WO2022144381A1 - Microbiome modulation of a host by delivery of dna payloads with minimized spread - Google Patents
Microbiome modulation of a host by delivery of dna payloads with minimized spread Download PDFInfo
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- WO2022144381A1 WO2022144381A1 PCT/EP2021/087774 EP2021087774W WO2022144381A1 WO 2022144381 A1 WO2022144381 A1 WO 2022144381A1 EP 2021087774 W EP2021087774 W EP 2021087774W WO 2022144381 A1 WO2022144381 A1 WO 2022144381A1
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Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
- C12N15/73—Expression systems using phage (lambda) regulatory sequences
Definitions
- the present invention relates to nucleic acids of interest for modulating the microbiome of a host, to vectors encoding said nucleic acids and to methods for modulating the microbiome of a host by delivering said nucleic acids of interest.
- the strategy relies on the delivery of DNA to target bacterial cells in a pure or mixed bacterial population by a viral capsid, by bacterial conjugation or by other methods so that one or several genes of interest will be expressed at a sufficient level to produce a desired effect.
- the effect can be a direct therapeutic effect on the bacteria itself in or on the host, by killing the bacteria and therefore reducing its colonization level or modifying its ratio compared to other bacteria in the population if multiple species or multiple strains are present; by modifying its genome, by modifying its metabolism or its composition (protein, lipids, sugars, metabolites, RNA, etc.).
- the effect can also be an indirect effect by leveraging the target bacteria to produce, display or secrete one or multiple molecule(s) such as prophylactic or therapeutic molecule(s) that will have a direct or indirect effect on the host or on other members of the host microbiome.
- exogenous DNA is transferred to progeny cells if the exogenous DNA is stably maintained in the cells in which it is delivered to, or is transferred to other bacteria via other gene transfer mechanism and then stably maintained in these other populations. More generally, the containment of the exogenous DNA payload once delivered in the bacterial populations is a concern.
- the present inventors have herein developed a new strategy that ensures that DNA payloads once delivered in target bacteria cannot replicate in the target bacteria but still express the gene(s) of interest at a level that is enough to exert the expected outcome on the bacteria or on the host, without the need of an antibiotic resistance selection marker on the DNA payload, and without the need of a selection step with an antibiotic.
- Plasmids carrying conditional origins of replication have a long history of use by microbiologists as a tool to genetically modify bacterial strains of interest, therefore creating stable genetically modified organisms. They are typically used to select for recombination events between a plasmid carrying such origins and the genome of a bacteria of interest.
- Such plasmids carry an antibiotic resistance selection marker and can be introduced into the bacteria by transformation, conjugation or any other method. Because they lack an autonomously replicating origin of replication, only the bacteria that have recombined the plasmid into their genome will stably maintain the selection marker and survive a selection step. The plasmid being stably integrated and maintained in progeny cells, the progeny cells will also be able to survive in presence of the selection marker.
- conditional origin of replication is based on the wild-type plasmid R6K and derivatives which belong to the IncX group of replicon, a group commonly found in a variety of bacterial isolates.
- the replication of these plasmids is dependent on binding of the pir encoded fl initiator protein to the origin of replication. This protein can be expressed from a different replicon (in trans) than the plasmid carrying the R6K origin of replication.
- the replication of the R6K ori plasmid is conditional on the expression of the p/rgene in trans. When delivered to a bacteria of interest, the plasmid will not replicate unless the p/rgene is present and expressed.
- conditional origins of replication relying on such system have a high chance of being replicated at a basal level - enough to spread in the population - or even at a full replication level if the inducer is present in the target population (for instance, Lacl-based origins will be active if lactose is present, which is very often the case in vivo, given modern age diet).
- the aim of the present invention is specifically to engineer and efficiently produce vehicles containing a DNA payload that can be transferred to a target bacterial population, not with the purpose of making and selecting recombination events between the DNA payload and the target bacterial genome to create stably genetically modified bacteria that can transfer the modification to progeny cells, but on the opposite with the purpose of limiting and/or preventing the creation of genetically modified progeny cells while still enabling a direct or indirect effect on the bacteria it is delivered into or its host via the efficient expression of genes of interest carried on the DNA payload.
- Desired effects to be obtained in targeted bacteria or the host include therapeutic effect, cosmetic effect, bioremediation effect, effects on plant growth or physiology, effects on animal growth or physiology as non limiting examples.
- the present inventors here demonstrate, for the first time, that it is possible to obtain an effect in vivo, such as a therapeutic effect, with the delivery of a non-replicative vector to a bacteria.
- the present inventors developed a novel conditional origin of replication particularly efficient for this application, that is based on a rarely occurring two-system components to limit recombination events in the target population, the primase and origin of replication of phage-like inducible elements, namely phage-inducible chromosomal islands ( P IC Is) , and they demonstrate for the first time that such type of conditional origin, even with the primase in trans, enables the efficient packaging of the DNA payload into the delivery vehicle, here a phage-derived particle or packaged phagemid.
- PICIs disclosed in Fillol-Salom etal. (2016) The ISME Journal 12:2114-2128 or in Fillol- Salom et al. (2019) Mol. Cell 75:1020-1030 are systems similar to P4-like elements that hijack Myoviridae, with the main difference that, according to current research, they do not modify the size of the capsid to accommodate their genomes. Since lambdoid PICIs are usually 10-13 kb long and the phages they hijack possess genomes close to 50 kb, this means that they are able to insert several copies of their small genome into a large capsid.
- PICIs are able to completely abolish phage production and only lead to the packaging of their genomes.
- PICIs sense when the lambdoid phage to be hijacked is being induced, they excise from the genome where they reside as prophage-like islands and they replicate their genomes. Replication is based on a single protein, the primase, containing primase and helicase activity, and a short DNA fragment, usually right after the primase gene, that is recognized as an origin of replication by the primase. Additionally, many different PICIs have been described, each one containing different primase-ori pairs.
- the present invention arises from the unexpected finding that not only a DNA payload devoid of antibiotic resistance marker and autonomously replicative origin of replication can be packaged at high-titer in phage-derived particles but also that these DNA payloads can be efficiently delivered to the target bacteria and that these DNA payloads, while non replicative, can exert the intended effect.
- the present inventors also demonstrated for the first time that a non replicative DNA payload expressing a nuclease or an engineered nuclease, such as a base-editor, can result in similar killing or base-editing efficiency as its replicative counterpart.
- the present invention thus concerns a method for in vivo modulating the microbiome of a host organism by delivering a nucleic acid of interest into a targeted receiver bacterial cell of said microbiome, said nucleic acid of interest producing a given effect on said targeted receiver bacterial cell, wherein said method comprises administering, in said host organism, a nucleic acid vector comprising said nucleic acid of interest, wherein said vector further comprises a conditional origin of replication which is inactive in the targeted receiver bacterial cell but is active in a donor bacterial cell, and said vector is devoid of antibiotic resistance marker, thereby delivering said nucleic acid of interest into the targeted receiver bacterial cell, and wherein, once delivered into said targeted receiver bacterial cell, said nucleic acid of interest produces said given effect on said targeted receiver bacterial cell while said vector is not replicated in said targeted receiver bacterial cell.
- the present invention also concerns a method for in vivo modulating the microbiome of a host organism by delivering a nucleic acid of interest into a targeted receiver bacterial cell of said microbiome, said nucleic acid of interest being expressed in said targeted receiver bacterial cell, thereby producing a given effect on said targeted receiver bacterial cell, wherein said method comprises administering, in said host organism, a nucleic acid vector comprising said nucleic acid of interest, wherein said vector further comprises a conditional origin of replication which is inactive in the targeted receiver bacterial cell but is active in a donor bacterial cell, and said vector is devoid of antibiotic resistance marker, thereby delivering said nucleic acid of interest into the targeted receiver bacterial cell, and wherein, once delivered into said targeted receiver bacterial cell, said nucleic acid of interest produces said given effect on said targeted receiver bacterial cell while said vector is not replicated in said targeted receiver bacterial cell.
- said given effect on said targeted receiver bacterial cell generates, directly or indirectly, a reaction in said organism hosting said targeted receiver bacterial cell.
- nucleic acid refers to a sequence of at least two nucleotides covalently linked together which can be single-stranded or double-stranded or contains portions of both single-stranded and double-stranded sequences.
- Nucleic acids of the present invention can be naturally occurring, recombinant or synthetic.
- the nucleic acid can be in the form of a circular sequence or a linear sequence or a combination of both forms.
- the nucleic acid can be DNA, both genomic or cDNA, or RNA or a combination of both.
- the nucleic acid may contain any combination of deoxyribonucleotides and ribonucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, 5-hydroxymethylcytosine and isoguanine.
- bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, 5-hydroxymethylcytosine and isoguanine.
- modified bases that can be used in the present invention are detailed in Chemical Reviews 2016, 116 (20) 12655- 12687.
- nucleic acid also encompasses any nucleic acid analogs which may contain other backbones comprising, without limitation, phosphoramide, phosphorothioate, phosphorodithioate, O-methylphosphoroamidite linkage and/or deoxyribonucleotides and ribonucleotides nucleic acids. Any combination of the above features of a nucleic acid is also encompassed by the present invention.
- peptide refers both to a short chain of at least 2 amino acids linked between each other and to a part of, a subset of, or a fragment of a protein which part, subset or fragment being not expressed independently from the rest of the protein.
- a peptide is a protein.
- a peptide is not a protein and peptide only refers to a part, a subset or a fragment of a protein.
- the peptide is from 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 amino acids to 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 40, 50, 100, 200 amino acids in size.
- the present invention relates to methods for in vivo modulating the microbiome of a host organism.
- microbiome is meant herein the aggregate of all microbiota that reside on or within an organism tissues and biofluids along with the corresponding anatomical sites in which they reside, including, for mammalian organisms, the skin, mammary glands, placenta, seminal fluid, vagina, uterus, ovarian follicles, lung, saliva, oral mucosa, conjunctiva, biliary tract, and gastrointestinal tract, blood, tumors, brain.
- the microbiome more specifically refers to the bacteria populations forming said microbiota.
- modulating the microbiome is meant herein exerting a modifying or controlling influence on the microbiome.
- modulating the microbiome encompases modulating the microbiome function and/or modulating the microbiome composition.
- modulating the microbiome composition is meant herein changing the composition of said microbiome, including removing specific species or strains of said microbiome, changing the proportion between different species or strains of said microbiome or replacing specific species or strains of said microbiome by other species or strains. Said modulation of the microbiome composition can be achieved directly or indirectly, typically by modifying said targeted bacterial cell, which can then have an effect, such as a killing effect, on other bacteria of the microbiome, which were not initially targeted by said vector.
- modulating the microbiome function is meant herein changing the function of specific species or strains of said microbiome, for example by making specific species or strains express particular molecules, or by making specific species or strains stop expressing particular molecules.
- host organism is meant herein any multicellular organism, including any animal or any plant. In a particular embodiment, said host organism is a host subject.
- host subject is meant herein any animal (e.g., a primate, e.g., a human) hosting said microbiome.
- the subject according to the invention is preferably a mammal, even more preferably a human.
- the term "subject” can also refer to non-human animals, in particular mammals such as dogs, cats, horses, cows, pigs, sheep, donkeys, rabbits, ferrets, gerbils, hamsters, chinchillas, rats, mice, guinea pigs and non-human primates, among others, or non-mammals such as poultry, that are in need of treatment.
- the human subject according to the invention may be a human at the prenatal stage, a new-born, a child, an infant, an adolescent or an adult at any age.
- a nucleic acid of interest is delivered into a targeted receiver bacterial cell of said microbiome or a group of targeted receiver bacterial cells of said microbiome, said nucleic acid of interest being comprised in a vector provided by a donor bacterial cell.
- donor bacterial cell is meant herein a bacterium that is capable of hosting a vector comprising a nucleic acid of interest, of producing a vector comprising said nucleic acid of interest and/or which is capable of transferring said vector comprising said nucleic acid to another bacterium.
- said vector may be a phagemid
- said donor bacterial cell may then be a bacterial cell able to produce said phagemid, more particularly in the form of a packaged phagemid.
- said vector may be a plasmid, more particularly a conjugative plasmid, and said donor bacterial cell may then be a bacterium that is capable of transferring said conjugative plasmid to another bacterium, in particular by conjugation.
- receiver bacterial cell is meant herein any bacterium from the host microbiome which is specifically targeted to be delivered with said nucleic acid of interest.
- the targeted receiver bacteria can be any bacteria, in particular present in an organism, more particularly in a mammal organism. It can be any commensal, symbiotic or pathogenic bacteria of the microbiota or microbiome.
- a microbiome may comprise a variety of endogenous bacterial species, any of which may be targeted in accordance with the present disclosure.
- the genus and/or species of targeted receiver bacterial cells may depend on the type of bacteriophages being used for preparing the vector and/or bacterial delivery vehicles. For example, some bacteriophages exhibit tropism for, or preferentially target, specific host species of bacteria. Other bacteriophages do not exhibit such tropism and may be used to target a number of different genus and/or species of endogenous bacterial cells.
- receiver bacterial cells include, without limitation, cells from bacteria of the genus Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Bordetella spp., Neisseria spp., Aeromonas spp., Franciesella spp., Corynebacterium spp., Citrobacter spp., Chlamydia spp., Hemophilus spp., Brucella spp., Mycobacterium spp., Legionella spp., Rhodococcus spp., Pseudomonas spp., Helicobacter spp., Vibrio spp., Bacillus spp., Erysipelothrix spp., Salmonella spp., Streptomyces spp., Streptococcus spp., Staphylococc
- the targeted receiver bacterial cell may be any one or more of the foregoing genus of bacteria.
