a monolith ion exchange column.
[64] In some embodiments, the method comprises processing on one column.
[65] In some embodiments, the liquid is loaded onto the column and then EV-containing eluate is eluted.
[66] In some embodiments, the method comprises processing on two columns.
[67] In some embodiments, the liquid is loaded onto a first column, then EV-containing eluate is eluted from the first column, and while EV-containing eluate is eluted from the first column, liquid is loaded onto the second column.
[68] In some embodiments, EV-containing eluate is eluted from the second column, and while EV-containing eluate is eluted from the second column, liquid is loaded onto the first column.
[69] In some embodiments, the loading and eluting steps on the first and second columns are alternated to process the liquid continuously.
[70] In some embodiments, bacterial culture media is filtered prior to performing the chromatography.
[71] In some embodiments, the chromatography enriches EV yield by greater than about 5-fold.
[72] In some embodiments, the chromatography enriches EV yield by about 6-fold.
[73] In some embodiments, the chromatography enriches EV yield by about 12-fold.
[74] In some embodiments, the yield of EVs from the chromatography is greater than about 50%.
[75] In some embodiments, the yield of EVs from the chromatography is greater than about 60%.
[76] In some embodiments, the method further comprises performing tangential flow filtration on the EV eluate.
[77] In some embodiments, the method further comprises drying the EV eluate. In some embodiments, the EV eluate is dried after the chromatography step. In some embodiments, the EV eluate is dried after the tangential flow filtration step.
[78] In some embodiments, the method further comprises milling the dried EV eluate.
[79] In some aspects, the disclosure provides an EV eluate produced by a method of processing a liquid that comprises extracellular vesicles (EVs) provided herein.
[80] In some aspects, the disclosure provides a method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs (e.g., as described herein); and
(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter (e.g., as described herein), thereby preparing output of the product harvest filter, wherein the output comprises EVs.
[81] In some aspects, the disclosure provides an output of a product harvest filter produced by a method provided herein.
[82] In some aspects, the disclosure provides a method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs (e.g., as described herein); and
(ii) performing chromatography on the bacterial culture media to prepare an eluate (e.g., as described herein), wherein the eluate comprises EVs.
[83] In some aspects, the disclosure provides an eluate produced by a method provided herein.
[84] In some aspects, the disclosure provides a method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs (e.g., as described herein);
(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter (e.g., as described herein), thereby preparing output of the product harvest filter, wherein the output comprises EVs; and
(iii) performing chromatography on the output of the product harvest filter to prepare an eluate (e.g., as described herein), wherein the eluate comprises EVs.
[85] In some aspects, the disclosure provides an eluate produced by a method provided herein.
[86] In some embodiments of an aspect provided herein, the method comprises EVs from a bacterial strain that is associated with mucus.
[87] In some embodiments of an aspect provided herein, the method comprises EVs from anaerobic bacteria.
[88] In some embodiments of an aspect provided herein, the anaerobic bacteria are obligate (e.g., strict) anaerobes.
[89] In some embodiments of an aspect provided herein, the anaerobic bacteria are facultative anaerobes.
[90] In some embodiments of an aspect provided herein, the anaerobic bacteria are aerotolerant anaerobes.
[91] In some embodiments of an aspect provided herein, the EVs are from monoderm bacteria.
[92] In some embodiments of an aspect provided herein, the EVs are from diderm bacteria.
[93] In some embodiments of an aspect provided herein, the EVs are from Gram negative bacteria.
[94] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae;
Sporomusaceae; Synergistaceae; Phrislensenellaceae or Akkermaniaceae family.
[95] In some embodiments of an aspect provided herein, the EVs are from Gram positive bacteria.
[96] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
[97] In some embodiments of an aspect provided herein, the EVs are from bacteria of the genus Prevotella.
[98] In some embodiments of an aspect provided herein, the EVs are from bacteria of the genus Veillonella.
[99] In some embodiments of an aspect provided herein, the EVs are from bacteria of the genus Parabacteroides.
[100] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Oscillospiraceae family.
[101] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Tannerellaceae family.
[102] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Prevotellaceae family.
[103] In some embodiments of an aspect provided herein, the EVs are from bacteria of the Veillonellaceae family.
[104] In some embodiments of an aspect provided herein, the EVs are from bacteria of class, order, family, genus, species and/or strain of bacteria provided in Table 1, Table 2, Table 3, and/or Table 4.
[105] In some aspects, the disclosure provides a product produced by a method provided herein.
BRIEF DESCRIPTION OF THE FIGURES
[106] Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity.
[107] Figures 2A and 2B are graphs showing comparisons of EV yields (EV product batches (-fold)) from batch culture versus perfusion culture yields over time (hours).
Figure 2A shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is coupled with growth. Figure 2B shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is not coupled with growth. In both Figures 2A and 2B, the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).
[108] Figure 3 is a schematic showing a set up for a two-filter system. Harvest volume transfers from the fermenter to the two filters: product harvest filter and medium exchange filter. Captured product from the product harvest filter transfers to a product reservoir and can be further processed, such as through a capture step(s). Metabolites and waste products that passed through the medium exchange filter transfer to a waste reservoir.
[109] Figure 4 is a graph showing a theoretical result of using a two-filter (dual membrane perfusion) system. Shifting flux from the “Product Harvest” filter to the “Medium Exchange” filter increases the Flux Ratio, and results in product concentration (-fold; upward sloping line) increasing and permeate volume (downward sloping line; volume (vol)/day) decreasing with increasing ratio.
DETAILED DESCRIPTION
[HO] The disclosure provides methods developed to harvest EVs (such as increased yields of EVs) from bacterial cultures. Yields can be increased, for example, by using perfusion culture systems instead of batch culture systems and/or by increasing the size of the cultures (such as to a commercial scale such as a 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater) (for batch or perfusion cultures). Such harvest methods include filter systems (such as two-filter systems) and chromatography techniques (such as monoliths) to decrease volumes, increase concentrations, and/or increase purity of EVs after medium (that contains EVs) is removed from cultures.
Perfusion
[Hl] The use of perfusion culture systems offers advantages over batch process culture systems of bacteria for EV production. Batch anaerobic fermentation can result in poor substrate utilization and high inhibitor production. A perfusion process improves productivity through removal of inhibitory waste products and control of microbial metabolism. Perfusion provides a cell free product stream ready for downstream processing.
[112] For example, as compared to batch culture, perfusion culture can increase EV yields by at least 10-fold, e.g., by at least 15-fold or 17-fold or 50-fold, after 72 hours of culturing.
[113] Such degrees of increased yields were unexpected. In perfusion, the growth rate of the bacterial cells is slower than the log phase growth rate of the cells in a batch culture. If a strain produces a large amount of EVs in log phase where growth rate is high (referred to as growth coupled), then less cell specific EV production would be expected in perfusion. If production of EVs is not tied to growth, then high or higher cell specific yields in perfusion may be produced where the growth rate is low. The high degree of increased yields using perfusion culture was not expected, and was seen for EV production by a strain in which EV production was coupled with growth, and also for a bacterial strain in which EV production was not coupled with growth.
[114] Considerations relating to perfusion culturing bacteria for EV production exist, particularly when perfusion culturing is performed at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater). The considerations can include the following factors. For anaerobic bacteria (such as strict anaerobes), anaerobic conditions need to be maintained during the culture period. Larger amounts of raw materials and prepared media are needed for perfusion cultures than batch processes, for example, up to ten times more per day.
[115] As media is flowed out of the perfusion culture (e.g., as the media is exchanged), a filter system (one-filter or two-filter system) removes metabolites, and waste products of the culture, yet does not remove the bacterial cells. The filter system also removes product (EVs). The volumes of media from a perfusion culture are greater than for a batch process (for example, up to ten times greater per day). Thus, in processing the media, filter area and flow rates need to be managed to ensure sufficient removal of product, metabolites, and waste products, and to avoid the need for expensive enlarged filter areas. Also, flow rates and volumes need to be managed to minimize filter sieving. A two-filter system can address one or more of these considerations.
[116] Additional further processing of the output of the perfusion culture can be performed. Such additional further processing can include filtration (such as with a two-filter system), chromatography, tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
Two-filter system
[117] In place of a single-filter system for processing a bacterial culture for EV production (such as by perfusion culture (particularly when perfusion culturing is performed at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater))), a two-filter system can be used to process the culture media. The two-filter system can be used as part of a continuous process (e.g., as opposed to a batch or intermittent process). The two filters can be run at the same time, e.g., but at different flow rates. One filter functions to collect product (e.g., EVs) (the product harvest filter). The pore size of the product harvest filter is selected to allow product (e.g., EVs) to pass through, such as a 0.5 micron pore size. Media, metabolites, and waste products can also pass through the product harvest filter. The second filter functions to collect media, metabolites, and waste products (the medium exchange filter), yet product (e.g., EVs) does not pass through. The pore size of the medium exchange filter is selected to allow media, metabolites, and waste products to pass through but to not allow product to pass through, such as a 0.05 micron (or smaller, such as 0.02 micron or 0.01 micron) pore size. Rather than selecting by pore size, the medium exchange filter can be selected based on size cut-off: such as a 750kD or 500kD size limits for what can pass through. Both the product harvest filter and the medium exchange filter can be made of the same material, such as PES (polyethersulfone).