- the targeted receiver bacteria can be selected from the group consisting of Yersinia spp., Escherichia spp., Klebsiella spp., Acinetobacter spp., Pseudomonas spp., Helicobacter spp., Vibrio spp, Salmonella spp., Streptococcus spp., Staphylococcus spp., Bacteroides spp., Clostridium spp., Shigella spp., Enterococcus spp., Enterobacter spp., Propionibacterium spp., Cutibacterium spp. and Listeria spp.
- targeted receiver bacterial cells of the present disclosure are anaerobic bacterial cells (e.g., cells that do not require oxygen for growth).
- Anaerobic bacterial cells include facultative anaerobic cells such as but not limited to Escherichia coli, Shewanella oneidensis and Listeria.
- Anaerobic bacterial cells also include obligate anaerobic cells such as, for example, Bacteroides and Clostridium species.
- anaerobic bacteria are most commonly found in the gastrointestinal tract.
- the targeted receiver bacteria are thus bacteria most commonly found in the gastrointestinal tract.
- the targeted receiver bacterial cells are, without limitation, Bacteroides thetaiotaomicron, Bacteroides fragilis, Bacteroides distasonis, Bacteroides vulgatus, Clostridium leptum, Clostridium coccoides, Staphylococcus aureus, Bacillus subtilis, Clostridium butyricum, Brevibacterium lactofermentum, Streptococcus agalactiae, Lactococcus lactis, Leuconostoc lactis, Actinobacillus actinomycetemcomitans, cyanobacteria, Escherichia coli, Helicobacter pylori, Selenomonas ruminatium, Shigella sonnei, Zymomonas mobilis, Mycoplasma mycoides, Treponema denticola, Bacillus thuringiensis, Staphylococcus lugdunensis
- the targeted bacteria of interest are selected from the group consisting of Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, Enterobacter cloacae, and Enterobacter aerogenes, and a mixture thereof.
- the targeted bacterial cells are, without limitation, Anaerotruncus, Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus, Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio, Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus, Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacter, Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus, Leuconostoc, Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter, Papillibacter, Proprionispira, Pseudobutyrivibrio, Pseudoramibacter, Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttle
- the targeted bacteria cells are, without limitation, Achromobacter xylosoxidans, Acidaminococcus fermentans, Acidaminococcus intestini, Acidaminococcus sp., Acinetobacter baumannii, Acinetobacter junii, Acinetobacter Iwoffii, Actinobacillus capsulatus, Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces radingae, Adlercreutzia equolifaciens, Aeromicrobium massiliense, Aggregatibacter actinomycetemcomitans, Akkermansia muciniphila, Aliagarivorans marinus, Alistipes finegoldii, Alistipes indistinctus, Alistipes inops, Alistipes onderdonkii, Alistipes putredinis, Alistipes senegal
- the targeted bacteria cells are those commonly found on the skin microbiota and are without limitation Acetobacter farinalis, Acetobacter malorum, Acetobacter orleanensis, Acetobacter sicerae, Achromobacter anxifer, Achromobacter denitrificans, Achromobacter marplatensis, Achromobacter spanius, Achromobacter xylosoxidans subsp.
- Aeromonas piscicola Aeromonas popoffii
- Aeromonas rivuli Aeromonas salmonicida subsp. pectinolytica
- Aeromonas salmonicida subsp. smithia Amaricoccus kaplicensis, Amaricoccus veronensis, Aminobacter aganoensis, Aminobacter ciceronei, Aminobacter lissarensis, Aminobacter niigataensis, Ancylobacter polymorphus, Anoxybacillus flavithermus subsp.
- anitratus Actinomyces odontolyticus, Actinomyces oris, Actinomyces turicensis, Actinomycetospora corticicola, Actinotignum schaalii, Aerococcus christensenii, Aerococcus urinae, Aeromicrobium flavum, Aeromicrobium massiliense, Aeromicrobium tamlense, Aeromonas sharmana, Aggregatibacter aphrophilus, Aggregatibacter segnis, Agrococcus baldri, Albibacter methylovorans, Alcaligenes faecalis subsp.
- Corynebacterium ammoniagenes Corynebacterium amycolatum, Corynebacterium aurimucosum, Corynebacterium aurimucosum, Corynebacterium coyleae, Corynebacterium durum, Corynebacterium macburgense, Corynebacterium glaucum, Corynebacterium glyciniphilum, Corynebacterium imitans, Corynebacterium jeikeium, Corynebacterium jeikeium, Corynebacterium kroppenstedtii, Corynebacterium lipophiloflavum, Corynebacterium massiliense, Corynebacterium mastitidis, Corynebacterium matruchotii, Corynebacterium minutissimum, Corynebacterium mucifaciens, Corynebacterium mustelae, Corynebacterium mycetoides, Corynebacterium pyru
- lactis Lactococcus lactis subsp. lactis, Lactococcus piscium, Lapillicoccus jejuensis, Lautropia mirabilis, Legionella beliardensis, Leptotrichia buccalis, Leptotrichia goodfellowii, Leptotrichia hofstadii, Leptotrichia hongkongensis, Leptotrichia shahii, Leptotrichia trevisanii, Leptotrichia wadei, Luteimonas terricola, Lysinibacillus fusiformis, Lysobacter spongiicola, Lysobacter xinjiangensis, Macrococcus caseolyticus, Marmoricola pocheonensis, Marmoricola scoriae, Massilia alkalitolerans, Massilia alkalitolerans, Massilia aurea, Massilia plicata, Massilia timonae,
- Propionibacterium acnes subsp. acnes Propionibacterium acnes subsp. elongatum, Propionibacterium granulosum, Propionimicrobium lymphophilum, Propionispira arcuata, Pseudokineococcus lusitanus, Pseudomonas aeruginosa, Pseudomonas chengduensis, Pseudonocardia benzenivorans, Pseudorhodoplanes sinuspersici, Psychrobacter sanguinis, Ramlibacter ginsenosidimutans, Rheinheimera aquimaris, Rhizobium alvei, Rhizobium daejeonense, Rhizobium larrymoorei, Rhizobium rhizoryzae, Rhizobium soli, Rhizobium taibaishanense, Rhizobium vignae, Rhodanobacter glycinis,
- the targeted bacteria cells are those commonly found in the vaginal microbiota and are, without limitation, Acinetobacter antiviralis, Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter johnsonii, Actinobaculum massiliense, Actinobaculum schaalii, Actinomyces europaeus, Actinomyces graevenitzii, Actinomyces israelii, Actinomyces meyeri, Actinomyces naeslundii, Actinomyces neuii, Actinomyces odontolyticus, Actinomyces turicensis, Actinomyces urogenitalis, Actinomyces viscosus, Aerococcus christensenii, Aerococcus urinae, Aerococcus viridans, Aeromonas encheleia, Aeromonas salmonicida, Afipi
- the targeted receiver bacteria are Klebsiella pneumoniae.
- the targeted receiver bacteria are Bacteroides thetaiotaomicron and/or Bacteroides faecis.
- the targeted receiver bacteria are Roseburia intestinalis.
- the targeted bacteria are Cutibacterium acnes more specifically the acne related Cutibacterium acnes from the phylogroup IA1 or RT4, RT5, RT8, RT9, RT10 or Clonal Complex(CC) CC1 , CC3, CC4, more specifically the ST1 , ST3, ST4.
- the targeted receiver bacteria are pathogenic bacteria.
- the targeted receiver bacteria can be virulent bacteria.
- the targeted receiver bacteria are involved in infections in the host.
- the targeted receiver bacteria are associated with the triggering, progression, or aggravation of auto-immune diseases in the host.
- the targeted receiver bacteria are associated with the triggering, progression or aggravation of tumors or metastasis in the host.
- the targeted receiver bacteria are associated with the triggering, progression or aggravation of neurodegenerative disease in the host.
- the targeted receiver bacteria are associated with the triggering, progression or aggravation of CNS related disease in the host.
- the targeted receiver bacteria are associated with the resistance of the host towards treatments against infection, tumor, neurodegenerative disease, CNS related disease, autoimmune disease, and/or cancer.
- the targeted receiver bacteria can be antibacterial resistant bacteria, including those selected from the group consisting of extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli, ESBL Klebsiella pneumoniae, vancomycin-resistant Enterococcus (VRE), methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant (MDR) Acinetobacter baumannii, MDR Enterobacter spp., and a combination thereof.
- the targeted receiver bacteria can be selected from the group consisting of extended-spectrum beta-lactamase-producing (ESBL) Escherichia coli strains.
- said targeted receiver bacteria are ESBL Escherichia coli and/or ESBL Klebsiella pneumoniae.
- the targeted receiver bacterium can be a bacterium of the microbiome of a given species, in particular a bacterium of the human microbiota.
- said nucleic acid of interest produces a given effect on said targeted receiver bacterial cell, as defined above.
- nucleic acid producing a given effect on said targeted receiver bacterial cell is meant herein that the delivery of said nucleic acid into said targeted receiver bacterial cell induces, directly or indirectly, a reaction into said targeted receiver bacterial cell (such as the expression of a RNA, the expression of a protein or the activation or the inhibition of an activity), wherein said reaction in said targeted receiver bacterial cell, preferably further generates, directly or indirectly, a reaction in said organism hosting said targeted receiver bacterial cell.
- the nucleic acid of interest is expressed in said targeted receiver bacterial cell, thereby producing said given effect.
- Expression of said nucleic acid of interest includes expression into a coding or non-coding RNA, or expression into a protein.
- the nucleic acid of interest is not expressed in said targeted receiver bacterial cell, and the presence of said nucleic acid of interest in said targeted receiver bacterial cell produces said given effect (for example by providing binding regions to molecules already present in said targeted receiver bacterial cell).
- said given effect may be selected from the group consisting of killing the receiver bacterial cell, making the receiver bacterial cell stop producing a given molecule, making the receiver bacterial cells reducing its level of production of a given molecule, and making the receiver bacterial cell produce a molecule of interest.
- said given effect is making the receiver bacterial cell produce a molecule of interest, in particular a host modulatory molecule.
- said given effect is making the receiver bacterial cell produce, as molecule of interest, transcription factors and/or modified nucleases, in particular to activate specific pathways or genes in the bacteria that are naturally turned off.
- said given effect is making the receiver bacterial cell produce a molecule of interest which increases or decreases, preferably temporarily, the fitness of said receiver bacterial cell to its environment, in particular compared to other members of the microbiome which are not receiver bacterial cells.
- said given effect is making the receiver bacterial cell produce, as molecule of interest, a molecule of interest which acts on the microbiome environment, in particular without generating an effect at the level of the host organism cells.
- host modulatory molecule or “HMM” is meant herein any molecule, produced by said receiver bacterial cell, that acts, directly or indirectly, at the level of the host organism.
- Said HMM may be of any nature.
- said HMM may be selected from the group consisting of non-coding nucleic acids, coding nucleic acids, proteins, lipids, sugars, LPS, metabolites and small molecules.
- non-coding nucleic acids typically include non-coding DNAs or non-coding RNAs, such as siRNAs.
- Examples of coding nucleic acids typically include coding DNAs or coding RNAs.
- proteins typically include cytokines, such as chemokines, interferons, interleukins, lymphokines, tumour necrosis factors and anti-inflammatory cytokines; surface layer proteins, such as SIpB, in particular from Propionibacterium freudenreichii; microbial antiinflammatory molecule (MAM), such as MAM from Faecalibacterium prausnitz; antibodies such as monoclonal antibodies, multispecific antibodies, chimeric antibodies, antibody fragments and derivatives thereof; nanobodies; enzymes, in particular enzymes leading to the production of other HMMs; peptides such as Immune Selective Anti-Inflammatory Derivatives (FEG, Salivary gland derived peptides), and mimic proteins or peptides derived from the microbiome that mimic antigens from cells of the subject.
- cytokines such as chemokines, interferons, interleukins, lymphokines, tumour necros
- Mimic peptides of particular interest are bacterial mimic peptides that are associated with auto-immune diseases, for example those mentioned in Negi et al. (2017) Pios One 12:e0180518, which are hereby incorporated by reference.
- Of particular interest are the gene sequences encoding any of the mimic peptides in S1 Table of Negi et al.
- Examples of lipids typically include SCFAs, such as butyrate.
- Examples of small molecules typically include cyclosporin, nonsteroidal antiinflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs) and ROS.
- NSAIDs nonsteroidal antiinflammatory drugs
- SAIDs steroidal anti-inflammatory drugs
- ROS ROS
- Said HMM may further have any effect.
- said HMM may be a molecule that will affect the immune system of the host, the host CNS and/or the host metabolism.
- said HMM may be selected from the group consisting of anticancer molecules, antibiotic molecules, anti-viral molecules, anti-parasite molecules, anti-protozoal molecules, anesthetic molecules, anticoagulant molecules, inhibitors of an enzyme, steroidal molecules, anti-inflammatory molecules, antihistamine molecules, immunosuppressant molecules, anti-neoplastic molecules, antigens, vaccines, antibodies, decongestant molecules, sedative molecules, analgesic molecules, antipyretic molecules, hormones, anti-hormone molecules, anticholinergic agents, antidepressant molecules, antipsychotic molecules, neurotoxin molecules, hypnotic molecules, tranquilizer molecules, anticonvulsant molecules, muscle relaxant molecules, anti-aging molecules, anti-neurodegeneration molecules, neuromodulators, antispasmodic molecules, muscle contractant molecules, channel blocker molecules, miotic molecules, anti-secretory molecules, anti-thrombotic molecules, diuretic molecules, cardiovascular active molecules, vasoactive molecules, vasodilating molecules, anticancer molecules, antibiotic
- Said HMM may further be of any origin.