[118] To reduce sieving of the product harvest filter, the medium exchange filter runs at a higher flux than the product harvest filter. For example, the flux ratio (medium exchange
filterproduct harvest filter) can be 5: 1, or 9: 1, or 10: 1. In addition to reducing sieving of the product harvest filter, this allows the product harvest filter to collect higher concentration product (EVs) in a smaller volume, such as 1/5, 1/9 or 1/10 the volume than if a single-filter system was used. For example, rather than further processing 120,000 liters, a smaller volume such as 12,000 liters is further processed. This reduced volume provides advantages for further processing of the product-containing volume (e.g., the output of the product harvest filter when a two-filter system is used).
[119] As shown herein, a two-filter (dual-membrane) perfusion system in place of a single-filter perfusion system reduces downstream volume and increases product concentration.
[120] Additional further processing of the output of the two-filter system can be performed. Such additional further processing can include chromatography, tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
Chromatography
[121] Downstream of growing a bacterial culture for EV production (such as by perfusion culture (particularly when perfusion culturing is performed at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater))), chromatography can be used to process culture media or a product-containing volume.
[122] Chromatography can be used to process a product-containing volume, such as after culture media containing product (e.g., EVs) is filtered, such as through a single-filter system or a two-filter system. Rather than using a conventional resin, a monolith can be used (e.g., such as a monolith supplied by Sartorius). In contrast to a conventional resin, a monolith is cast as a single block and is inserted into a chromatographic housing. The monolith is characterized by a highly interconnected network of channels.
[123] Considerations relating to chromatography of culture media (e.g., from a perfusion culture) or a product-containing volume, such as after culture media containing product is filtered (such as through a single-filter system or a two-filter system) exist, particularly when culturing is performed at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater). Considerations include the feasibility and specificity of chromatography to capture the product (e.g., EVs); the large size of the chromatography matrices (such as monoliths); binding capacity; process volumes (including load, buffers and waste); and pool volume management.
[124] One or two columns (such as monoliths (such as monolith ion exchange columns)) can be used to process culture media (e.g., from a perfusion culture) or a product-containing volume, such as after culture media containing product (e.g., EVs) is filtered, such as through a single-filter system or a two-filter system. In a system in which one column is used, the culture media or product-containing volume is loaded onto the column and then the product is eluted. In a system in which two columns are used, culture media or product-containing volume is loaded onto a first column. While product is being eluted from the first column, culture media or product-containing volume is loaded onto a second column. Once the second column has been loaded and product has been eluted from the first column, culture media or product-containing volume is loaded onto the first column while product is being eluted from the second column. The loading and eluting steps on the first and second columns can continue to be alternated to process culture media or product-containing volume continuously. This allows for continuous capture, such as to achieve pure and highly concentrated product (this may be considered a process intermediate if additional further processing is performed). As demonstrated herein for capture directly from perfusion culture media, pH can affect column loading capacity, such as by three-fold. Enrichment factors can be greater than about 5-fold, e.g., 6- or 12-fold. Yields from the chromatography can be, for example, greater than about 50%, e.g., greater than about 60%.
[125] Additional further processing of the output of the chromatography can be performed. Such additional further processing can include tangential flow filtration, drying (such as by lyophilization or spray drying), and/or milling of a dried product.
Definitions
[126] Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.
[127] The term “about” when used before a numerical value indicates that the value may vary within a reasonable range, such as within ±10%, ±5% or ±1% of the stated value.
[128] “Extracellular vesicles” (EVs) may be naturally-produced vesicles derived from bacteria. EVs are comprised of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate moieties, and are isolated from culture supernatant. The natural production of these vesicles can be artificially enhanced (for example, increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (for example, by media or temperature alterations). Further,
EV compositions may be modified to reduce, increase, add, or remove bacterial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (for example, lymph node), absorption (for example, gastrointestinal), and/or yield (for example, thereby altering the efficacy). As used herein, the term “purified EV composition” or “EV composition” refers to a preparation of EVs that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components. Extracellular vesicles may also be obtained from mammalian cells and from can be obtained from microbes such as archaea, fungi, microscopic algae, protozoans, and parasites.
[129] “Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include, DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).
[130] The term “isolated” or “enriched” encompasses a microbe, an EV (such as a bacterial EV) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. Isolated bacteria or EVs may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated bacteria or EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure, for example, substantially free of other components.
[131] “Metabolite” as used herein refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any
cellular or bacterial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or bacterial metabolic reaction.
[132] As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying,” and “purified” refer to an EV (such as an EV from bacteria) preparation or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (for example, whether in nature or in an experimental setting), or during any time after its initial production. An EV preparation or compositions may be considered purified if it is isolated at or after production, such as from one or more other bacterial components, and a purified microbe or bacterial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “purified.” In some embodiments, purified EVs are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. EV compositions (or preparations) are, for example, purified from residual habitat products.
[133] As used herein, the term “purified EV composition” or “EV composition” refers to a preparation that includes EVs from bacteria that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the EVs in any process used to produce the preparation. It also refers to a composition that has been significantly enriched or concentrated. In some embodiments, the EVs are concentrated by 2-fold, 3-fold, 4-fold, 5- fold, 10-fold, 100-fold, 1000-fold, 10,000-fold or more than 10,000-fold.
[134] “ Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (for example, a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of
the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
Bacterial Extracellular Vesicles
[135] Bacteria propagated as sources of EVs can be selected based on assays in the art that identify bacteria with properties of interest. For example, in some embodiments, bacteria are selected for the ability to modulate host immune response and/or affect cytokine levels.
[136] In some embodiments, EVs are selected from a bacterial strain that is associated with mucus. In some embodiments, the mucus is associated with the gut lumen. In some embodiments, the mucus is associated with the small intestine. In some embodiments, the mucus is associated with the respiratory tract.
[137] In some embodiments, EVs are selected from a bacterial strain that is associated with an epithelial tissue, such as oral cavity, lung, nose, or vagina.
[138] In some embodiments, the EVs are from bacteria that are human commensals.
[139] In some embodiments, the EVs are from human commensal bacteria that originate from the human small intestine.
[140] In some embodiments, the EVs are from human commensal bacteria that originate from the human small intestine and are associated there with the outer mucus layer.
[141] Examples of taxonomic groups (such as class, order, family, genus, species and/or strain) of bacteria that can be used as a source of EVs described herein are provided in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere throughout the specification. In some embodiments, the bacterial strain is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification). In some embodiments, the EVs are from an oncotrophic bacteria. In some embodiments, the EVs are from an immunostimulatory bacteria. In some embodiments, the EVs are from an immunosuppressive bacteria. In some embodiments, the EVs are from an immunomodulatory bacteria. In certain embodiments, EVs are generated from a combination of bacterial strains provided herein. In some embodiments, the combination is a combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45 or 50 bacterial strains. In some embodiments, the combination includes EVs from bacterial strains provided
herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification and/or bacterial strains having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain provided herein (for example, listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification. In some embodiments, bacteria from a taxonomic group (for example, class, order, family, genus, species or strain)) listed in Table 1, Table 2, Table 3, and/or Table 4 and/or elsewhere in the specification can be used as a source of EVs.
[142] In some embodiments, the EVs are obtained from Gram negative bacteria.
[143] In some embodiments, the Gram negative bacteria belong to the class Negativicutes. The Negativicutes represent a unique class of microorganisms as they are the only diderm members of the Firmicutes phylum. These anaerobic organisms can be found in the environment and are normal commensals of the oral cavity and GI tract of humans. Because these organisms have an outer membrane, the yields of EVs from this class were investigated. It was found that on a per cell basis these bacteria produce a high number of vesicles (10-150 EVs/cell). The EVs from these organisms are broadly stimulatory and highly potent in in vitro assays. Investigations into their therapeutic applications in several oncology and inflammation in vivo models have shown their therapeutic potential. The Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae. and Sporomusaceae . The Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus. Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestine, and Propionospora sp.
[144] In some embodiments, the EVs are obtained from Gram positive bacteria.
[145] In some embodiments, the EVs are from aerotol erant bacteria.
[146] In some embodiments, the EVs are from monoderm bacteria.
[147] In some embodiments, the EVs are from diderm bacteria.
[148] In some, the EVs are from bacteria of the family: Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; or Akkermaniaceae .
[149] In some embodiments, the EVs are from bacteria of the family Oscillospiraceae ; Clostridiaceae; Lachnospiraceae; or Christensenellaceae .
[150] In some embodiments, the EVs are from bacteria of the genus Prevotella.
[151] In some embodiments, the EVs are from bacteria of the genus Veillonella.
[152] In some embodiments, the EVs are from bacteria of the mis Parabacteroides.
[153] In some embodiments, the EVs are from a bacterial strain of the Oscillospiraceae family.
[154] In some embodiments, the EVs are from a bacterial strain of the Tannerellaceae family.
[155] In some embodiments, the EVs are from a bacterial strain of the Prevotellaceae family.
[156] In some embodiments, the EVs are from a bacterial strain of the Veillonellaceae family.
[157] In some embodiments, the EVs are obtained from aerobic bacteria.
[158] In some embodiments, the EVs are obtained from anaerobic bacteria. In some embodiments, the anaerobic bacteria comprise obligate anaerobes. In some embodiments, the anaerobic bacteria comprise facultative anaerobes.
[159] In some embodiments, the EVs are obtained from acidophile bacteria.
[160] In some embodiments, the EVs are obtained from alkaliphile bacteria.
[161] In some embodiments, the EVs are obtained from neutral ophile bacteria.
[162] In some embodiments, the EVs are obtained from fastidious bacteria.
[163] In some embodiments, the EVs are obtained from nonfasti di ous bacteria.
[164] In some embodiments, bacteria from which EVs are obtained are lyophilized.
[165] In some embodiments, bacteria from which EVs are obtained are gamma irradiated (for example, at 17.5 or 25 kGy).
[166] In some embodiments, bacteria from which EVs are obtained are UV irradiated.
[167] In some embodiments, bacteria from which EVs are obtained are heat inactivated
(for example, at 50°C for two hours or at 90°C for two hours).
[168] In some embodiments, bacteria from which EVs are obtained are acid treated.
[169] In some embodiments, bacteria from which EVs are obtained are oxygen sparged
(for example, at 0.1 vvm for two hours).
[170] In some embodiments, the EVs are lyophilized.
[171] In some embodiments, the EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
[172] In some embodiments, the EVs are UV irradiated.
[173] In some embodiments, the EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
[174] In some embodiments, the EVs are acid treated.
[175] In some embodiments, the EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
[176] The phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria. For example, in the methods of EVs preparation provided herein, EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
[177] EVs can be isolated from a batch culture of bacteria.
[178] EVs can be isolated from a perfusion culture of bacteria.
[179] In certain embodiments, the EVs described herein are obtained from obligate anaerobic bacteria. Examples of obligate anaerobic bacteria include gram-negative rods (including the genera of Bacteroides, Prevotella, Porphyromonas, Fusobacterium, Bilophila and Sutterella sppf, gram -positive cocci (primarily Peptostreptococcus sppf, gram -positive spore-forming (Clostridium sppf, non-spore-forming bacilli (Actinomyces,
Propioni bacterium, Eubacterium, Lactobacillus and Bifidobacterium sppf, and gramnegative cocci (mainly Veillonella spp. ). In some embodiments, the obligate anaerobic bacteria are of a genus selected from the group consisting of Agathobaculum, Atopobium, Blautia, Burkholderia, Dielma, Longicatena, Paraclostridium, Turicibacter, and Tyzzerella.
[180] The Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae . The Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, mA Acidaminococcus. Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
[181] In some embodiments, the EVs are from bacteria of the Negativicutes class.
[182] In some embodiments, the EVs are from bacteria of the Veillonellaceae family.
[183] In some embodiments, the EVs are from bacteria of the Selenomonadaceae family.
[184] In some embodiments, the EVs are from bacteria of the Acidaminococcaceae family.
[185] In some embodiments, the EVs are from bacteria of the Sporomusaceae family.
[186] In some embodiments, the EVs are from bacteria of the Megasphaera genus.
[187] In some embodiments, the EVs are from bacteria of the Selenomonas genus.
[188] In some embodiments, the EVs are from bacteria of the Propionospora genus.
[189] In some embodiments, the EVs are from bacteria of the Acidaminococcus genus.
[190] In some embodiments, the EVs are from Megasphaera sp. bacteria.
[191] In some embodiments, the EVs are from Selenomonas felix bacteria.
[192] In some embodiments, the EVs are from Acidaminococcus intestini bacteria.
[193] In some embodiments, the EVs are from Propionospora sp. bacteria.
[194] The Oscillospriraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.
[195] In some embodiments, the EVs are from bacteria of the Clostridia class.
[196] In some embodiments, the EVs are from bacteria of the Oscillospriraceae family.
[197] In some embodiments, the EVs are from bacteria of the Faecalibacterium genus.
[198] In some embodiments, the EVs are from bacteria of the Fournierella genus.
[199] In some embodiments, the EVs are from bacteria of the Harryflintia genus.
[200] In some embodiments, the EVs are from bacteria of the Agathobaculum genus.
[201] In some embodiments, the EVs are from Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
[202] In some embodiments, the EVs are from Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
[203] In some embodiments, the EVs are from Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
[204] In some embodiments, the EVs are from Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
[205] In some embodiments, the EVs described herein are obtained from bacterium of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.
[206] In some embodiments, the EVs described herein are obtained from a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Lactococcus lactis cremoris, Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxytoca, and Veillonella tobetsuensis.
[207] In some embodiments, the EVs described herein are obtained from a Prevotella bacteria. In some embodiments, the EVs described herein are obtained from a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella
pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, and Prevotella veroralis.
[208] In some embodiments, the EVs described herein are obtained from a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3. In some embodiments, the EVs described herein are obtained from a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
[209] The Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae . The Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, mA Acidaminococcus. Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
[210] The Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae . The Negativicutes class includes the genera Megasphaera, Selenomonas, Propionospora, and Acidaminococcus. Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
[211] In some embodiments, the bacteria from which the EVs are obtained are of the Negativicutes class.
[212] In some embodiments, the bacteria from which the EVs are obtained are of the Veillonellaceae family.
[213] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonadaceae family.
[214] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.
[215] In some embodiments, the bacteria from which the EVs are obtained are of the Sporomusaceae family.
[216] In some embodiments, the bacteria from which the EVs are obtained are of the Megasphaera genus.
[217] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonas genus.
[218] In some embodiments, the bacteria from which the EVs are obtained are of the Propionospora genus.
[219] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcus genus.
[220] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
[221] In some embodiments, the bacteria from which the EVs are obtained are Selenomonas felix bacteria.
[222] In some embodiments, the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.
[223] In some embodiments, the bacteria from which the EVs are obtained are Propionospora sp. bacteria.
[224] The Oscillospriraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.
[225] In some embodiments, the bacteria from which the EVs are obtained are of the Clostridia class.
[226] In some embodiments, the bacteria from which the EVs are obtained are of the Oscillospriraceae family.
[227] In some embodiments, the bacteria from which the EVs are obtained are of the Faecalibacterium genus.
[228] In some embodiments, the bacteria from which the EVs are obtained are of the Fournierella genus.
[229] In some embodiments, the bacteria from which the EVs are obtained are of the Harryflintia genus.
[230] In some embodiments, the bacteria from which the EVs are obtained are of the Agathobaculum genus.
[231] In some embodiments, the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
[232] In some embodiments, the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
[233] In some embodiments, the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
[234] In some embodiments, the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
[235] In some embodiments, the bacteria from which the EVs are obtained are bacteria of a genus selected from the group consisting of Escherichia, Klebsiella, Lactobacillus, Shigella, and Staphylococcus.
[236] In some embodiments, the bacteria from which the EVs are obtained are a species selected from the group consisting of Blautia massiliensis, Paraclostridium benzoelyticum, Dielma fastidiosa, Longicatena caecimuris, Lactococcus lactis cremoris, Tyzzerella nexilis, Hungatella effluvia, Klebsiella quasipneumoniae subsp. Similipneumoniae, Klebsiella oxytoca, and Veillonella tobetsuensis.
[237] In some embodiments, the bacteria from which the EVs are obtained are a Prevotella bacteria selected from the group consisting of Prevotella albensis, Prevotella amnii, Prevotella bergensis, Prevotella bivia, Prevotella brevis, Prevotella bryantii, Prevotella buccae, Prevotella buccalis, Prevotella copri, Prevotella dentalis, Prevotella denticola, Prevotella disiens, Prevotella histicola, Prevotella intermedia, Prevotella maculosa, Prevotella marshii, Prevotella melaninogenica, Prevotella micans, Prevotella multiformis, Prevotella nigrescens, Prevotella oralis, Prevotella oris, Prevotella oulorum, Prevotella pallens, Prevotella salivae, Prevotella stercorea, Prevotella tannerae, Prevotella timonensis, Prevotella jejuni, Prevotella aurantiaca, Prevotella baroniae, Prevotella colorans, Prevotella corporis, Prevotella dentasini, Prevotella enoeca, Prevotella falsenii, Prevotella fusca, Prevotella heparinolytica, Prevotella loescheii, Prevotella multisaccharivorax, Prevotella nanceiensis, Prevotella oryzae, Prevotella paludivivens, Prevotella pleuritidis, Prevotella ruminicola, Prevotella saccharolytica, Prevotella scopos, Prevotella shahii, Prevotella zoogleoformans, and Prevotella veroralis.
[238] In some embodiments, the bacteria from which the EVs are obtained are a strain of bacteria comprising a genomic sequence that is at least 90%, at least 91%, at least 92%, at
least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the genomic sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3. In some embodiments, the bacteria from which the EVs are obtained are a strain of bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence of the strain of bacteria deposited with the ATCC Deposit number as provided in Table 3.