- said HMM may be selected from the group consisting of host endogenous molecules, host exogenous molecules expressed naturally by other organisms, and synthetic compounds.
- host endogenous molecule is meant herein any molecule naturally produced by the host subject, in particular by a healthy host subject.
- host exogenous molecule expressed naturally by other organisms is meant herein any molecule which is not produced by the host subject (or by a subject of the same species as the host species) but which is naturally produced by another organism, in particular an organism from another species, from another gender, from another family, from another class or from another kingdom.
- said host exogenous molecule expressed naturally by other organisms may be a molecule produced by bacteria, in particular by microbiota.
- the nucleic acid of interest encodes a bacteriocin or a lysin, which can be a proteinaceous toxin produced by receiver bacteria to kill or inhibit growth of other bacteria.
- Bacteriocins are categorized in several ways, including producing strain, common resistance mechanisms, and mechanism of killing. Such bacteriocin had been described from gram negative bacteria (e.g. microcins, colicin-like bacteriocins and tailocins) and from gram positive bacteria (e.g. Class I, Class II, Class III or Class IV bacteriocins).
- the nucleic acid of interest encodes a toxin selected in the group consisting of microcins, colicin-like bacteriocins, tailocins, Class I, Class II, Class III and Class IV bacteriocins.
- the corresponding immunity polypeptide i.e. anti-toxin
- the corresponding immunity polypeptide may be used to protect receiver bacterial cells (see review by Cotter et aL, Nature Reviews Microbiology 1 1 : 95, 2013).
- synthetic compound is meant herein any molecule which is neither naturally produced by the host subject (or by a subject of the same species as the host species) nor by another organism, in particular an organism from another species, from another gender, from another family, from another class or from another kingdom.
- Said molecule of interest may further be produced by said targeted receiver bacterial cell in any form.
- said HMM may be selected from the group consisting of secreted molecules, intracellular molecules and membrane-displayed molecules.
- the production of said molecule of interest by said targeted receiver bacterial cell may require the delivery of a nucleic acid of interest which includes one or more type(s) of gene(s) or group(s) of genes.
- said nucleic acid of interest may be selected from the group consisting of a gene encoding said molecule of interest, in particular said HMM, several genes encoding a protein complex that is the molecule of interest, in particular the HMM, a gene or group of genes encoding enzyme(s) of a metabolic pathway leading to the production of the molecule of interest, in particular of the HMM, a coding nucleic acid which is the molecule of interest, in particular the HMM, and a non-coding nucleic acid which is the molecule of interest, in particular the HMM.
- said given effect is making the receiver bacterial cell stop producing a given molecule.
- receiver bacterial cell stop producing a given molecule By “making the receiver bacterial cell stop producing a given molecule” is meant herein reducing or abolishing the production of said given molecule by said bacterial cell and/or making the receiver bacterial cell produce a variant of said given molecule.
- said given molecule the production of which is to be stopped has a negative effect on said host organism.
- said given molecule the production of which is to be stopped affects the fitness of said receiver bacterial cell to its environment.
- making the receiver bacterial cell stop producing said given molecule increases or decreases, preferably temporarily, the fitness of said receiver bacterial cell to its environment, in particular compared to other members of the microbiome which are not receiver bacterial cell.
- said given molecule may be selected from the group consisting of a toxin, a toxic factor, a virulence protein, a virulence factor, a protein encoded by an antibiotic resistance gene, a protein encoded by a remodeling gene or by a modulatory gene.
- said given effect is to selectively remove antibiotic resistance from antibiotic resistant bacterial strains.
- said nucleic acid of interest is a gene or group of genes encoding one or more exogenous enzyme(s) which result(s) in a genetic modification.
- said nucleic acid of interest is a gene encoding a base-editor or a prime-editor.
- the genetic modification is made with one or more of the following enzymes and systems.
- CBE Cytosine base editors
- ABE Adenosine base editors
- ABE Adenine Base Editor
- Adenine Thymine Base Editor that convert A:T into T :A (W02020181202)
- Thymine Adenine Base Editor that convert T:A into A:T (W02020181193, WG2020181178, WG2020181195)
- Base editors differ in the base modification enzymes. CBE rely on ssDNA cytidine deaminase among which: APOBEC1 , rAPOBECI , APOBEC1 mutant or evolved version (evoAPOBECI ), and APOBEC homologs (APOBEC3A (eA3A), Anc689), Cytidine deaminase 1 (CDA1), evoCDAI , FERNY, evoFERNY.
- CDA1 Cytidine deaminase 1
- TadA* is an evolved version of TadA, an E.coli tRNA adenosine deaminase enzyme, able to convert adenosine into Inosine on ssDNA.
- TadA* include TadA-8a-e and TadA-7.10.
- nickase active Cas9 nCas9 D10A
- Non-limiting examples of DNA based editor proteins include BE1 , BE2, BE3, BE4, BE4- GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Cas12a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRY- CBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas9
- Cytosine Guanine Base Editors consist of a nickase CRISPR fused to: a. A cytosine deaminase (rAPOBEC) and base excision repair proteins (e.g. rXRCCI ) (Chen etal. (2020) Biorxiv “Precise and programmable C:G to G:C base editing in genomic DNA”). b. A rat APOBEC1 variant (R33A) protein and an E. co/z-derived uracil DNA N- glycosylase (ellNG) (Kurt et al. (2020) Nat. Biotechnol. “CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells”).
- rAPOBEC cytosine deaminase
- rXRCCI base excision repair proteins
- Cytosine Adenine Base Editors consist of a Cas9 nickase, a cytidine deaminase (e.g. AID), and a uracil-DNA glycosylase (Ung) (Zhao etal. (2020) Nature Biotechnol. “New base editors change C to A in bacteria and C to G in mammalian cells”).
- ACBE include a nucleic acid programmable DNA-binding protein and an adenine oxidase (W02020181 180).
- ATBE consist of a Cas9 nickase and one or more adenosine deaminase or an oxidase domain (WG2020181202).
- TABE consist of a Cas9 nickase and an adenosine methyltransferase, a thymine alkyltransferase, or an adenosine deaminase domain (W02020181193, W02020181 178, W02020181 195).
- Base editor molecules can also consist of two or more of the above listed editor enzymes fused to a Cas protein (e.g. combination of an ABE and CBE). These biomolecules are named dual base editors and enable the editing of two different bases (Grunewald et al. (2020) Nature Biotechnol. “A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing”; Li et al. (2020) Nature Biotechnol. “Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors”).
- Prime editors as described in Anzalone et a/. (2019) Nature 576:149-157, which is hereby incorporated by reference, consist of nCas9 fused to a reverse transcriptase used in combination with a prime editing RNA (pegRNA, a guide RNA that includes a template region for reverse transcription).
- pegRNA prime editing RNA
- Prime Editing allows introduction of insertions, deletions (indels) and 12 base-to-base conversions.
- Prime editing relies on the ability of a reverse transcriptase (RT), fused to a Cas nickase variant, to convert RNA sequence brought by a prime editing guide RNA (pegRNA) into DNA at the nick site generated by the Cas protein.
- RT reverse transcriptase
- pegRNA prime editing guide RNA
- Prime editing systems include:
- a Cas nickase variant such as Cas9-H840A fused to a reverse transcriptase domain such as M-MLV RT or its mutant version (M-MLV RT(D200N), M-MLV RT(D200N/L603W), M- MLV RT(D200N/L603W/T330P/ T306K/W313F)
- a prime editing guide RNA (pegRNA)
- pegRNA a prime editing guide RNA
- the prime editing system can include the expression of an additional sgRNA targeting the Cas nickase activity towards the non-edited DNA strand ideally only after the resolution of the edited strand flap by designing the sgRNA to anneal with the edited strand but not with the original strand.
- Non-limiting examples of prime editing systems include PE1 , PE1 -M1 , PE1 -M2, PE1 -M3, PE1 -M6, PE1 -M15, PE1 -M3inv, PE2, PE3, PE3b.
- CRISPEY Cas9 Retron precise Parallel Editing via homologY
- the SCRIBE strategy a retron system expressed in combination with a recombinase promoting the recombination of single stranded DNA, also known as single stranded annealing proteins (SSAPs) (Farzadfard & Lu (2014) Science 346:1256272).
- SSAPs single stranded annealing proteins
- Such recombinases include but are not limited to phage recombinases such as lambda red, recET, Sak, Sak4, and newly described SSAPs described in Wannier et al. (2020) Proc Natl Acad Sci U S A 117(24) : 13689- 13698 which is hereby incorporated by reference.
- the CRISPR system is included in the nucleic acid of interest.
- the CRISPR system contains two distinct elements, i.e. i) an endonuclease, in this case the CRISPR associated nuclease (Cas or "CRISPR associated protein") and ii) a guide RNA.
- the guide RNA may be in the form of a chimeric RNA which consists of the combination of a CRISPR (RNAcr) bacterial RNA and a RNAtracr (trans-activating RNA CRISPR) (Jinek et al. (2012) Science 337(6096):816-21 ).
- the guide RNA combines the targeting specificity of the RNAcr corresponding to the "spacing sequences" that serve as guides to the Cas proteins, and the conformational properties of the RNAtracr in a single transcript.
- the target genomic sequence can be permanently modified or interrupted. The modification is advantageously guided by a repair matrix.
- the CRISPR system includes two main classes depending on the nuclease mechanism of action. Class 1 is made of multi-subunit effector complexes and includes type I, III and IV.
- Class 2 is made of single-unit effector modules, like Cas9 nuclease, and includes type II (ll-A,ll-B,ll-C,ll-C variant), V (V-A,V-B,V-C,V-D,V-E,V-U1 ,V-U2,V-U3,V-U4,V-U5) and VI (VI- A,VI-B1 ,VI-B2,VI-C,VI-D)
- the nucleic acid of interest may comprise a nucleic acid sequence encoding Cas protein.
- CRISPR enzymes are available for use as a sequence of interest on the plasmid.
- the CRISPR enzyme is a Type II CRISPR enzyme.
- the CRISPR enzyme catalyzes DNA cleavage.
- the CRISPR enzyme catalyzes RNA cleavage.
- the CRISPR enzyme does not make a double strand break.
- the CRISPR enzyme makes a single strand break or nicks.
- the CRISPR enzyme does not make any break in the DNA or RNA.
- a Cas13-deaminase fusion is used to base edit an RNA.
- the CRISPR enzymes may be coupled to a sgRNA.
- the sgRNA targets a gene encoding a given molecule as defined above.
- Non-limiting examples of Cas proteins as part of a multi-subunit effector or as a singleunit effector include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Casi o, Cas1 1 (SS), Cas12a (Cpf1 ), Cas12b (C2c1 ), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a (C2c2), Cas13b (C2c6), Cas13c (C2c7), Cas13d, Csa5, Csc1 , Csc2, Cse1 , Cse2, Csy1 , Csy2, Csy3, Csf1 , Csf2, Csf3, C
- the invention encompasses fusion proteins comprising a Cas9 (e.g., a Cas9 nickase) domain and a deaminase domain.
- the fusion protein comprises Cas9 and a cytosine deaminase enzyme, such as APOBEC enzymes, or adenosine deaminase enzymes, such as ADAT enzymes, for example as disclosed in U.S. Patent Publ. 2015/0166980, which is hereby incorporated by reference.
- the deaminase is an ACF1/ASE deaminase.
- the APOBEC deaminase is selected from the group consisting of APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, and APOBEC3H deaminase.
- the fusion protein comprises a Cas9 domain, a cytosine deaminase domain, and a uracil glycosylase inhibitor (UGI) domain.
- the deaminase is an adenosine deaminase that deaminate adenosine in DNA, for example as disclosed in U.S. Patent 10,113,163, which is hereby incorporated by reference.
- the fusion proteins further comprise an inhibitor of base repair, such as, a nuclease dead inosine specific nuclease (dISN), for example as disclosed in U.S. Patent 10,113,163.
- DISN nuclease dead inosine specific nuclease
- the invention encompasses fusion proteins comprising a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase, programmed with a prime editing guide RNA (pegRNA) that both specifies the target site and encodes the desired edit, for example as described in Anzalone et al. (2019) Nature 576:149-157, which is hereby incorporated by reference.
- pegRNA prime editing guide RNA
- the CRISPR enzyme is any Cas protein, in particular any Cas9 protein, for instance any naturally occurring bacterial Cas9 as well as any variants, chimeras, homologs or orthologs thereof.
- Cas9 is meant a protein Cas9 (also called Csn1 or Csx12) or a functional protein, peptide or polypeptide fragment thereof, i.e. capable of interacting with the guide RNA(s) and of exerting the enzymatic activity (nuclease) which allows it to perform the double-strand cleavage of the DNA of the target genome.
- Cas9 can thus denote a modified protein, for example truncated to remove domains of the protein that are not essential for the predefined functions of the protein, in particular the domains that are not necessary for interaction with the gRNA(s).
- Cas9 the entire protein or a fragment thereof
- the sequence encoding Cas9 can be obtained from any known Cas9 protein (Fonfara et al. (2014) Nucleic Acids Res 42(4):2577-90; Shmakov et al. (2017) Nat Rev Microbiol 15(3):169-182).