[239] The Negativicutes class includes the families Veillonellaceae, Selenomonadaceae, Acidaminococcaceae, and Sporomusaceae . The Negativicutes class includes the genera
Exemplary Negativicutes species include, but are not limited to, Megasphaera sp., Selenomonas felix, Acidaminococcus intestini, and Propionospora sp.
[240] In some embodiments, the bacteria from which the EVs are obtained are of the Negativicutes class.
[241] In some embodiments, the bacteria from which the EVs are obtained are of the Veillonellaceae family.
[242] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonadaceae family.
[243] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcaceae family.
[244] In some embodiments, the bacteria from which the EVs are obtained are of the Sporomusaceae family.
[245] In some embodiments, the bacteria from which the EVs are obtained are of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Christensenellaceae; or Akkermaniaceae family.
[246] In some embodiments, the bacteria from which the EVs are obtained are of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
[247] In some embodiments, the bacteria from which the EVs are obtained are of the Megasphaera genus.
[248] In some embodiments, the bacteria from which the EVs are obtained are of the Selenomonas genus.
[249] In some embodiments, the bacteria from which the EVs are obtained are of the Propionospora genus.
[250] In some embodiments, the bacteria from which the EVs are obtained are of the Acidaminococcus genus.
[251] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera sp. bacteria.
[252] In some embodiments, the bacteria from which the EVs are obtained are Selenomonas felix bacteria.
[253] In some embodiments, the bacteria from which the EVs are obtained are Acidaminococcus intestini bacteria.
[254] In some embodiments, the bacteria from which the EVs are obtained are Propionospora sp. bacteria.
[255] The Oscillospriraceae family within the Clostridia class of microorganisms are common commensal organisms of vertebrates.
[256] In some embodiments, the bacteria from which the EVs are obtained are of the Clostridia class.
[257] In some embodiments, the bacteria from which the EVs are obtained are of the Oscillospriraceae family.
[258] In some embodiments, the bacteria from which the EVs are obtained are of the Faecalibacterium genus.
[259] In some embodiments, the bacteria from which the EVs are obtained are of the Fournierella genus.
[260] In some embodiments, the bacteria from which the EVs are obtained are of the Harryflintia genus.
[261] In some embodiments, the bacteria from which the EVs are obtained are of the Agathobaculum genus.
[262] In some embodiments, the bacteria from which the EVs are obtained are Faecalibacterium prausnitzii (for example, Faecalibacterium prausnitzii Strain A) bacteria.
[263] In some embodiments, the bacteria from which the EVs are obtained are Fournierella massiliensis (for example, Fournierella massiliensis Strain A) bacteria.
[264] In some embodiments, the bacteria from which the EVs are obtained are Harryflintia acetispora (for example, Harryflintia acetispora Strain A) bacteria.
[265] In some embodiments, the bacteria from which the EVs are obtained are Agathobaculum sp. (for example, Agathobaculum sp. Strain A) bacteria.
[266] In some embodiments, the bacteria from which the EVs are obtained are a strain of Agathobaculum sp. In some embodiments, the Agathobaculum sp. strain is a strain comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, CRISPR sequence) of the Agathobaculum sp. Strain A (ATCC Deposit Number PTA-125892). In some embodiments, the Agathobaculum sp. strain is the Agathobaculum sp. Strain A (ATCC Deposit Number PTA- 125892).
[267] In some embodiments, the bacteria from which the EVs are obtained are of the class Bacteroidia [phylum Bacteroidota\. In some embodiments, the bacteria from which the EVs are obtained are bacteria of order Bacteroidales. In some embodiments, the bacteria from which the EVs are obtained are of the family Porphyromonoadaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Prevotellaceae . In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Bacteroidia wherein the bacteria is diderm and the bacteria stain Gram negative.
[268] In some embodiments, the bacteria from which the EVs are obtained are bacteria of the class Clostridia [phylum Firmicutes\. In some embodiments, the bacteria from which the EVs are obtained are of the order Eubacteriales. In some embodiments, the bacteria from which the EVs are obtained are of the family Oscillispiraceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Lachnospiraceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Peptostreptococcaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Clostridiales family XIII/ Incertae sedis 41. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia that
stain Gram positive. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Clostridia wherein the cell envelope structure of the bacteria is monoderm and the bacteria stain Gram positive.
[269] In some embodiments, the bacteria from which the EVs are obtained are of the class Negativicutes [phylum Firmicutes\. In some embodiments, the bacteria from which the EVs are obtained are of the order Veillonellales. In some embodiments, the bacteria from which the EVs are obtained are of the family Veillonelloceae. In some embodiments, the bacteria from which the EVs are obtained are of the order Selenomonadales. In some embodiments, the bacteria from which the EVs are obtained are bacteria of the family Selenomonadaceae . In some embodiments, the bacteria from which the EVs are obtained are of the family Sporomusaceae . In some embodiments, t the bacteria from which the EVs are obtained are of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are of the bacteria from which the EVs are obtained are the EVs are from bacteria of the class Negativicutes wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
[270] In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia [phylum Synergistota\. In some embodiments, the bacteria from which the EVs are obtained are of the order Synergistales. In some embodiments, the bacteria from which the EVs are obtained are of the family Synergistaceae . In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm. In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia that stain Gram negative. In some embodiments, the bacteria from which the EVs are obtained are of the class Synergistia wherein the cell envelope structure of the bacteria is diderm and the bacteria stain Gram negative.
[271] In some embodiments, the bacteria from which the EVs are obtained are from one strain of bacteria, for example, a strain provided herein.
[272] In some embodiments, the bacteria from which the EVs are obtained are from one strain of bacteria (for example, a strain provided herein) or from more than one strain provided herein.
[273] In some embodiments, the bacteria from which the EVs are obtained are Lactococcus lactis cremoris bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the
Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368). In some embodiments, the bacteria from which the EVs are obtained are Lactococcus bacteria, for example, Lactococcus lactis cremoris Strain A (ATCC designation number PTA-125368).
[274] In some embodiments, the bacteria from which the EVs are obtained are of the Prevotella genus. In some embodiments, the bacteria from which the EVs are obtained are Prevotella histicola bacteria.
[275] In some embodiments, the bacteria from which the EVs are obtained are Prevotella bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella Strain B 50329 (NRRL accession number B 50329). In some embodiments, the bacteria from which the EVs are obtained are Prevotella bacteria, for example, Prevotella Strain B 50329 (NRRL accession number B 50329).
[276] In some embodiments, the bacteria from which the EVs are obtained are Prevotella histicola bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Prevotella histicola Strain C deposited as ATCC designation number PTA-126140. In some embodiments, the bacteria from which the EVs are obtained are Prevotella histicola bacteria, for example Prevotella histicola Strain C deposited as ATCC designation number PTA-126140).
[277] In some embodiments, the bacteria from which the EVs are obtained are Bifidobacterium bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Bifidobacterium bacteria deposited as ATCC designation number PTA-125097. In some embodiments, the bacteria from which the EVs are obtained are Bifidobacterium bacteria, for example, Bifidobacterium bacteria deposited as ATCC designation number PTA-125097.
[278] In some embodiments, the bacteria from which the EVs are obtained are Veillonella bacteria, for example, a strain comprising at least 90% or at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Veillonella bacteria deposited as ATCC designation number PTA-125691. In some embodiments, the bacteria from which the EVs are obtained are Veillonella bacteria, for example, Veillonella bacteria deposited as ATCC designation number PTA-125691.
[279] In some embodiments, the bacteria from which the EVs are obtained are Ruminococcus gnavus bacteria. In some embodiments, the Ruminococcus gnavus bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC
designation number PTA-126695. In some embodiments, the Ruminococcus gnavus bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695. In some embodiments, the Ruminococcus gnavus bacteria are Ruminococcus gnavus bacteria deposited as ATCC designation number PTA-126695.
[280] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera sp. bacteria. In some embodiments, the Megasphaera sp. bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770. In some embodiments, the Megasphaera sp. bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Megasphaera .s/ bacteria deposited as ATCC designation number PTA- 126770. In some embodiments, the Megasphaera sp. bacteria are Megasphaera sp. bacteria deposited as ATCC designation number PTA-126770.
[281] In some embodiments, the bacteria from which the EVs are obtained are Fournierella massiliensis bacteria. In some embodiments, the Fournierella massiliensis bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696. In some embodiments, the Fournierella massiliensis bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696. In some embodiments, the Fournierella massiliensis bacteria are Fournierella massiliensis bacteria deposited as ATCC designation number PTA-126696.
[282] In some embodiments, the bacteria from which the EVs are obtained are Harryflintia acetispora bacteria. In some embodiments, the Harryflintia acetispora bacteria are a strain comprising at least 90% (or at least 97%) genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694. In some embodiments, the Harryflintia acetispora bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of the Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694. In some embodiments, the Harryflintia acetispora bacteria are Harryflintia acetispora bacteria deposited as ATCC designation number PTA-126694.
[283] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce metabolites, for example, the bacteria produce butyrate, iosine, proprionate, or tryptophan metabolites.
[284] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce butyrate. In some embodiments, the bacteria are from the genus Blautia;
Christensella; Copracoccus; Eubacterium; Lachnosperacea; Megasphaera; or Roseburia.
[285] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce iosine. In some embodiments, the bacteria are from the genus Bifidobacterium; Lactobacillus; or Olsenella.
[286] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce proprionate. In some embodiments, the bacteria are from the genus Akkermansia; Bacteriodes; Dialister; Eubacterium; Megasphaera; Parabacteriodes;
Prevotella; Ruminococcus; or Veillonella.
[287] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce tryptophan metabolites. In some embodiments, the bacteria are from the genus Lactobacillus or Peptostreptococcus.
[288] In some embodiments, the bacteria from which the EVs are obtained are bacteria that produce inhibitors of histone deacetylase 3 (HDAC3). In some embodiments, the bacteria are from the species Bariatricus massiliensis, Faecalibacterium prausnitzii, Megasphaera massiliensis or Roseburia intestinalis.
[289] In some embodiments, the bacteria are from the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus; Enhydrobacter; Exiguobacterium; Faecalibacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus;
Pseudomonas; Rhizobium; or Sphingomonas. In some embodiments, the bacteria are from the genus Cutibacterium. In some embodiments, the bacteria are from the species Cutibacterium avidum. In some embodiments, the bacteria are from the genus Lactobacillus. In some embodiments, the bacteria are from the species Lactobacillus gasseri. In some embodiments, the bacteria are from the genus Dysosmobacter . In some embodiments, the bacteria are from the species Dysosmobacter welbionis.
[290] In some embodiments, the bacteria from which the EVs are obtained are of the genus Alloiococcus; Bacillus; Catenibacterium; Corynebacterium; Cupriavidus;
Enhydrobacter; Exiguobacterium; Faecalibacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus; Pseudomonas; Rhizobium; or Sphingomonas.
[291] In some embodiments, the bacteria from which the EVs are obtained are of the Cutibacterium genus. In some embodiments, the bacteria from which the EVs are obtained are Cutibacterium avidum bacteria.
[292] In some embodiments, the bacteria from which the EVs are obtained are of the genus Leuconostoc.
[293] In some embodiments, the bacteria from which the EVs are obtained are of the genus Lactobacillus.
[294] In some embodiments, the bacteria from which the EVs are obtained are of the genus Akkermansia; Bacillus; Blautia; Cupriavidus; Enhydrobacter; Faecalibacterium; Lactobacillus; Lactococcus; Micrococcus; Morganella; Propionibacterium; Proteus; Rhizobium; or Streptococcus.
[295] In some embodiments, the bacteria from which the EVs are obtained are Leuconostoc holzapfelii bacteria.
[296] In some embodiments, the bacteria from which the EVs are obtained are Akkermansia muciniphila; Cupriavidus metallidurans; Faecalibacterium prausnitzii; Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; Lactobacillus sakei; or Streptococcus pyogenes bacteria.
[297] In some embodiments, the bacteria from which the EVs are obtained are Lactobacillus casei; Lactobacillus plantarum; Lactobacillus paracasei; Lactobacillus plantarum; Lactobacillus rhamnosus; or Lactobacillus sakei bacteria.
[298] In some embodiments, the EVs described herein are obtained from a genus selected from the group consisting of Acinetobacter; Deinococcus; Helicobacter; Rhodococcus;
Weissella cibaria; Alloiococcus; Atopobium; Catenibacterium; Corynebacterium; Exiguobacterium; Geobacillus; Methylobacterium; Micrococcus; Morganella; Proteus; Rhizobium; Rothia; Sphingomonas; Sphingomonas; and Leuconostoc.
[299] In some embodiments, the EVs described herein are obtained from a species selected from the group consisting of Acinetobacter baumanii; Deinococcus radiodurans; Helicobacter pylori; Rhodococcus equi; Weissella cibaria; Alloiococcus otitis; Atopobium vaginae; Catenibacterium mituokai; Corynebacterium glutamicum; Exiguobacterium aurantiacum; Geobacillus stearothermophilus; Methylobacterium jeotgali; Micrococcus luteus; Morganella morganii; Proteus mirabilis; Rhizobium leguminosarum; Rothia amarae; Sphingomonas paucimobilis; and Sphingomonas koreens.
[300] In some embodiments, the EVs are from Leuconostoc holzapfelii bacteria. In some embodiments, the EVs are from Leuconostoc holzapfelii Ceb-kc-003 (KCCM11830P) bacteria.
[301] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera sp. bacteria (for example, from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387).
[302] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number NCIMB 42787, NCIMB 43388 or NCIMB 43389).
[303] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number DSM 26228).
[304] In some embodiments, the bacteria from which the EVs are obtained are Parabacteroides distasonis bacteria (for example, from the strain with accession number NCIMB 42382).
[305] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria (for example, from the strain with accession number NCIMB 43388 or NCIMB 43389), or a derivative thereof. See, for example, WO 2020/120714. In some embodiments, the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria from the strain with accession number NCIMB 43388 or NCIMB 43389. In some embodiments, the Megasphaera massiliensis bacteria is the strain with accession number NCIMB 43388 or NCIMB 43389.
[306] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787, or a derivative thereof. See, for example, WO 2018/229216. In some embodiments, the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity)
to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera massiliensis bacteria strain deposited under accession number NCIMB 42787. In some embodiments, the Megasphaera massiliensis bacteria is the strain deposited under accession number NCIMB 42787.
[307] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera spp. bacteria from the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387, or a derivative thereof. See, for example, WO 2020/120714. In some embodiments, the Megasphaera sp. bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Megasphaera sp. from a strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387. In some embodiments, the Megasphaera sp. bacteria is the strain with accession number NCIMB 43385, NCIMB 43386 or NCIMB 43387.
[308] In some embodiments, the bacteria from which the EVs are obtained are Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382, or a derivative thereof. See, for example, WO 2018/229216. In some embodiments, the Parabacteroides distasonis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of the Parabacteroides distasonis bacteria deposited under accession number NCIMB 42382. In some embodiments, the Parabacteroides distasonis bacteria is the strain deposited under accession number NCIMB 42382.
[309] In some embodiments, the bacteria from which the EVs are obtained are Megasphaera massiliensis bacteria deposited under accession number DSM 26228, or a derivative thereof. See, for example, WO 2018/229216. In some embodiments, the Megasphaera massiliensis bacteria is a strain comprising at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (for example, at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity)
to the nucleotide sequence (for example, genomic sequence, 16S sequence, and/or CRISPR sequence) of Megasphaera massiliensis bacteria deposited under accession number DSM 26228. In some embodiments, the Megasphaera massiliensis bacteria is the strain deposited under accession number DSM 26228.
[310] In some embodiments, the bacteria from which the EVs are obtained are modified (for example, engineered) to reduce toxicity or other adverse effects, to enhance delivery) (for example, oral delivery) of the EVs (for example, by improving acid resistance, muco- adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (for example, M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the EVs (for example, either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the EVs (for example, through modified production of polysaccharides, pili, fimbriae, adhesins). In some embodiments, the engineered bacteria described herein are modified to improve EV manufacturing (for example, higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times). For example, in some embodiments, the engineered bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may results in the overexpression and/or underexpression of one or more genes. The engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
* The abbreviation given in the parenthetical is for the species in the row in which it is listed.
Modified EVs
[315] In some aspects, the EVs described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
[316] In some embodiments, the therapeutic moiety is a cancer-specific moiety. In some embodiments, the cancer-specific moiety has binding specificity for a cancer cell (for example, has binding specificity for a cancer-specific antigen). In some embodiments, the cancer-specific moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the cancer-specific moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the cancer-specific moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In some embodiments, the cancer-specific moiety is a bipartite fusion protein that has two parts: a first part that binds to and/or is linked to the bacterium and a second part that is capable of binding to a cancer cell (for example, by having binding specificity for a cancer-specific antigen). In some embodiments, the first part is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the first part has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen). In some embodiments, the first and/or second part comprises an antibody or antigen binding fragment thereof. In some embodiments, the first and/or second part comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the first and/or second part comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptorbinding fragment thereof. In certain embodiments, co-administration of the cancer-specific moiety with the EVs (either in combination or in separate administrations) increases the targeting of the EVs to the cancer cells.
[317] In some embodiments, the EVs described herein are engineered such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (for example, a magnetic bead). In some embodiments, the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria. In some embodiments, the magnetic and/or paramagnetic moiety is linked to and/or a part of an EV-binding moiety that that binds to the EV. In some embodiments, the EV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the EV-binding moiety has binding specificity for the EV (for example, by having binding specificity for a bacterial antigen). In some embodiments, the EV-binding moiety comprises an antibody or antigen binding fragment thereof. In some embodiments, the EV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the EV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fragment thereof. In certain embodiments, co-administration of the magnetic and/or paramagnetic moiety with the EVs (either together or in separate administrations) can be used to increase the targeting of the EVs (for example, to cancer cells and/or a part of a subject where cancer cells are present.
Production of Bacterial Extracellular Vesicles (EVs)
[318] In certain aspects, the EVs from bacteria described herein are prepared using any method known in the art.