- Cas9 proteins useful in the present disclosure include, but are not limited to, Cas9 proteins of Streptococcus pyogenes (SpCas9), Streptococcus thermophiles (St1 Cas9, St3Cas9), Streptococcus mutans, Staphylococcus aureus (SaCas9), Campylobacter jejuni (CjCas9), Francisella novicida (FnCas9) and Neisseria meningitides (NmCas9).
- Cpf 1 (Cas12a) the entire protein or a fragment thereof
- the sequence encoding Cpf 1 (Cas12a) can be obtained from any known Cpf1 (Cas12a) protein (Koonin et al. (2017) Current Opinion in Microbiology 37:67-78).
- Cpf1 (Cas12a) proteins useful in the present disclosure include, but are not limited to, Cpf1 (Cas12a) proteins of Acidaminococcus sp, Lachnospiraceae bacteriu and Francisella novicida.
- Cas13a (the entire protein or a fragment thereof) can be obtained from any known Cas13a (C2c2) protein (Abudayyeh et al. (2017) Nature 550:280).
- Cas13a (C2c2) proteins useful in the present disclosure include, but are not limited to, Cas13a (C2c2) proteins of Leptotrichia wadei (LwaCas13a).
- Cas13d The sequence encoding Cas13d (the entire protein or a fragment thereof) can be obtained from any known Cas13d protein (Yan et al. (2016) Mol Ce//70(2):327-339).
- Cas13d proteins useful in the present disclosure include, but are not limited to, Cas13d proteins of Eubacterium siraeum and Ruminococcus sp.
- programmable nucleases can be used. These include an engineered TALEN (Transcription Activator-Like Effector Nuclease) and variants, engineered zinc finger nuclease (ZFN) variants, natural, evolved or engineered meganuclease or recombinase variants, and any combination or hybrids of programmable nucleases.
- TALEN Transcription Activator-Like Effector Nuclease
- ZFN zinc finger nuclease
- the programmable nucleases provided herein may be used to selectively modify DNA encoding a gene of interest such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene (cf. WO2014124226 and US2015/0064138).
- RNA base editing is based on the same principle as DNA base editing: an enzyme catalyzing the conversion of a RNA base into another must be brought close to the target base to perform its conversion locally.
- the enzyme used for RNA editing is an adenosine deaminase from ADAR family that converts Adenosine into Inosine in dsRNA structure.
- ADARDD ADAR deaminase domain
- RNA editing More recently the ability of some CRISPR-Cas systems to bind RNA molecules was repurposed into RNA editing.
- dPspCasI 3b catalytically dead Cas13b enzyme fused to a hyperactive mutant of ADAR2 deaminase domain (ADAR2DD-E488Q for REPAIRvI and ADAR2DD-E488Q-T375G for REPAI Rv2)
- ADAR2DD-E488Q for REPAIRvI
- ADAR2DD-E488Q-T375G for REPAI Rv2 Rex et al improved specificity and efficiency compare to previous RNA editing strategies.
- Non-limiting examples of RNA based editor proteins include REPAIRvI , REPAIRv2
- the modification is made in a gene selected in the group consisting of an antibiotic resistance gene, virulence factor or protein gene, toxin factor or protein gene, a gene expressing a bacterial receptor, a membrane protein, a structural protein, a secreted protein, and a gene expressing resistance to a drug in general.
- the modification is made to target and inactivate a virulence factor.
- a virulence factor can be any substance produced by a pathogen that alters host-pathogen interaction by increasing the degree of damage done to the host. Virulence factors are used by pathogens in many ways, including, for example, in cell adhesion or colonization of a niche in the host, to evade the host's immune response, to facilitate entry to and egress from host cells, to obtain nutrition from the host, or to inhibit other physiological processes in the host. Virulence factors can include enzymes, endotoxins, adhesion factors, motility factors, factors involved in complement evasion, and factors that promote biofilm formation. For example, such targeted virulence factor gene can be E.
- coli virulence factor gene such as, without limitation, EHEC- HlyA, Stx1 (VT1 ), Stx2 (VT2), Stx2a (VT2a), Stx2b (VT2b), Stx2c (VT2c), Stx2d (VT2d), Stx2e (VT2e) and Stx2f (VT2f), Stx2h (VT2h), fimA, fimF, fimH, neuC, kpsE, sfa, foe, iroN, aer, iha, papC, papGI, papGII, papGIII, hlyC, cnf1 , hra, sat, ireA, usp ompT, ibeA, malX, fyuA, irp2, traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA,
- such targeted virulence factor gene can be Shigella dysenteriae virulence factor gene such as, without limitation, stx1 and stx2.
- such targeted virulence factor gene can be Yersinia pestis virulence factor gene such as, without limitation, yscF (plasmid-borne (pCDI) T3SS external needle subunit).
- yscF plasmid-borne (pCDI) T3SS external needle subunit
- such targeted virulence factor gene can be Francisella tularensis virulence factor gene such as, without limitation, fslA.
- such targeted virulence factor gene can be Bacillus anthracis virulence factor gene such as, without limitation, pag (Anthrax toxin, cell-binding protective antigen).
- such targeted virulence factor gene can be Vibrio cholera virulence factor gene such as, without limitation, ctxA and ctxB (cholera toxin), tcpA (toxin co-regulated pilus), and toxT (master virulence regulator).
- Vibrio cholera virulence factor gene such as, without limitation, ctxA and ctxB (cholera toxin), tcpA (toxin co-regulated pilus), and toxT (master virulence regulator).
- such targeted virulence factor gene can be Pseudomonas aeruginosa virulence factor genes such as, without limitation, pyoverdine (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdl, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdE, pvdR, pvdT, opmQ), siderophore pyochelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF and pchG, and toxins (e.g., exoll, exoS and exoT).
- pyoverdine e.g., sigma factor pvdS, bio
- such targeted virulence factor gene can be Klebsiella pneumoniae virulence factor genes such as, without limitation, fimA (adherence, type I fimbriae major subunit), and cps (capsular polysaccharide).
- Klebsiella pneumoniae virulence factor genes such as, without limitation, fimA (adherence, type I fimbriae major subunit), and cps (capsular polysaccharide).
- such targeted virulence factor gene can be Acinetobacter baumannii virulence factor genes such as, without limitation, ptk (capsule polymerization) and epsA (assembly).
- such targeted virulence factor gene can be Salmonella enterica Typhi virulence factor genes such as, without limitation, MIA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with bile tolerance, including efflux pump genes acrA, acrB and tolC.
- such targeted virulence factor gene can be Fusobacterium nucleatum virulence factor genes such as, without limitation, FadA and TIGIT.
- such targeted virulence factor gene can be Bacteroides fragilis virulence factor genes such as, without limitation, bft.
- the modification is made in an antibiotic resistance gene such as, without limitation, GyrB, ParE, ParY, AAC(1 ), AAC(2'), AAC(3), AAC(6'), ANT(2"), ANT(3"), ANT(4'), ANT(6), ANT(9), APH(2"), APH(3"), APH(3'), APH(4), APH(6), APH(7"), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1 , CTX-M, KPC, SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXA p-lactamase, mecA, Omp36, OmpF, PIB, bla (blal, blaR1 ) and mec (mecl, mecR1 ) operons, Chlor
- the antibiotic is selected from the group consisting of penicillins such as penicillin G, penicillin K, penicillin N, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin, cioxacillin, dicloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin; cephalosporins such as cefacetrile, cefadroxil, cephalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefa
- the modification is made in a bacterial toxin gene.
- Bacterial toxins can be classified as either exotoxins or endotoxins. Exotoxins are generated and actively secreted; endotoxins remain part of the bacteria. The response to a bacterial toxin can involve severe inflammation and can lead to sepsis.
- Such toxin can be for example Botulinum neurotoxin, Tetanus toxin, Staphylococus toxins, Diphteria toxin, Anthrax toxin, Alpha toxin, Pertussis toxin, Shiga toxin, Heat-stable enterotoxin (E. coli ST), colibactin, BET (B. fragilis toxin) or any toxin described in Henkel et al., (Toxins from Bacteria in EXS. 2010; 100: 1-29).
- said toxin is Shiga toxin.
- the modification is made in a mimic peptide gene sequence so that the homology with the human peptide sequence is reduced, and therefore results in the mimic peptide being not recognized anymore by the host immune system.
- Mimic peptides of particular interest are bacterial mimic peptides that are associated with auto-immune diseases, for example those mentioned in Negi et al. (2017) Pios One 12:e0180518 vent which are hereby incorporated by reference.
- Of particular interest are the gene sequences encoding any of the mimic peptides in S1 Table of Negi et al.
- the mimic peptide is from Proteobacteria or Firmicutes.
- the gene sequences encoding 24 gut bacterial peptides identified by Negi et al. with homology to four human peptides from Low molecular weight phosphotyrosine protein phosphatase, Aldehyde dehydrogenase family 3 member B1 , Maleylacetoacetate isomerase and Uracil-DNA glycosylase. These gene sequences can be modified to reduce the homology with the human sequences and prevent cross-reactivity of those recognized by the host immune system with the human counterpart.
- the genetic modification is in the Bacteroides faecis or Bacteroides thetaiotaomicron beta-galactosidase gene.
- the Bacteroides faecis or Bacteroides thetaiotaomicron beta-galactosidase protein with the genetic modification shows lower homology with human MYH6 cardiac peptide as compared to the Bacteroides faecis or Bacteroides thetaiotaomicron beta-galactosidase protein without the genetic modification.
- the genetic modification is performed in the peptides fragment recognized as epitope by the human immune system leading to a weaker or absence of epitope recognition by the human immune system.
- the genetic modification is in human commensal bacteria encoding a Ro60 ortholog gene.
- the Ro60 protein resulting from the genetic modification shows lower homology with human Ro60 peptide as compared to the original protein.
- the genetic modification is performed in the DNA sequence corresponding to peptides fragment recognized as epitope by the human immune system leading to a weaker or absence of epitope recognition by the human immune system.
- the human bacterial commensal targeted for genetic modification are: Propionibacterium propionicum, Corynebacterium amycolatum, Actinomyces massiliensis, Bacteroides thetaiotaomicron. Even more preferably the human bacterial commensal targeted for genetic modification is Propionibacterium propionicum.
- the genetic modification is in human commensal bacterial DNA sequence encoding a peptide that mimic insulin B 9-25, a self-epitope involved in type 1 diabetes.
- the genetic mutation reduces homology to the insulin B9-25 epitope SHLVEALYLVCGERGFF (SEQ ID NO: 1 ).
- the target bacteria belong to the Firmicutes phylum.
- the target gene in the target bacteria is part of the transketolase N superfamily.
- the genetic modification is in Roseburia intestinalis encoding a peptide that mimic the epitope of the autoantigen p2-glycoprotein I (P2GPI), a self-epitope involved in antiphospholipid syndrome (APS).
- P2GPI autoantigen p2-glycoprotein I
- APS antiphospholipid syndrome
- the genetic mutation is reducing homology to the T cell (P2GPI) epitope KVSFFCKNKEKKCSY (SEQ ID NO: 2) and/or B cell epitope VSRGGMRKFIC (SEQ ID NO: 3).
- the genetic modification can be in the translated or untranslated regions of a gene.
- the genetic modification can be in the promoter region of a gene or within any other region involved in gene regulation.
- the genetic modification results in the change in at least 1 , 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 500, etc. amino acids to a different amino acid.
- the genetic modification introduces a stop codon.
- the genetic modification is outside protein coding sequences, within RNA, or within regulatory sequences.
- the genetic modification does not integrate a phage genome or exogenous DNA into the host bacterial chromosome or endogenous plasmid(s).
- the genetic modification does not result in expression of an exogenous protein from an integrated exogenous DNA in the host bacterial chromosome or endogenous plasmid(s).
- the genetic modification does not involve either NHEJ or HR endogenous repair mechanism of the host bacteria.
- said given effect is killing the receiver bacterial cell.
- said nucleic acid of interest is a gene encoding a nuclease.
- the nucleic acid of interest is a programmable nuclease circuit to be delivered to the targeted bacteria.
- This programmable nuclease circuit may be able to mediate in vivo sequence-specific elimination of bacteria that contain a target gene of interest (e.g. a gene that is harmful to humans).
- Some embodiments of the present disclosure relate to engineered variants of different CRISPR-Cas systems classes and types, such as the Type II CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR- associated) system of Streptococcus pyogenes, as disclosed above.
- programmable nucleases that can be used include other CRISPR-Cas systems, engineered TALEN (Transcription Activator- Like Effector Nuclease) variants, engineered zinc finger nuclease (ZFN) variants, natural, evolved or engineered meganuclease or recombinase variants, and any combination or hybrids of programmable nucleases, as disclosed above.
- the programmable nuclease circuit provided herein may be used to selectively cleave DNA encoding a gene of interest such as, for example, a toxin gene, a virulence factor gene, an antibiotic resistance gene, a remodeling gene or a modulatory gene (cf. WO2014124226 and US2015/0064138).
- said nuclease is the Cpf 1 nuclease.
- said nuclease is the Cas9 nuclease
- said nuclease is the Mad4 nuclease, as defined above.
- said nuclease is the Mad7 nuclease, as defined above.
- said nuclease is the Cms1 nuclease, as defined above.