[319] In some embodiments, the EVs are prepared without an EV purification step. For example, in some embodiments, bacteria described herein are killed using a method that leaves the EVs intact and the resulting bacterial components, including the EVs, are used in the methods and compositions described herein. In some embodiments, the bacteria are killed using an antibiotic (for example, using an antibiotic described herein). In some embodiments, the bacteria are killed using UV irradiation. In some embodiments, the bacteria are heat- killed.
[320] In some embodiments, the EVs described herein are purified from one or more other bacterial components. Methods for purifying EVs from bacteria are known in the art. In some embodiments, EVs are prepared from bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): eO 134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (for example, at 10,000 x g for 30
min at 4°C, at 15,500 x g for 15 min at 4°C). In some embodiments, the culture supernatants are then passed through filters to exclude intact bacterial cells (for example, a 0.22 pm filter). In some embodiments, the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS. In some embodiments, filtered supernatants are centrifuged to pellet bacterial EVs (for example, at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C). In some embodiments, the EVs are further purified by resuspending the resulting EV pellets (for example, in PBS), and applying the resuspended EVs to an Optiprep (iodixanol) gradient or gradient (for example, a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (for example, at 200,000 x g for 4-20 hours at 4°C). EV bands can be collected, diluted with PBS, and centrifuged to pellet the EVs (for example, at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C). The purified EVs can be stored, for example, at -80°C or -20°C until use. In some embodiments, the EVs are further purified by treatment with DNase and/or proteinase K.
[321] For example, in some embodiments, cultures of bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria. Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells. Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C. Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C. The resulting pellets contain bacteria EVs and other debris. Using ultracentrifugation, filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains bacterial EVs and other debris such as large protein complexes. In some embodiments, using a filtration technique, such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
[322] Alternatively, EVs can be obtained from bacteria cultures continuously during growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (for example, XCell ATF from Repligen). The ATF system retains intact cells (>0.22 pm) in the bioreactor, and allows smaller components (for example, EVs, free proteins) to pass through a filter for collection. For example, the system may be configured so that the <0.22 pm filtrate is then passed through a second filter
of 100 kDa, allowing species such as EVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor. Alternatively, the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. EVs collected by this method may be further purified and/or concentrated by ultracentrifugation or filtration as described above for filtered supernatants.
[323] EVs obtained by methods provided herein may be further purified by size-based column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35- 60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfate precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, for example, 4-24 hours at 4°C.
[324] In some embodiments, to confirm sterility and isolation of the EV preparations, EVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 um filter to exclude intact cells. To further increase purity, isolated EVs may be DNase or proteinase K treated.
[325] In some embodiments, for preparation of EVs used for in vivo injections, purified EVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing EVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v). In some
embodiments, for preparation of EVs used for in vivo injections, EVs in PBS are sterile- filtered to < 0.22 pm.
[326] In certain embodiments, to make samples compatible with further testing (for example, to remove sucrose prior to TEM imaging or in vitro assays), samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (for example, Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
[327] In some embodiments, the sterility of the EV preparations can be confirmed by plating a portion of the EVs onto agar medium used for standard culture of the bacteria used in the generation of the EVs and incubating using standard conditions.
[328] In some embodiments, select EVs are isolated and enriched by chromatography and binding surface moieties on EVs. In some embodiments, select EVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
[329] In some embodiments, EVs are analyzed, for example, as described in Jeppesen, et al. Cell 177:428 (2019).
[330] In some embodiments, EVs are lyophilized.
[331] In some embodiments, EVs are gamma irradiated (for example, at 17.5 or 25 kGy).
[332] In some embodiments, EVs are UV irradiated.
[333] In some embodiments, EVs are heat inactivated (for example, at 50°C for two hours or at 90°C for two hours).
[334] In some embodiments, EVs are acid treated.
[335] In some embodiments, EVs are oxygen sparged (for example, at 0.1 vvm for two hours).
[336] The phase of growth can affect the amount or properties of bacteria and/or EVs produced by bacteria. For example, in the methods of EV preparation provided herein, EVs can be isolated, for example, from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
[337] The growth environment (for example, culture conditions) can affect the amount of EVs produced by bacteria. For example, the yield of EVs can be increased by an EV inducer, as provided in Table 5.
Table 5: Culture Techniques to Increase EV Production
[338] In the methods of EVs preparation provided herein, the method can optionally include exposing a culture of bacteria to an EV inducer prior to isolating EVs from the bacterial culture. The culture of bacteria can be exposed to an EV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
Methods of Making Enhanced Bacteria
[339] In certain aspects, provided herein are methods of making engineered bacteria for the production of the EVs described herein. In some embodiments, the engineered bacteria are modified to enhance certain desirable properties. For example, in some embodiments, the engineered bacteria are modified to enhance the immunomodulatory and/or therapeutic effect of the EVs (for example, either alone or in combination with another therapeutic agent), to reduce toxicity and/or to improve bacterial and/or EV manufacturing (for example, higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times). The engineered bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet light mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, CRISPR/Cas9, or any combination thereof.
[340] In some embodiments of the methods provided herein, the bacterium is modified by directed evolution. In some embodiments, the directed evolution comprises exposure of the bacterium to an environmental condition and selection of bacterium with improved survival and/or growth under the environmental condition. In some embodiments, the method comprises a screen of mutagenized bacteria using an assay that identifies enhanced bacterium. In some embodiments, the method further comprises mutagenizing the bacteria (for example, by exposure to chemical mutagens and/or UV radiation) or exposing them to a therapeutic agent (for example, antibiotic) followed by an assay to detect bacteria having the desired phenotype (for example, an in vivo assay, an ex vivo assay, or an in vitro assay).
Exemplary Embodiments
1. A method of producing extracellular vesicles (EVs), the method comprising growing EV- producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises
culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
2. The method of embodiment 1, wherein the perfusion culture increases EV yields by at least about 10-fold, e.g., by at least about 15-fold or by at least about 17-fold or by at least about 50-fold, as compared to a batch culture of the same bacteria.
3. The method of embodiment 1 or 2, wherein the perfusion culture increases EV yields after 24, 48, or 72 hours of culturing, as compared to a batch culture of the same bacteria.
4. The method of any one of embodiments 1 to 3, wherein EV production of the bacteria is coupled to growth in batch culture.
5. The method of any one of embodiments 1 to 4, wherein EV production of the bacteria is not coupled to growth in batch culture.
6. The method of any one of embodiments 1 to 5, wherein the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
7. The method of any one of embodiments 1 to 6, wherein a filter system (one-filter or two- filter system) removes EVs, metabolites, and waste products of (e.g., from) the culture media.
8. The method of embodiment 7, wherein the filter system is a two-filter system.
9. The method of any one of embodiments 1 to 6, wherein the method further comprises filtering the culture media.
10. The method of any one of embodiments 1 to 9, wherein the method further comprises performing chromatography on the culture media.
11. The method of any one of embodiments 1 to 10, wherein the method further comprises performing tangential flow filtration on the culture media.
12. The method of any one of embodiments 1 to 11, wherein the method further comprises drying the culture media.
13. The method of embodiment 12, wherein the method further comprises milling the dried culture media.
14. A method of processing bacterial culture media that comprises extracellular vesicles (EVs), the method comprising passing bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter (e.g., and passing the bacterial culture media that comprises EVs through the product harvest filter produces an output of the product harvest filter) and the second filter is a medium exchange filter.
15. The method of embodiment 14, wherein the bacterial culture media is from a perfusion culture.
16. The method of embodiment 15, wherein the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter scale or 20,000 liter scale or greater).
17. The method of any one of embodiments 14 to 16, wherein the product harvest filter and the medium exchange filter comprise the same material.
18. The method of any one of embodiments 14 to 17, wherein the product harvest filter comprises PES (polyethersulfone).
19. The method of any one of embodiments 14 to 18, wherein the medium exchange filter comprises PES (polyethersulfone).
20. The method of any one of embodiments 14 to 19, wherein the product harvest filter and the medium exchange filter comprise PES (polyethersulfone).
21. The method of any one of embodiments 14 to 20, wherein EVs, media, waste and metabolites pass through the product harvest filter.
22. The method of any one of embodiments 14 to 21, wherein the pore size of the product harvest filter is about 0.5 micron.
23. The method of any one of embodiments 14 to 22, wherein media, waste and metabolites pass through the medium exchange filter.
24. The method of any one of embodiments 14 to 23, wherein the pore size of the medium exchange filter is less than about 0.5 micron.
25. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter is about 0.05 micron.
26. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter is about 0.02 micron.
27. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter is about 0.01 micron.
28. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter comprises a size cut off of 750kD (kilodalton).
29. The method of any one of embodiments 14 to 24, wherein the pore size of the medium exchange filter comprises a size cut off of 500kD.
30. The method of any one of embodiments 14 to 29, wherein the medium exchange filter runs at a higher flux than the product harvest filter.
31. The method of any one of embodiments 14 to 30, wherein the flux ratio (medium exchange filterproduct harvest filter) is about 5: 1.
32. The method of any one of embodiments 14 to 30, wherein the flux ratio (medium exchange filterproduct harvest filter) is about 9: 1.
33. The method of any one of embodiments 14 to 30, wherein the flux ratio (medium exchange filterproduct harvest filter) is about 10: 1.
34. The method of any one of embodiments 14 to 33, wherein the flux ratio (medium exchange filterproduct harvest filter) reduces sieving of the product harvest filter (e.g., as compared to the amount of sieving if the flux ratio was 1 : 1 or if the product harvest filter was used alone).
35. The method of any one of embodiments 14 to 34, wherein the volume of the output of the product harvest filter is about l/5x the volume than if a single-filter system was used (e.g., the volume of the output of the product harvest filter is about l/5x the volume as compared to the volume that would result from a single-filter system).
36. The method of any one of embodiments 14 to 34, wherein the volume of the output of the product harvest filter is about l/9x the volume than if a single-filter system was used.
37. The method any one of embodiments 14 to 34, wherein the volume of the output of the product harvest filter is about l/10x the volume than if a single-filter system was used.
38. The method of any one of embodiments 14 to 37, wherein the output of the product harvest filter comprises a higher concentration of EVs than if a single-filter system was used.
39. The method of any one of embodiments 14 to 38, wherein the output of the product harvest filter comprises a concentration of EVs that is at least about 5x higher than if a singlefilter system was used.
40. The method of any one of embodiments 14 to 38, wherein the output of the product harvest filter comprises a concentration of EVs that is at least about 9x higher than if a singlefilter system was used.
41. The method of any one of embodiments 14 to 38, wherein the output of the product harvest filter comprises a concentration of EVs that is at least about lOx higher than if a single-filter system was used.
42. The method of any one of embodiments 14 to 41, wherein the method further comprises performing chromatography on the output of the product harvest filter.
43. The method of any one of embodiments 14 to 42, wherein the method further comprises performing tangential flow filtration on the output of the product harvest filter.
44. The method of any one of embodiments 14 to 43, wherein the method further comprises drying the output of the product harvest filter.
45. The method of embodiment 44, wherein the method further comprises milling the dried output of the product harvest filter.
46. A method of processing a liquid that comprises extracellular vesicles (EVs) to prepare an EV eluate, the method comprising performing chromatography on the liquid.
47. The method of embodiment 46, wherein the liquid comprises bacterial culture media.
48. The method of embodiment 47, wherein the bacterial culture media is from a perfusion culture.
49. The method of embodiment 48, wherein the perfusion culture is at commercial scale (such as 1,000 liter scale or greater, such as 5,000 liter or 20,000 liter scale or greater).
50. The method of embodiment 46, wherein the liquid comprises a product-containing volume.
51. The method of embodiment 50, wherein the product-containing volume is output from a single-filter system.
52. The method of embodiment 50, wherein the product-containing volume is output from a two-filter system.
53. The method of embodiment 50, wherein the product-containing volume is output from a product harvest filter.
54. The method of embodiment 53, wherein the product harvest filter comprises PES (polyethersulfone).
55. The method of embodiment 53 or 54, wherein the pore size of the product harvest filter is about 0.5 micron.
56. The method of any one of embodiments 46 to 55, wherein the chromatography comprises a chromatography column.
57. The method of embodiment 57, wherein the chromatography column comprises a monolith.
58. The method of embodiment 56 or 57, wherein the chromatography column comprises a monolith ion exchange column.
59. The method of any one of embodiments 56 to 58, wherein the method comprises processing on one column.
60. The method of any one of embodiments 56 to 59, wherein the liquid is loaded onto the column and an EV-containing eluate is eluted.
61. The method any one of embodiments 56 to 58, wherein the method comprises processing on two columns.
62. The method of any one of embodiments 56 to 58 or 61, wherein the liquid is loaded onto a first column, the EV-containing eluate is eluted from the first column, wherein while EV-containing eluate is eluted from the first column, liquid is loaded onto the second column.
63. The method of any one of embodiments 56 to 58, 61, or 62, wherein EV-containing eluate is eluted from the second column, wherein while EV-containing eluate is eluted from the second column, liquid is loaded onto the first column.
64. The method of any one of embodiments 56 to 58 or 61 to 63, wherein the loading and eluting steps on the first and second columns are alternated to process the liquid continuously.
65. The method of any one of embodiments 46 to 64, wherein bacterial culture media is filtered prior to performing the chromatography.
66. The method of any one of embodiments 46 to 65, wherein the chromatography enriches EV yield by greater than about 5-fold (e.g., as compared to EV yield from the liquid in the absence of chromatography).
67. The method of any one of embodiments 46 to 66, wherein the chromatography enriches EV yield by about 6-fold.
68. The method of any one of embodiments 46 to 66, wherein the chromatography enriches EV yield by about 12-fold.
69. The method of any one of embodiments 46 to 68, wherein the yield of EVs from the chromatography is greater than about 50%.
70. The method of any one of embodiments 46 to 69, wherein the yield of EVs from the chromatography is greater than about 60%.
72. The method of any one of embodiments 46 to 70, wherein the method further comprises performing tangential flow filtration on the EV eluate.
73. The method of any one of embodiments 46 to 72, wherein the method further comprises drying the EV eluate.
74. The method of embodiment 73, wherein the method further comprises milling the dried EV eluate.
75. A method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs; and
(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter, thereby preparing output of the product harvest filter, wherein the output comprises EVs.
76. A method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs; and
(ii) performing chromatography on the bacterial culture media to prepare an eluate, wherein the eluate comprises EVs.
77. A method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs;
(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter, thereby preparing output of the product harvest filter, wherein the output comprises EVs; and
(iii) performing chromatography on the output of the product harvest filter to prepare an eluate, wherein the eluate comprises EVs.
78. The method of any one of embodiments 1 to 77, wherein the method comprises EVs from a bacterial strain that is associated with mucus.
79. The method of any one of embodiments 1 to 77, wherein the method comprises EVs from anaerobic bacteria.
80. The method of embodiment 79, wherein the anaerobic bacteria are obligate (e.g., strict) anaerobes.
81. The method of embodiment 79, wherein the anaerobic bacteria are facultative anaerobes.
82. The method of embodiment 79, wherein the anaerobic bacteria are aerotolerant anaerobes.
83. The method of any one of embodiments 1 to 77, wherein the EVs are from monoderm bacteria.
84. The method of any one of embodiments 1 to 77, wherein the EVs are from diderm bacteria.
85. The method of any one of embodiments 1 to 77, wherein the EVs are from Gram negative bacteria.
86. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Prevotellaceae; Veillonellaceae; Tannerellaceae; Rikenellaceae; Selenomonadaceae; Sporomusaceae; Synergistaceae; Phrislensenellaceae or Akkermaniaceae family.
87. The method of any one of embodiments 1 to 77, wherein the EVs are from Gram positive bacteria.
88. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Oscillospiraceae ; Clostridiaceae; or Lachnospiraceae family.
89. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the genus Prevotella.
90. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the genus Veillonella.
91. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the genus Parabacteroides.
92. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Oscillospiraceae family.
93. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Tannerellaceae family.
94. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Prevotellaceae family.
95. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of the Veillonellaceae family.
96. The method of any one of embodiments 1 to 77, wherein the EVs are from bacteria of class, order, family, genus, species and/or strain of bacteria provided in Table 1, Table 2, Table 3, and/or Table 4.
EXAMPLES
Example 1: EV manufacturing platform
[341] Figure l is a schematic showing a process/manufacturing platform for EVs to improve productivity. Such a system provides culture intensification and clarification, using a high density perfusion process at 20,000L-scale process, and could increase manufacturing plant output >50-fold and includes using a two-filter system (such as hollow fiber product separation). Next, purification and concentration provide continuous capture to achieve pure and highly concentrated process intermediates that contain EVs, and can include continuous chromatography capture and tangential flow filtration. The output can be further processed by drying (such as spray drying or lyophilization) of EVs, and can undergo post-processing, such as milling.
Example 2: Perfusion culture yields
[342] Figures 2A and 2B are graphs showing comparisons of EV yields (EV product batches (-fold)) from batch culture versus perfusion culture yields over time (hours). Figure 2A shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is coupled with growth. Figure 2B shows the batch yields (batch yield; dashed line) and perfusion EV yield (total product; upper solid line with circles) for a first strain of bacteria for which EV production is not coupled with growth. In both Figures 2A and 2B, the lower solid line with circles is the EV yield recovered from the perfusion culture (permeated product).
[343] Total EV productivity was significantly improved (total product; upper solid line with circles in both graphs).
[344] Media exchange rate was high.
[345] Current filter sieving limited EV recovered in the filtrate, this provides high potential for improvement. See lower solid line with circles in both graphs.
Example 3: Two-filter system
[346] Figure 3 is a schematic showing a set up for a two-filter system. Harvest volume transfers from the fermenter to the two filters: product harvest filter and medium exchange filter. Captured product from the product harvest filter transfers to a product reservoir and can be further processed, such as through a capture step(s). Metabolites, and waste products that passed through the medium exchange filter transfer to a waste reservoir.
[347] As shown in the figure, a 9: 1 flux ratio (Medium Exchange: Product Harvest) yields lOx the product in 1/1 Oth the volume.
[348] “Medium Exchange” filter handles the bulk of perfusion and is impermeable to product.
[349] “Product Harvest” filter operates at low flux to reduce sieving.