- antibiotic resistant strains are targetly killed by programming the nuclease to perform a DNA cleavage, e.g. a double strand DNA break in an antibiotic resistance gene located on the chromosome of the target bacteria or on a plasmid with addictive systems (toxin/antitoxin).
- a DNA cleavage e.g. a double strand DNA break in an antibiotic resistance gene located on the chromosome of the target bacteria or on a plasmid with addictive systems (toxin/antitoxin).
- Other sequences of interest preferably programmable, can be delivered to targeted bacteria to kill it.
- the nucleic acid of interest may encode holins or toxins.
- said nucleic acid of interest further makes the receiver bacterial cell produce a molecule of interest, as disclosed above, in particular a host modulatory molecule, as disclosed above, before being killed or just after being killed as a bacterial host for instance.
- the nucleic acid of interest may be under the control of a promoter.
- a promoter may be classified as strong or weak according to its affinity for RNA polymerase.
- the strength of a promoter may depend on whether initiation of transcription occurs at that promoter with high or low frequency. Different promoters with different strengths may be used in the present invention leading to different levels of gene/protein expression (e.g. the level of expression initiated from an mRNA originating from a weak promoter is lower than the level of expression initiated from a strong promoter).
- a promoter sequence may be selected from a large number of known bacterial genes expressed by various bacterial species. Also, methods of prokaryotic promoter prediction exist, and can be based on DNA stability analysis as described in Kanhere and Bansal (BMC Bioinformatics 2005, 6:1 ). The choice of promoter on the vector according to the present invention can thus be made based on the bacteria to target.
- the nucleic acid of interest may be positioned under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the nucleic acid of interest in its natural environment.
- Examples of bacterial promoters for use in accordance with the present invention include, without limitation, positively regulated E. coli promoters such as positively regulated o 70 promoters (e.g., inducible pBad/araC promoter, Lux cassette right promoter, modified lambda Prm promote, plac Or2-62 (positive), pBad/AraC with extra REN sites, pBad, P(Las) TetO, P(Las) CIO, P(Rhl), Pu, FecA, pRE, cadC, hns, pLas, pLux), a “s” promoter (e.g., Pdps), o 32 promoters (e.g., heat shock) and o 54 promoters (e.g., glnAp2); negatively regulated E.
- positively regulated E. coli promoters such as positively regulated o 70 promoters (e.g., inducible pBad/
- coli promoters such as negatively regulated o 70 promoters (e.g., Promoter (PRM+), modified lambda Prm promoter, TetR - TetR-4C P(Las) TetO, P(Las) CIO, P(Lac) IQ, RecA_DlexO_DLac01 , dapAp, FecA, Pspac-hy, pel, plux-cl, plux-lac, CinR, CinL, glucose controlled, modified Pr, modified Prm+, FecA, Pcya, rec A (SOS), Rec A (SOS), EmrR regulated, Betl_regulated, pLacJux, pTet_Lac, pLac/Mnt, pTet/Mnt, LsrA/cl, pLux/cl, Lacl, LaclQ, pLacIQI, pLas/cl, pLas/Lux, pLux/Las
- subtilis promoters such as repressible B. subtilis o A promoters (e.g., Gram-positive IPTG-inducible, Xyl, hyper-spank), o promoters, and the BioFAB promoters disclosed in Mutalik VK et al (Nature Methods, 2013, 10: 354-360, see in particular the supplementary data) as well as on the BioFAB website (http://biofab.synberc.org/data).
- Other inducible microbial promoters and/or bacterial promoters may be used in accordance with the present invention.
- An inducible promoter for use in accordance with the present disclosure may be induced by (or repressed by) one or more physiological condition(s), such as changes in pH, temperature, radiation, osmotic pressure, saline gradients, cell surface binding, and the concentration of one or more extrinsic or intrinsic inducing agent(s).
- the extrinsic inducer or inducing agent may comprise, without limitation, amino acids and amino acid analogs, saccharides and polysaccharides, nucleic acids, protein transcriptional activators and repressors, cytokines, toxins, petroleum-based compounds, metal containing compounds, salts, ions, enzyme substrate analogs, hormones or combinations thereof.
- Particularly preferred bacterial promoters for use in accordance with the present invention may be selected from constitutive promoters regulated by o70 such as the promoters of the Anderson collection (http://parts.igem.org/Promoters/Catalog/Anderson): BBa_J23100, BBa_J23101 , BBa_J23102, BBa_J23103, BBa_J23104, BBa_J23105, BBa_J23106, BBa_J23107, BBa_J23108, BBa_J23109, BBa_J23110, BBa_J23111 , BBa_J231 12, BBa_J23113, BBa_J23114, BBa_J231 15, BBa_J231 16, BBa_J23117, BBa_J23118, and BBa J23119.
- the promoters of the Anderson collection http://parts.igem.org/Promoters/Catalog
- promoters are the promoters disclosed in Stanton et al. (2014) Nat. Chem. Biol. 10:99-105, incorporated herein by reference, including in particular TetR, lcaR(A), AmtR, Betl, SrpR, Orf2, BM3R1 , ButR, PhlF, PsrA, HlyllR, AmeR, LmrA, QacR, ScbR, McbR, LitR, HapR, SmcR, TarA and variants thereof.
- said promoter is SrpR and/or PhlF, or a variant thereof.
- a promoter may or may not be used in conjunction with an "enhancer,” which refers to a ds-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence downstream of the promoter.
- the enhancer may be located at any functional location before or after the promoter.
- the vector may comprise a terminator sequence, or terminator.
- a terminator is a nucleic acid sequence that causes transcription to stop.
- a terminator may be unidirectional or bidirectional. It is comprised of a DNA sequence involved in specific termination of an RNA transcript by an RNA polymerase.
- a terminator sequence prevents transcriptional activation of downstream nucleic acid sequences by upstream promoters.
- a terminator that ends the production of an RNA transcript is contemplated.
- a terminator may be necessary in vivo to achieve desirable gene/protein expression levels.
- terminator The most commonly used type of terminator is a forward terminator. When placed downstream of a nucleic acid of interest that is usually transcribed, a forward transcriptional terminator will cause transcription to abort.
- bidirectional transcriptional terminators are provided, which usually cause transcription to terminate on both the forward and reverse strand.
- reverse transcriptional terminators are provided, which usually terminate transcription on the reverse strand only.
- terminators usually fall into two categories (1 ) rho-independent terminators and (2) rho-dependent terminators.
- Rho-independent terminators are generally composed of palindromic sequence that forms a stem loop rich in G-C base pairs followed by a string of uracil bases.
- Terminators for use in accordance with the present invention include any terminator of transcription described herein or known to one of ordinary skill in the art.
- Examples of terminators include, without limitation, the termination sequences of genes such as, for example, the bovine growth hormone terminator, and viral termination sequences such as, for example, the TO terminator, the TE terminator, lambda Tl and the T1 T2 terminator found in bacterial systems.
- the termination signal may be a sequence that cannot be transcribed or translated, such as those resulting from a sequence truncation.
- Terminators for use in accordance with the present invention also include terminators disclosed in Chen YJ et al (2013, Nature Methods, 10: 659-664), and the BioFAB terminators disclosed in Cambray G et al (Nucl Acids Res, 2013, 41 (9): 5139-5148).
- vector refers to a nucleic acid molecule, typically DNA or RNA that serves to transfer a passenger nucleic acid sequence, i.e. DNA or RNA, into a receiver or target cell.
- a vector may comprise an origin of replication, a selectable marker, and optionally a suitable site for the insertion of a gene such as the multiple cloning site.
- plasmids bacteriophage genomes, phagemids, phageplasmids, virus genomes, cosmids, and artificial chromosomes.
- a vector may be referred to as a payload.
- the vector used in the context of the invention may be a plasmid (e.g, a conjugative plasmid capable of transfer into a host cell), phage, phagemid or prophage.
- the payload can be a phagemid or phasmid obtained from natural, evolved or engineered bacteriophage genome.
- the payload can also be composed only in part of phagemid or phasmid obtained from natural, evolved or engineered bacteriophage genome.
- the payload is the delivery vehicle as bacteria are naturally competent to take up a payload from the environment on their own.
- phagemid and “phasmid” are equivalent and refer to a vector that derives from both a plasmid and a bacteriophage genome.
- a phagemid of the disclosure comprises a phage packaging site and an origin of replication (ori), as disclosed below.
- the term “packaged phagemid” refers to a phagemid which is encapsidated in a bacteriophage scaffold, bacterial virus particle or capsid. Particularly, it refers to a bacteriophage scaffold, bacterial virus particle or capsid devoid of a bacteriophage genome.
- the packaged phagemid may be produced with a helper phage strategy, well known from the man skilled in the art.
- the helper phage comprises all the genes coding for the structural and functional proteins that are indispensable for the phagemid according to the invention to be encapsidated.
- the packaged phagemid may be produced with a satellite virus strategy, also known from the man skilled in the art.
- Satellite virus are subviral agent and are composed of nucleic acid that depends on the co-infection of a host cell with a helper virus for all the morphogenetic functions, whereas for all its episomal functions (integration and immunity, multicopy plasmid replication) the satellite is completely autonomous from the helper.
- the satellite genes can encode proteins that promote capsid size reduction of the helper phage, as described for the P4 Sid protein that controls the P2 capsid size to fit its smaller genome.
- said vector when said vector is a packaged phagemid, said vector does not comprise any element derived from the organism from which the conditional origin of replication is derived.
- the packaging site of said vector is not derived from the organism from which the conditional origin of replication is derived.
- Vectors can include, without limitation, plasmid vectors and recombinant phage vectors.
- the skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform and select host cells comprising any of the isolated nucleotides or nucleic acid sequences of the invention.
- the term “coniugative plasmid” refers to a plasmid that is transferred from one bacterial cell to another during conjugation and a “donor bacterium”, as used herein, is then a bacterium that is capable of transferring a conjugative plasmid to another bacterium.
- the vector used in the context of the invention is devoid of antibiotic resistance marker.
- Antibiotic resistance genes are well known in the art and include but are not limited to ampicillin resistance (Amp), chloramphenicol resistance (Cm), tetracycline resistance (Tet), kanamycin resistance (Kan), hygromycin resistance (Qiyg or hph genes), and zeomycin resistance (Zeo).
- the vector used in the context of the invention comprises an auxotrophic marker.
- auxotrophic markers in bacteria have previously been described, for example, in U.S. Pat. Nos. 4,920,048, 5,691 ,185, 6,291 ,245, 6,413,768, and 6,752,994; U.S. Patent Publication No. 20050186666; Struhl et al. (1976) PNAS USA 73; 1471 -1475; MacCormick et al., (1995) FEMS Microbiol. Lett. 127:105-109; Dickely et al. (1995) Mol. Microbiol. 15:839-847; Sorensen et al. (2000) AppL Environ.
- said auxotrophic marker is ThyA.
- said vector does not comprise any restriction site recognized by restriction enzymes which are frequently encoded by said targeted receiver bacterial cell.
- said vector comprises no more than 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 restriction site(s) recognized by restriction enzymes which are frequently encoded by said targeted bacterial cell or a population or a group of targeted bacterial cell(s).
- restriction site and “restriction enzyme site” are equivalent and refer to locations on a nucleic acid containing specific sequences of nucleotides, which are recognized by restriction enzymes.
- the nucleic acid comprises specific sequences which are bound and cleaved by restriction enzymes.
- Restriction sites are generally palindromic sequences of 4-8 base pairs in length. More precisely, the restriction site refers to a particular sequence and a modification state, so as to be bound and cleaved by restriction enzymes. In particular, it refers to a particular unmodified sequence, so as to be bound and cleaved by restriction enzymes.
- the restriction enzyme is of type I, II or III. Alternatively, it may refer to a particular modified sequence, so as to be bound and cleaved by restriction enzymes, for instance a methylated, hydroxymethylated and glucosyl- hydroxymethylated DNA. In this context, the restriction enzyme is of type IV.
- restriction site As used herein, “recognized by” with respect to a restriction site and a restriction enzyme means that the restriction site is cleaved by the restriction enzyme.
- N means that the nucleotide can be A, C, G or T
- B means that the nucleotide can be C, G or T
- Y means that the nucleotide can be C or T
- W means that the nucleotide can be A or T
- R means that the nucleotide can be A or G
- D means A, G or T.
- restriction enzyme and “restriction endonuclease” are equivalent and refer to an enzyme that cuts nucleic acids at or near restriction sites. Restriction enzymes are commonly classified into four types (types I to type IV). The REBASE database allow to list the restriction sites that a given bacterium can recognize according to the restriction enzymes that it expresses.
- the vector according to the invention preferably included into a delivery vehicle, preferably a bacteriophage capsid, preferably comprises no more than 100 restriction sites. In a preferred embodiment, the vector according to the invention, preferably included in a delivery vehicle, comprises no more than 10 restriction sites. In a most preferred embodiment, the vector according to the invention, preferably included in a delivery vehicle, does not comprise any restriction site.
- the present invention also concerns a nucleic acid vector, as defined above, for use in in vivo delivery of a nucleic acid of interest, as defined above, into a targeted receiver bacterial cell, said nucleic acid of interest producing a given effect on said targeted receiver bacterial cell, wherein said vector comprises:
- conditional origin of replication which is inactive in the targeted receiver bacterial cell but is active in a donor bacterial cell, and wherein said vector is devoid of antibiotic resistance marker.
- the vector of the invention comprises a conditional origin of replication which is inactive in the targeted receiver bacterial cell but is active in a donor bacterial cell.