[350] This two-filter system (dual-membrane perfusion) can extend operating time. In addition to reducing sieving of the product harvest filter, this system allows the product harvest filter to collect higher concentration product (EVs) in a smaller volume than if a single filter system was used.
Example 4: Dual membrane perfusion results
[351] Figure 4 is a graph showing a theoretical result of using a two-filter (dual membrane perfusion) system. Overall perfusion rate remains constant at 6 volumes/day.
[352] Shifting flux from the “Product Harvest” filter to the “Medium Exchange” filter increases the Flux Ratio, and results in product concentration (-fold; upward sloping line) increasing and permeate volume (downward sloping line; volume (vol)/day) decreasing with increasing ratio.
[353] Dual-membrane perfusion reduces downstream volume and increases product concentration.
Example 5: Monolith ion exchange chromatography
[354] Harvest volume directly from a perfusion culture showed that a multi-column capture directly from perfusion had acceptable capacity.
[355] pH also affects and can improve capacity (column volume), enrichment, and yield. See Table 6 where the performance of two pH conditions (pH A and pH B) were evaluated.
Incorporation by Reference
[357] All publications patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Equivalents
[358] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Claims
1. A method of producing extracellular vesicles (EVs), the method comprising growing EV- producing bacteria in a perfusion culture (e.g., wherein the perfusion culture comprises culture media that comprises EVs) (e.g., under conditions that support bacteria growth and EV production).
2. The method of claim 1, wherein the method further comprises filtering the culture media.
3. The method of claim 1 or 2, wherein the method further comprises performing chromatography on the culture media.
4. The method of any one of claims 1 to 3, wherein the method further comprises performing tangential flow filtration on the culture media.
5. A method of processing bacterial culture media that comprises extracellular vesicles (EVs), the method comprising passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter.
6. The method of claim 5, wherein the product harvest filter comprises PES (polyethersulfone).
7. The method of claim 5 or 6, wherein the medium exchange filter comprises PES (polyethersulfone).
8. The method of any one of claims 5 to 7, wherein the medium exchange filter runs at a higher flux than the product harvest filter.
9. The method of any one of claims 5 to 8, wherein the method further comprises performing chromatography on the output of the product harvest filter.
10. The method of any one of claims 5 to 9, wherein the method further comprises performing tangential flow filtration on the output of the product harvest filter.
11. A method of processing a liquid that comprises extracellular vesicles (EVs) to prepare an EV eluate, the method comprising performing chromatography on the liquid.
12. The method of claim 11, wherein the liquid comprises bacterial culture media.
13. The method of claim 11 or 12, wherein the method further comprises performing tangential flow filtration on the EV eluate.
14. A method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs; and
(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter, thereby preparing output of the product harvest filter, wherein the output comprises EVs.
15. A method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs; and
(ii) performing chromatography on the bacterial culture media to prepare an eluate, wherein the eluate comprises EVs.
16. A method comprising:
(i) growing EV-producing bacteria in a perfusion culture (e.g., under conditions that support bacteria growth and EV production), wherein the perfusion culture comprises bacterial culture media that comprises EVs;
(ii) passing the bacterial culture media that comprises EVs through a two-filter system, wherein the first filter is a product harvest filter and the second filter is a medium exchange filter, thereby preparing output of the product harvest filter, wherein the output comprises EVs; and
(iii) performing chromatography on the output of the product harvest filter to prepare an eluate, wherein the eluate comprises EVs.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202263414284P | 2022-10-07 | 2022-10-07 | |
US63/414,284 | 2022-10-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2024076723A1 true WO2024076723A1 (en) | 2024-04-11 |
Family
ID=88697857
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2023/034623 WO2024076723A1 (en) | 2022-10-07 | 2023-10-06 | Processing extracellular vesicles |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2024076723A1 (en) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018229216A1 (en) | 2017-06-14 | 2018-12-20 | 4D Pharma Research Limited | Compositions comprising a bacterial strain of the genus megasphera and uses thereof |
WO2019060629A1 (en) * | 2017-09-21 | 2019-03-28 | Codiak Biosciences, Inc. | Production of extracellular vesicles in single-cell suspension using chemically-defined cell culture media |
WO2020120714A1 (en) | 2018-12-12 | 2020-06-18 | 4D Pharma Research Limited | Compositions comprising bacterial strains |
WO2020191369A1 (en) * | 2019-03-21 | 2020-09-24 | Codiak Biosciences, Inc. | Process for preparing extracellular vesicles |
WO2021146616A1 (en) * | 2020-01-17 | 2021-07-22 | Codiak Biosciences, Inc. | Cholesterol assays for quantifying extracellular vesicles |
-
2023
- 2023-10-06 WO PCT/US2023/034623 patent/WO2024076723A1/en unknown
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018229216A1 (en) | 2017-06-14 | 2018-12-20 | 4D Pharma Research Limited | Compositions comprising a bacterial strain of the genus megasphera and uses thereof |
WO2019060629A1 (en) * | 2017-09-21 | 2019-03-28 | Codiak Biosciences, Inc. | Production of extracellular vesicles in single-cell suspension using chemically-defined cell culture media |
WO2020120714A1 (en) | 2018-12-12 | 2020-06-18 | 4D Pharma Research Limited | Compositions comprising bacterial strains |
WO2020191369A1 (en) * | 2019-03-21 | 2020-09-24 | Codiak Biosciences, Inc. | Process for preparing extracellular vesicles |
WO2021146616A1 (en) * | 2020-01-17 | 2021-07-22 | Codiak Biosciences, Inc. | Cholesterol assays for quantifying extracellular vesicles |
Non-Patent Citations (8)
Title |
---|
"Guide to Huge Computers", 1994, ACADEMIC PRESS |
ATSCHUL, S. F. ET AL., J MOLEC BIOL, vol. 215, no. 403, 1990 |
CARILLO ET AL., SIAM J APPLIED MATH, vol. 48, 1988, pages 1073 |
DEVEREUX, J. ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, 1984, pages 387 |
G. NORHEIM ET AL., PLOS ONE., vol. 10, no. 9, 2015, pages 0134353 |
JEPPESEN ET AL., CELL, vol. 177, 2019, pages 428 |
PEARSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 2444 |
S. BIN PARK ET AL., PLOS ONE., vol. 6, no. 3, 2011, pages 17629 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Guo et al. | A novel rapid and continuous procedure for large-scale purification of magnetosomes from Magnetospirillum gryphiswaldense | |
WO2021100640A1 (en) | Modified cyanobacteria, method for manufacturing modified cyanobacteria, and method for manufacturing protein | |
WO2024031921A1 (en) | Pichia pastoris with enhanced lactoferrin expression, method for constructing same, and use thereof | |
CN111607607B (en) | Method for improving formation of Citrobacter williamsii biofilm | |
WO2021257788A1 (en) | Methods for manufacturing adas | |
CN109536427B (en) | Lactobacillus engineering bacterium with improved acid stress resistance | |
Oh et al. | A rapid method for RNA preparation from Gram-positive bacteria | |
WO2024076723A1 (en) | Processing extracellular vesicles | |
WO2013110891A1 (en) | Use of an acellular empirical culture medium for the growth of intracellular bacteria | |
CN114807196B (en) | Fluorescent marking method for tracking soil-borne plant pathogenic bacteria drug-resistant gene | |
CN111139208B (en) | High-yield engineering bacterium for producing ivermectin and preparation method and application thereof | |
KR101583047B1 (en) | Removal of Heavy Metals Using Spores | |
CN110713958B (en) | Clostridium butyricum and application thereof | |
CN105985939B (en) | A kind of its purposes of poly (hydroxyalkanoate) pellet degradation enzyme and (R) -3HB production method | |
Durand et al. | Drancourtella massiliensis gen. nov., sp. nov. isolated from fresh healthy human faecal sample from South France | |
CN109666618B (en) | Lactobacillus engineering bacterium with improved viability in acid stress environment | |
TWI284150B (en) | Plasmid-free clone of E. coli strain DSM 6601 | |
CN115976032B (en) | Gene for expressing camel lactoferrin antibacterial peptide, antibacterial peptide and application | |
PIKOLI et al. | Diversity Analysis of an Extremely Acidic Soil in a Layer of Coal Mine Detected the Occurrence of Rare Actinobacteria | |
Malik et al. | Characterization of thermoplasma species cultured from sampling on Tangkuban Perahu, Indonesia | |
CN111944779B (en) | Trehalose synthesis dual-function enzyme coding gene TvTPS/TPP and application thereof | |
CN116162584A (en) | Application of split gene in regulating threonine synthesis and cell morphology and growth | |
WO2024024427A1 (en) | Method for determining outer membrane detachment in cyanobacterium, device for determining outer membrane detachment in cyanobacterium, and program | |
Hamzah et al. | EXTRACTION AND PURIFICATION OF UROPATHOGENIC E. COLI OUTER MEMBRANE VESICLES (OMVS) ISOLATED FROM URINE SPECIMEN PATIENT SUFFERING URINARY TRACT INFECTION (UTI) | |
CN117343942B (en) | PagA recombinant protein and preparation method thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 23801583 Country of ref document: EP Kind code of ref document: A1 |