- condition origin of replication refers to an origin of replication whose functionality may be controlled by the presence of a specific molecule.
- conditional origin of replication is an origin of replication, the replication of which depends upon the presence of one or more given protein, peptid, RNA, nucleic acid, molecule or any combination thereof.
- the replication of said origin of replication may further depend on a process, such as transcription, to activate said replication.
- conditional origin of replication is inactive in the targeted receiver bacterial cell because of the absence of said given protein, peptid, RNA, nucleic acid, molecule or any combination thereof in said receiver bacterial cell.
- said conditional origin of replication is active in said donor bacterial cell because said donor bacterial cell expresses said given protein, peptid, RNA, nucleic acid, molecule or any combination thereof.
- said protein, peptid, RNA nucleic acid, molecule or any combination thereof is expressed in trans in said donor bacterial cell.
- said protein, peptid, RNA, nucleic acid, molecule or any combination thereof is not encoded on the same nucleic acid molecule as the one comprising the origin of replication.
- said protein, peptid, RNA, nucleic acid, molecule or any combination thereof is encoded on a chromosome or on a plasmid.
- said plasmid comprises an antibiotic resistance marker.
- said plasmid is devoid of antibiotic resistance marker.
- conditional origin of replication is inactive in the targeted receiver bacterial cell because of the absence of said given protein, peptid, RNA, nucleic acid, molecule or any combination thereof in said receiver bacterial cell, said conditional origin of replication may be selected depending on the specific receiver bacterial cell to be targeted.
- conditional origin of replication used according to the present invention may originate from plasmids, bacteriophages or PICIs which preferably share the following characteristics: they contain in their origin of replication repeat sequences, or iterons, and they code for at least one protein interacting with said origin of replication (/.e. Rep, protein O, protein P, pri) which is specific to them.
- conditional replication systems of the following plasmids and bacteriophages RK2, R1 , pSC101 , F, Rts1 , RSF1010, P1 , P4, lambda, phi82, phiSO.
- said conditional origin of replication is selected from the group consisting of the R6KA DNA replication origin and derivatives thereof, the IncPa oriV origin of replication and derivatives thereof, ColE1 origins of replication modified to be under an inducible promoter, and origins of replication from phage-inducible chromosomal islands (PICIs) and derivatives thereof.
- said conditional origin of replication is an origin of replication present in less than 50%, or less than 40%, less than 30%, less than 20%, less than 10% or less than 5% of the bacteria of the host microbiome.
- said conditional origin of replication comprises or consists of a sequence less than 80% identical, in particular less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% identical to the sequences of the origins of replication of the bacteria of the host microbiome, in particular of the bacteria representing more than 50%, more particularly more than 60%, more than 70%, more than 80%, more than 90% or more than 95% of the host microbiome.
- phage-inducible chromosomal islands are mobile genetic elements having a conserved gene organization, and encode a pair of divergent regulatory genes, including a PICI master repressor.
- PICIs encode a small set of genes including an integrase (int) gene; right of rpr, and transcribed in the opposite direction, the PICIs encode an excision function (xis), and a replication module consisting of a primase homolog (pri) and optionally a replication initiator (rep), which are sometimes fused, followed by a replication origin (ori), next to these genes, and also transcribed in the same direction, PICIs encode genes involved in phage interference, and optionally, a terminase small subunit homolog (terS).
- int integrase
- rep replication initiator
- ori replication origin
- said conditional origin of replication is an origin of replication derived from phage-inducible chromosomal islands (PICIs).
- the present inventors showed that it is possible to derive novel conditionally replicative plasmids, in particular based on the primase-helicase and origin of replication from PICIs. These origins may be relatively rare in target strains, and more advantageously the primase-ori pair may be unique for each PICI, significantly reducing the possibility of undesired recombination or payload spread events. They can further be modified to further limit recombination chances and remove restriction sites to bypass target bacteria defense systems.
- said conditional origin of replication is derived from the origin of replication from the PICI of the Escherichia coli strain CFT073, disclosed in Fillol-Salom et al. (2016) The ISME Journal 12:21 14-2128.
- said conditional origin of replication is the primase ori from the PICI of the Escherichia coli strain CFT073, typically of sequence SEQ ID NO: 4.
- said conditional origin of replication is the primase ori from he PICI of the Escherichia coli strain CFT073, devoid of at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 1 1 , at least 12, at least 13, at least 14, at least 15 or at least 16 restriction site(s) selected from the group consisting of GAAABCC, GCCGGC, RCCGGY, GCNGC, TWCANNNNTGG (SEQ ID NO: 5), TGGCCA, ACCYAC, YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD, GGCYAC, RGCCGGYYD, and VGCCGGYBD.
- said conditional origin of replication is the primase ori from the PICI of the Escherichia coli strain CFT073, devoid of the restriction site GAAABCC.
- said conditional origin of replication is of sequence SEQ ID NO: 6.
- said conditional origin of replication is the primase ori from he PICI of the Escherichia coli strain CFT073 devoid of the restriction sites selected from the group consisting of GAAABCC, GCCGGC, RCCGGY, GCNGC, TWCANNNNNNTGG (SEQ ID NO: 5), TGGCCA, ACCYAC, YGGCCR, AGACC, GCWGC, GGGANGC, GKAGATD, GCCGGYYD, GGCYAC, RGCCGGYYD, and VGCCGGYBD.
- said conditional origin of replication is of sequence SEQ ID NO: 7.
- said origin of replication is derived from phageinducible chromosomal islands (PICIs)
- said conditional origin of replication is active in said donor bacterial cell because said donor bacterial cell expresses a rep protein, in particular a primase-helicase, in particular a primase-helicase of sequence SEQ ID NO: 8, typically encoded by a nucleic acid comprising or consisting of the sequence SEQ ID NO: 9.
- the vector of the invention comprises or consists of the sequence SEQ ID NO: 10. In another particular embodiment, the vector of the invention comprises or consists of the sequence SEQ ID NO: 11 .
- said origin of replication may be derived from a microorganism which is different from the one that is used to encode the structural elements of the capsid packaging said phagemid.
- said vector is located inside a bacterial delivery vehicle.
- the vector located inside a delivery vehicle is a phagemid and the delivery vehicle is a bacterial virus particle or a capsid.
- delivery vehicle » refers to any vehicle that allows the transfer of a vector or payload into a bacterium.
- delivery vehicle encompassed by the present invention including, without limitation, bacteriophage scaffold, virus scaffold, bacterial virus particle, chemical based delivery vehicle (e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes), protein-based or peptide-based delivery vehicle, lipid-based delivery vehicle, nanoparticle-based delivery vehicles, non-chemical-based delivery vehicles (e.g., transformation, electroporation, sonoporation, optical transfection), particle-based delivery vehicles (e.g., gene gun, magnetofection, impalefection, particle bombardment, cell-penetrating peptides) or donor bacteria (conjugation).
- chemical based delivery vehicle e.g., cyclodextrin, calcium phosphate, cationic polymers, cationic liposomes
- protein-based or peptide-based delivery vehicle e.g., lipid-based delivery vehicle, nanoparticle-based delivery vehicles, non-chemical-based delivery vehicles (e.
- the delivery vehicle can refer to a bacteriophage derived scaffold and can be obtained from a natural, evolved or engineered capsid.
- the delivery vehicle is the vector or payload as bacteria are naturally competent to take up a payload from the environment on their own.
- the present disclosure is directed to a bacterial delivery vehicle containing the vector or payload as described herein.
- the bacterial delivery vehicles are typically prepared from bacterial virus.
- the bacterial delivery vehicles are typically chosen in order to be able to introduce the vector into the targeted bacteria.
- Bacterial viruses from which the bacterial delivery vehicles disclosed herein may be derived, include bacteriophages.
- the bacteriophage is selected from the Order Caudovirales consisting of, based on the taxonomy of Krupovic et al, Arch Virol, 2015, the family Myoviridae, the family Podoviridae, the family Siphoviridae, and the family Ackermannviridae.
- Bacteriophages may be selected from the family Myoviridae (such as, without limitation, genus Cp220virus, Cp8virus, Ea214virus, Felixol virus, Mooglevirus, Suspvirus, Hp1 virus, P2virus, Kayvirus, P100virus, Etavirus, Spo1 virus, Tsarbombavirus, Twortvirus, Cc31 virus, Jd18virus, Js98virus, Kp15virus, Moonvirus, Rb49virus, Rb69virus, S16virus, Schizot4virus, Sp18virus, T4virus, Cr3virus, Se1 virus, V5virus, Abouovirus, Agatevirus, Agrican357virus, Ap22virus, Arv1 virus, B4virus, Bastillevirus, Bc431 virus, Bcep78virus, Bcepmuvirus, Biquartavirus, Bxz1 virus, Cd119virus, Cp51 virus, Cvml Ovirus, Eah2virus, Elvirus, Hapunavirus, J
- Bacteriophages may be selected from the family Podoviridae (such as, without limitation, genus Fri1 virus, Kp32virus, Kp34virus, Phikmvvirus, Pradovirus, Sp6virus, T7virus, Cp1 virus, P68virus, Phi29virus, Nona33virus, Pocjvirus, TI201 1 virus, Bcep22virus, Bpp1 virus, Cba41 virus, Dfl12virus, Ea92virus, Epsilon15virus, F116virus, G7cvirus, Jwalphavirus, Kf1 virus, Kpp25virus, Lit1 virus, Luz24virus, Luz7virus, N4virus, Nonanavirus, P22virus, Pagevirus, Phieco32virus, Prtbvirus, Sp58virus, Una961 virus and Vp5virus).
- family Podoviridae such as, without limitation, genus Fri1 virus, Kp32virus, Kp34virus, Phikmvvirus, Pradovirus, Sp6virus, T7virus
- Bacteriophages may be selected from the family Siphoviridae (such as, without limitation, genus Camvirus, Likavirus, R4virus, Acadianvirus, Coopervirus, Pg 1 virus, Pipefishvirus, Rosebushvirus, Brujitavirus, Che9cvirus, Hawkeyevirus, Plotvirus, Jerseyvirus, Kl gvirus, Sp31 virus, Lmd1 virus, Una4virus, Bongovirus, Reyvirus, Buttersvirus, Charlievirus, Redivirus, Baxtervirus, Nymphadoravirus, Bignuzvirus, Fishburnevirus, Phayoncevirus, Kp36virus, Roguel virus, Rtpvirus, T1 virus, Tlsvirus, Ab18virus, Amigovirus, Anatolevirus, Andromedavirus, Attisvirus, Barnyardvirus, Bernal13virus, Biseptimavirus, Bronvirus, C2virus, C5virus, Cba181 virus, Cbastvirus, Cecivirus, Che8virus, Chivirus, Cjw1 virus
- Bacteriophages may be selected from the family Ackermannviridae (such as, without limitation, genus Ag3virus, Limestonevirus, Cba120virus and Vi1 virus).
- the bacteriophage is not part of the order Caudovirales but from families with unassigned order such as, without limitation, family Tectiviridae (such as genus Alphatectivirus, Betatectivirus), family Corticoviridae (such as genus Corticovirus), family Inoviridae (such as genus Fibrovirus, Habenivirus, Inovirus, Lineavirus, Plectrovirus, Saetivirus, Vespertiliovirus), family Cystoviridae (such as genus Cystovirus), family Leviviridae (such as genus Allolevivirus, Levivirus), family Microviridae (such as genus Alpha3microvirus, G4microvirus, Phix174microvirus, Bdellomicrovirus, Chlamydiamicrovirus, Spiromicrovirus) and family Plasmaviridae (such as genus Plasmavirus).
- family Tectiviridae such as genus Alphatectivirus, Betatectivirus
- the bacteriophage is targeting Archea not part of the Order Caudovirales but from families with unassigned order such as, without limitation, Ampullaviridae, FuselloViridae, Globuloviridae, Guttaviridae, Lipothrixviridae, Pleolipoviridae, Rudiviridae, Salterprovirus and Bicaudaviridae.
- Bacteria of the genus Actinomyces can be infected by the following phages: Av-I, Av-2, Av-3, BF307, CTI, CT2, CT3, CT4, CT6, CT7, CT8 and 1281.
- Bacteria of the genus Bacillus can be infected by the following phages: A, aizl, Al-K-I, B, BCJAI, BCI, BC2, BLLI, BLI, BP142, BSLI, BSL2, BSI, BS3, BS8, BS15, BS18, BS22, BS26, BS28, BS31 , BS104, BS105, BS106, BTB, B1715V1 , C, CK-I, Coll, Corl, CP-53, CS-I, CSi, D, D, D, D5, entl, FP8, FP9, FSi, FS2, FS3, FS5, FS8, FS9, G, GH8, GT8, GV-I, GV-2, GT-4, g3, gl2, gl3, gl4, gl6, gl7, g21 , g23, g24, g29, H2,
- Bacillus-specific phages are defective: DLP10716, DLP-11946, DPB5, DPB12, DPB21 , DPB22, DPB23, GA-2, M, No. IM, PBLB, PBSH, PBSV, PBSW, PBSX, PBSY, PBSZ, phi, SPa, type 1 and p.
- Bacteria of the genus Bacteroides can be infected by the following phages: ad I2, Baf- 44, Baf-48B, Baf-64, Bf-I, Bf-52, B40-8, Fl, pi, ⁇ pAI, cpBrOI, cpBrO2, 11 , 67.1 , 67.3, 68.1 , mt- Bacteroides (3), Bf42, Bf71 , HN-Bdellovibrio (1 ) and BF-41.
- Bacteria of the genus Bordetella can be infected by the following phages: 134 and NN- Bordetella (3).
- Bacteria of the genus Borrelia can be infected by the following phages: NN-Borrelia (1 ) and NN-Borrelia (2).
- Bacteria of the genus Burkholderia can be infected by the following phages: CP75, NN- Burkholderia (1 ) and 42. [238] Bacteria of the genus Campylobacter can be infected by the following phages: C type,
- NTCC12675 NTCC12676, NTCC12677, NTCC12678, NTCC12679, NTCC12680,
- Bacteria of the genus Enterococcus can be infected by the following phages: DF78, Fl, F2, 1 , 2, 4, 14, 41 , 867, DI, SB24, 2BV, 182, 225, C2, C2F, E3, E62, DS96, H24, M35, P3, P9, SBIOI, S2, 2BII, 5, 182a, 705, 873, 881 , 940, 1051 , 1057, 21096C, NN-Enterococcus (1 ), PEI, Fl, F3, F4, VD13, 1 , 200, 235 and 341 .
- Bacteria of the genus Erysipelothrix can be infected by the following phage: NN- Eiysipelothrix (1 ).
- Bacteria of the genus Fusobacterium can be infected by the following phages: NN- Fusobacterium (2), fv83-554/3, fv88-531/2, 227, fv2377, fv2527 and fv8501 .
- Bacteria of the genus Haemophilus can be infected by the following phages: HPI, S2 and N3.
- Bacteria of the genus Helicobacter can be infected by the following phages: HPI and AA - Helicobacter (1 ).
- Bacteria of the genus Lepitospira can be infected by the following phages: LEI, LE3, LE4 and ⁇ NN-Leptospira (1 ).
- Bacteria of the genus Morganella can be infected by the following phage: 47.
- Bacteria of the genus Neisseria can be infected by the following phages: Group I, group II and NPI.
- Bacteria of the genus Nocardia can be infected by the following phages: MNP8, NJ-L, NS-8, N5 and TtiN-Nocardia.
- Bacteria of the genus Proteus can be infected by the following phages: Pm5, 1 Svir, 2/44, 4/545, 6/1004, 13/807, 20/826, 57, 67b, 78, 107/69, 121 , 9/0, 22/608, 30/680, Pml, Pm3, Pm4, Pm6, Pm7, Pm9, PmIO, Pml I, Pv2, TTI, (pm, 7/549, 9B/2, 10A/31 , 12/55, 14, 15, 16/789, 17/971 , 19A/653, 23/532, 25/909, 26/219, 27/953, 32A/909, 33/971 , 34/13, 65, 5006M, 7480b, VI, 13/3a, Clichy 12, TT2600, (px7, 1/1004, 5/742, 9, 12, 14, 22, 24/860, 2600/D52, Pm8 and 24/2514.
- Bacteria of the genus Providencia can be infected by the following phages: PL25, PL26, PL37, 921 1/9295, 9213/921 lb, 9248, 7/R49, 7476/322, 7478/325, 7479, 7480, 9000/9402 and 9213/921 la.
- Bacteria of the genus Rickettsia can be infected by the following phage: NN-Rickettsia.
- Bacteria of the genus Serratia can be infected by the following phages: A2P, PS20, SMB3, SMP, SMP5, SM2, V40, V56, ic, 0CP-3, 0CP-6, 3M, 10/la, 20A, 34CC, 34H, 38T, 345G, 345P, 501 B, SMB2, SMP2, BC, BT, CW2, CW3, CW4, CW5, Lt232, L2232, L34, L.228, SLP, SMPA, V.43, a, (pCWI, 0CP6-1 , 0CP6-2, 0CP6-5, 3T, 5, 8, 9F, 10/1 , 2OE, 32/6, 34B, 34CT, 34P, 37, 41 , 56, 56D, 56P, 6OP, 61/6, 74/6, 76/4, 101/8900, 226, 227, 228, 229F, 286, 289, 290F, 512, 764
- Bacteria of the genus Treponema can be infected by the following phage: NN- Treponema (1).
- Bacteria of the genus Yersinia can be infected by the following phages: H, H-I, H-2, H-3, H-4, Lucas 110, Lucas 303, Lucas 404, YerA3, YerA7, YerA20, YerA41, 3/M64-76, 5/G394-76, 6/C753-76, 8/C239-76, 9/F18167, 1701, 1710, PST, 1/F2852-76, D'Herelle, EV, H, Kotljarova, PTB, R, Y, YerA41, ⁇ YerO3-12, 3, 4/C1324-76, 7/F783-76, 903, 1/M6176 and Yer2AT.
- the bacteriophage is selected in the group consisting of Salmonella virus SKML39, Shigella virus AG3, Dickeya virus Limestone, Dickeya virus RC2014, Escherichia virus CBA120, Escherichia virus PhaxI, Salmonella virus 38, Salmonella virus Det7, Salmonella virus GG32, Salmonella virus PM10, Salmonella virus SFP10, Salmonella virus SH19, Salmonella virus SJ3, Escherichia virus ECML4, Salmonella virus Marshall, Salmonella virus Maynard, Salmonella virus SJ2, Salmonella virus STML131, Salmonella virus ViI, Erwinia virus Ea2809, Klebsiella virus 0507KN21, Serratia virus IME250, Serratia virus MAM1, Campylobacter virus CP21, Campylobacter virus CP220, Campylobacter virus CPt10, Campylobacter virus IBB35, Campylobacter virus CP81, Campylobacter
- Mycobacterium virus SWU1 Mycobacterium virus Ta17a, Mycobacterium virus Tiger,
- the bacterial virus particles typically target E. coli and include the capsid of a bacteriophage selected in the group consisting of BW73, B278, D6, D108, E, El, E24, E41, FI-2, FI-4, FI-5, HI8A, Ffl8B, i, MM, Mu, 025, PhI-5, Pk, PSP3, Pl, PlD, P2, P4, Sl, W ⁇ , ⁇ K13, ⁇ l, ⁇ 2, ⁇ 7, ⁇ 92, 7 A, 8 ⁇ , 9 ⁇ , 18, 28-1, 186, 299, ⁇ H-Escherichia (2), AB48, CM, C4, C16, DD-VI, E4, E7, E28, FIl, FI3, H, Hl, H3, H8, K3, M, N, ND-2, ND-3, ND4, ND-5, ND6, ND-7, Ox- I, Ox-2, Ox-3, Ox-4, Ox-5,
- the present invention thus also concerns a bacterial delivery vehicle, as defined above, for use in in vivo delivery of a nucleic acid of interest into a targeted receiver bacterial cell, as defined above, wherein said bacterial delivery vehicle comprises the vector of the invention.
- Donor bacterial cell refers to a bacterial cell hosting a vector or a plasmid, to a production cell line or to a bacterium that is capable of transferring a conjugative plasmid to another bacterium.
- the present invention also concerns a donor bacterial cell comprising the vector of the invention or producing the bacterial delivery vehicle of the invention, wherein said donor bacterial cell stably comprises the vector of the invention and is able to replicate said vector.
- said donor bacterial cell when the conditional origin of replication of said vector is an origin of replication, the replication of which depends upon the presence of a given protein, peptid, nucleic acid, RNA, molecule or any combination thereof, said donor bacterial cell expresses said protein, peptid, nucleic acid, RNA, molecule or any combination thereof.
- said protein, peptid, nucleic acid, RNA, molecule or any combination thereof is expressed in trans, as defined in the section “Conditional origin of replication” above.
- said donor bacterial cell stably comprises a nucleic acid encoding said protein, peptid, nucleic acid, RNA, molecule or any combination thereof.
- said origin of replication is derived from phage-inducible chromosomal islands (PICIs)
- said conditional origin of replication is active in said donor bacterial cell because said donor bacterial cell expresses a rep protein, in particular a primase-helicase, in particular a primase-helicase of sequence SEQ ID NO: 8.
- said donor bacterial cell stably comprises a nucleic acid encoding said rep protein, in particular said primase-helicase, said nucleic acid typically comprising or consisting of the sequence SEQ ID NO: 9.
- said donor bacterial cell is a production cell line, in particular a cell line producing packaged phagemids including the vector of the invention.
- a satellite phage and/or helper phage may be used to promote the packaging of the vector in the delivery vehicles disclosed herein.
- Helper phages provide functions in trans and are well known to the man skilled in the art.
- the helper phage comprises all the genes coding for the structural and functional proteins that are indispensable for the phagemid to be packaged, (i.e. the helper phage provides all the necessary gene products for the assembly of the delivery vehicle).
- the helper phage may contain a defective origin of replication or packaging signal, or completely lack the latter, and hence it is incapable of self-packaging, thus only bacterial delivery particles carrying the vector or plasmid will be produced.
- Helper phages may be chosen so that they cannot induce lysis of the bacterial cells used for the delivery particle production.
- Some bacteriophages are defective and need a helper phage for payload packaging.
- STF, gpJ and gpH proteins may be provided in a plasmid under the control of an inducible promoter or expressed constitutively.
- the phage wild-type sequence may or not contain a deletion of the gene or sequence supplied in trans.
- chimeric or modified phage sequences encoding a new function like an engineered STF, gpJ or gpH protein, may be directly inserted into the desired position in the genome of the helper phage, hence bypassing the necessity of providing the modified sequence in trans.
- Methods for both supplying a sequence or protein in trans in the form of a plasmid, as well as methods to generate direct genomic insertions, modifications and mutations are well known to those skilled in the art.
- said helper phage comprises a nucleic acid sequence encoding a chimeric STF comprising or consisting of the sequence SEQ ID NO: 12, said nucleic acid sequence typically comprising or consisting of the sequence SEQ ID NO: 13, and said helper phage optionally further comprises a nucleic acid sequence encoding a chimeric gpJ variant comprising or consisting of the sequence SEQ ID NO: 14, said nucleic acid sequence typically comprising or consisting of the sequence SEQ ID NO: 15.
- said helper phage is a lambda prophage wherein (i) the nucleic acid encoding a wild-type STF protein has been replaced by a nucleic acid sequence encoding a chimeric STF comprising or consisting of the sequence SEQ ID NO: 12, said nucleic acid sequence typically comprising or consisting of the sequence SEQ ID NO: 13, (ii) the nucleic acid encoding a wild-type gpJ protein has been replaced by a nucleic acid sequence encoding a chimeric gpJ variant comprising or consisting of the sequence SEQ ID NO: 14, said nucleic acid sequence typically comprising or consisting of the sequence SEQ ID NO: 15, and (iii) the Cos site has been removed, and wherein optionally (iv) the helper prophage contains a mutation which prevents spontaneous cell lysis, such as the Sam7 mutation and (v) the helper prophage contains a thermosensitive version of the master cl repress
- the donor bacterial cell of the invention comprises the abovedefined helper phage.
- the vector used in the method of modulation of the invention may be administered as such, in a bacterial delivery vehicle or through a donor bacterial cell delivering said vector to the receiver bacterial cell.
- Said vector, bacterial delivery vehicle or donor bacterial cell may be more particularly administered in the form of a pharmaceutical or cosmetic composition comprising said vector, bacterial delivery vehicle or donor bacterial cell and a pharmaceutically acceptable carrier.
- the vector, bacterial delivery vehicle or donor bacterial cell may be formulated as a pharmaceutical or cosmetic preparation or compositions comprising at least one vector, bacterial delivery vehicle or donor bacterial cell, and at least one pharmaceutically or cosmetically acceptable carrier, diluent or excipient, and optionally one or more further pharmaceutically or cosmetically active compounds.
- a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular or subcutaneous injection or intravenous infusion), for topical administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, etc.
- said composition is for oral administration.
- Such administration forms may be solid, semi-solid or liquid, depending on the manner and route of administration.
- formulations for oral administration may be provided with an enteric coating that will allow the vector, bacterial delivery vehicle or donor bacterial cell, in the formulation to resist the gastric environment and pass into the intestines.
- vector formulations, bacterial delivery vehicle formulations or donor bacterial cell formulations for oral administration may be suitably formulated for delivery into any desired part of the gastrointestinal tract.
- suitable suppositories may be used for delivery into the gastrointestinal tract.
- the pharmaceutical or veterinary composition according to the invention may further comprise a pharmaceutically acceptable vehicle.
- the cosmetic composition of the invention may further comprise a cosmetically acceptable vehicle.
- a solid pharmaceutically or cosmetically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
- Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins.
- the pharmaceutical or veterinary or cosmetic composition may be prepared as a sterile solid composition that may be suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
- the pharmaceutical or veterinary or cosmetic compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
- the particles according to the disclosure can also be administered orally either in liquid or solid composition form.
- compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
- forms useful for enteral administration include sterile solutions, emulsions, and suspensions.
- the vectors, bacterial delivery vehicles or donor bacterial cells disclosed herein may be dissolved or suspended in a pharmaceutically or cosmetically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
- a pharmaceutically or cosmetically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
- the liquid vehicle can contain other suitable pharmaceutical or cosmetic additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
- suitable examples of liquid vehicles for oral and enteral administration include water (partially containing additives as above, e.g.
- cellulose derivatives preferably sodium carboxymethyl cellulose solution
- alcohols including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
- the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
- Sterile liquid vehicles are useful in sterile liquid form compositions for enteral administration.
- the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically or cosmetically acceptable propellant.
- the invention encompasses pharmaceutical or veterinary or cosmetic composition formulated for delayed or gradual enteric release.
- formulations or pharmaceutical or cosmetic preparations of the invention are formulated for delivery of the vector into the distal small bowel and/or the colon.
- the formulation can allow the vector to pass through stomach acid and pancreatic enzymes and bile, and reach undamaged to be viable in the distal small bowel and colon.
- the pharmaceutical or veterinary or cosmetic composition is micro-encapsulated, formed into tablets and/or placed into capsules, preferably enteric-coated capsules.
- the pharmaceutical or veterinary or cosmetic compositions are formulated for delayed or gradual enteric release, using cellulose acetate (CA) and polyethylene glycol (PEG).
- the pharmaceutical or veterinary or cosmetic compositions are formulated for delayed or gradual enteric release using a hydroxypropylmethylcellulose (HPMC), a microcrystalline cellulose (MCC) and magnesium stearate
- the pharmaceutical or veterinary compositions are formulated for delayed or gradual enteric release using e.g., a poly(meth)acrylate, e.g. a methacrylic acid copolymer B, a methyl methacrylate and/or a methacrylic acid ester, or a polyvinylpyrrolidone (PVP).
- a poly(meth)acrylate e.g. a methacrylic acid copolymer B, a methyl methacrylate and/or a methacrylic acid ester
- PVP polyvinylpyrrolidone
- the pharmaceutical or veterinary or cosmetic compositions are formulated for delayed or gradual enteric release using a release-retarding matrix material such as: an acrylic polymer, a cellulose, a wax, a fatty acid, shellac, zein, hydrogenated vegetable oil, hydrogenated castor oil, polyvinylpyrrolidone, a vinyl acetate copolymer, a vinyl alcohol copolymer, polyethylene oxide, an acrylic acid and methacrylic acid copolymer, a methyl methacrylate copolymer, an ethoxyethyl methacrylate polymer, a cyanoethyl methacrylate polymer, an aminoalkyl methacrylate copolymer, a poly(acrylic acid), a poly(methacrylic acid), a methacrylic acid alkylamide copolymer, a poly(methyl methacrylate), a poly(methacrylic acid anhydride), a methyl methacrylate polymer
- the pharmaceutical or veterinary compositions are formulated for delayed or gradual enteric release as described in U.S. Pat. App. Pub. 201 10218216, which describes an extended release pharmaceutical composition for oral administration, and uses a hydrophilic polymer, a hydrophobic material and a hydrophobic polymer or a mixture thereof, with a microenvironment pH modifier.
- the hydrophobic polymer can be ethylcellulose, cellulose acetate, cellulose propionate, cellulose butyrate, methacrylic acid-acrylic acid copolymers or a mixture thereof.
- the hydrophilic polymer can be polyvinylpyrrolidone, hydroxypropylcellulose, methylcellulose, hydroxypropylmethyl cellulose, polyethylene oxide, acrylic acid copolymers or a mixture thereof.
- the hydrophobic material can be a hydrogenated vegetable oil, hydrogenated castor oil, carnauba wax, candellia wax, beeswax, paraffin wax, stearic acid, glyceryl behenate, cetyl alcohol, cetostearyl alcohol or and a mixture thereof.
- the microenvironment pH modifier can be an inorganic acid, an amino acid, an organic acid or a mixture thereof.
- the microenvironment pH modifier can be lauric acid, myristic acid, acetic acid, benzoic acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, adipic acid, sebacic acid, fumaric acid, maleic acid; glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, sodium dihydrogen citrate, gluconic acid, a salicylic acid, tosylic acid, mesylic acid or malic acid or a mixture thereof.
- the pharmaceutical or veterinary or cosmetic compositions are a powder that can be included into a tablet or a suppository.
- a formulation or pharmaceutical or cosmetic preparation of the invention can be a 'powder for reconstitution' as a liquid to be drunk or otherwise administered.
- the pharmaceutical or veterinary or cosmetic compositions can be administered in a cream, gel, lotion, liquid, feed, or aerosol spray.
- a bacteriophage is immobilized to a solid surface using any substance known in the art and any technology known in the art, for example, but not limited to immobilization of bacteriophages onto polymeric beads using technology as outlined in U.S. Pat. No. 7,482,115, which is incorporated herein by reference. Phages may be immobilized onto appropriately sized polymeric beads so that the coated beads may be added to aerosols, creams, gels or liquids.
- the size of the polymeric beads may be from about 0.1 pm to 500 pm, for example 50 pm to 100 pm.
- the coated polymeric beads may be incorporated into animal feed, including pelleted feed and feed in any other format, incorporated into any other edible devise used to present phage to the animals, added to water offered to animals in a bowl, presented to animals through water feeding systems.
- the compositions are used for treatment of surface wounds and other surface infections using creams, gels, aerosol sprays and the like.
- the pharmaceutical or veterinary or cosmetic compositions can be administered by inhalation, in the form of a suppository or pessary, topically (e.g., in the form of a lotion, solution, cream, ointment or dusting powder), epi- or transdermally (e.g., by use of a skin patch), orally (e.g., as a tablet, which may contain excipients such as starch or lactose), as a capsule, ovule, elixirs, solutions, or suspensions (each optionally containing flavoring, coloring agents and/or excipients), or they can be injected parenterally (e.g., intravenously, intramuscularly or subcutaneously).
- a suppository or pessary topically (e.g., in the form of a lotion, solution, cream, ointment or dusting powder), epi- or transdermally (e.g., by use of a skin patch), orally (e.g.,
- compositions may be used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
- compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
- a bacteriophage and/or polypeptide of the present invention is administered topically, either as a single agent, or in combination with other antibiotic treatments, as described herein or known in the art.
- the pharmaceutical or veterinary or cosmetic compositions can also be dermally or transdermally administered.
- the pharmaceutical or veterinary or cosmetic composition can be combined with one or a combination of carriers, which can include but are not limited to, an aqueous liquid, an alcohol base liquid, a water soluble gel, a lotion, an ointment, a nonaqueous liquid base, a mineral oil base, a blend of mineral oil and petrolatum, lanolin, liposomes, proteins carriers such as serum albumin or gelatin, powdered cellulose carmel, and combinations thereof.
- a topical mode of delivery may include a smear, a spray, a bandage, a time-release patch, a liquid-absorbed wipe, and combinations thereof.
- the pharmaceutical or veterinary or cosmetic composition can be applied to a patch, wipe, bandage, etc., either directly or in a carrier(s).
- the patches, wipes, bandages, etc. may be damp or dry, wherein the phage and/or polypeptide (e.g., a lysin) is in a lyophilized form on the patch.
- the carriers of topical compositions may comprise semi-solid and gel-like vehicles that include a polymer thickener, water, preservatives, active surfactants, or emulsifiers, antioxidants, sun screens, and a solvent or mixed solvent system.
- U.S. Pat. No. 5,863,560 discloses a number of different carrier combinations that can aid in the exposure of skin to a medicament, and its contents are incorporated herein.
- the pharmaceutical or veterinary or cosmetic composition is conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray, or nebuliser with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3-heptafluoropropane, carbon dioxide, or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane or 1 ,1 ,1 ,2,3,3,3-h
- the dosage unit may be determined by providing a valve to deliver a metered amount.
- the pressurized container, pump, spray, or nebuliser may contain a solution or suspension of the active compound, e.g., using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g., sorbitan trioleate.
- a lubricant e.g., sorbitan trioleate.
- Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the bacteriophage and/or polypeptide of the invention and a suitable powder base such as lactose or starch.
- compositions of the invention can be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment, or dusting powder.
- Compositions of the invention may also be administered by the ocular route.
- the compositions of the invention can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride.
- they may be formulated in an ointment such as petrolatum.
- compositions of the present invention may vary depending on the particular use. The determination of the appropriate dosage or route of administration is within the skill of an ordinary physician. Animal experiments can provide reliable guidance for the determination of effective doses in human therapy.
- the pharmaceutical or veterinary composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.
- nasal sprays for transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used.
- the active compounds can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.
- composition of the invention may further comprise at least one additional active ingredient, for instance a prebiotic and/or a probiotic and/or an antibiotic, and/or another antibacterial or antibiofilm agent, and/or any agent enhancing the targeting of the vector to a bacteria and/or the delivery of the vector into a bacteria.
- additional active ingredient for instance a prebiotic and/or a probiotic and/or an antibiotic, and/or another antibacterial or antibiofilm agent, and/or any agent enhancing the targeting of the vector to a bacteria and/or the delivery of the vector into a bacteria.
- a "prebiotic” refers to an ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that may confer benefits upon the host.
- a prebiotic can be a comestible food or beverage or ingredient thereof.
- a prebiotic may be a selectively fermented ingredient.
- Prebiotics may include complex carbohydrates, amino acids, peptides, minerals, or other essential nutritional components for the survival of the bacterial composition.
- Prebiotics include, but are not limited to, amino acids, biotin, fructooligosaccharide, galacto-oligosaccharides, hemicelluloses (e.g., arabinoxylan, xylan, xyloglucan, and glucomannan), inulin, chitin, lactulose, mannan oligosaccharides, oligofructose- enriched inulin, gums (e.g., guar gum, gum arabic and carrageenan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), transgalactooligosaccharide, pectins (e.g., xylogalactouronan, citrus pectin, apple pectin, and rhamnogalacturonan-l), dietary fibers (e.g., soy fiber, sugarbeet fiber, pea
- a “probiotic” refers to a dietary supplement based on living microbes which, when taken in adequate quantities, has a beneficial effect on the host organism by strengthening the intestinal ecosystem.
- Probiotic can comprise a non-pathogenic bacterial or fungal population, e.g., an immunomodulatory bacterial population, such as an anti-inflammatory bacterial population, with or without one or more prebiotics. They contain a sufficiently high number of living and active probiotic microorganisms that can exert a balancing action on gut flora by direct colonisation.
- probiotic is taken to mean any biologically active form of probiotic, preferably including but not limited to lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria or saccharomycetes but even other microorganisms making up the normal gut flora, or also fragments of the bacterial wall or of the DNA of these microorganisms.
- lactobacilli bifidobacteria
- streptococci enterococci
- propionibacteria or saccharomycetes but even other microorganisms making up the normal gut flora, or also fragments of the bacterial wall or of the DNA of these microorganisms.
- Probiotics include, but are not limited to lactobacilli, bifidobacteria, streptococci, enterococci, propionibacteria, saccharomycetes, lactobacilli, bifidobacteria, or proteobacteria.
- said probiotic is not affected by the vector of the invention.
- said vehicle when said vector is comprised in a bacterial delivery vehicle, said vehicle may bind to said probiotic but said probiotic is not affected by said vector.
- said vehicle when said vector is comprised in a bacterial delivery vehicle, said vehicle does not bind to said probiotic and said probiotic is not affected by said vector.
- the effect of said vector induces or increases a synergy with the effect of the additional active ingredient.
- said vector enables said probiotic to engraft into said host organism.
- the antibiotic can be selected from the group consisting of penicillins such as penicillin G, penicillin K, penicillin N, penicillin O, penicillin V, methicillin, benzylpenicillin, nafcillin, oxacillin, cioxacillin, dicloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin, bacampicillin, metampicillin, talampicillin, epicillin, carbenicillin, ticarcillin, temocillin, mezlocillin, and piperacillin; cephalosporins such as cefacetrile, cefadroxil, cephalexin, cefaloglycin, cefalonium, cephaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cef
- the modulating method of the invention is for treating and/or preventing a disease in said host subject.
- said disease is caused or mediated by bacteria.
- the diseases or disorders caused or mediated by bacteria may be selected from the group consisting of skin chronic inflammation such as acne (acne vulgaris), progressive macular hypomelanosis, abdominal cramps, acute epiglottitis, arthritis, bacteraemia, bloody diarrhea, botulism, Brucellosis, brain abscess, cardiomyopathy, chancroid venereal disease, Chlamydia, Crohn’s disease, conjunctivitis, cholecystitis, colorectal cancer, polyposis, dysbiosis, Lyme disease, diarrhea, diphtheria, duodenal ulcers, endocarditis, erysipelothricosis, enteric fever, fever, glomerulonephritis, gastroenteritis, gastric ulcers, Guillain-Barre syndrome tetanus, gonorrhoea, gingivitis, inflammatory bowel diseases, irritable bowel syndrome, leptospirosis, leprosy, liste
- the infection caused by bacteria may be selected from the group consisting of infections, preferably intestinal infections such as esophagitis, gastritis, enteritis, colitis, sigmoiditis, rectitis, and peritonitis, urinary tract infections, vaginal infections, female upper genital tract infections such as salpingitis, endometritis, oophoritis, myometritis, parametritis and infection in the pelvic peritoneum, respiratory tract infections such as pneumonia, intra-amniotic infections, odontogenic infections, endodontic infections, fibrosis, meningitis, bloodstream infections, nosocomial infection such as catheter-related infections, hospital acquired pneumonia, postpartum infection, hospital acquired gastroenteritis, hospital acquired urinary tract infections, or a combination thereof.
- the infection according to the invention is caused by a bacterium presenting an antibiotic resistance.
- the infection is caused by a bacterium as listed above in the targeted bacteria.
- the disclosure also concerns a pharmaceutical or veterinary composition of the invention for the treatment of a metabolic disorder including, for example, obesity, type 2 diabetes and nonalcoholic fatty liver disease.
- a metabolic disorder including, for example, obesity, type 2 diabetes and nonalcoholic fatty liver disease.
- the pharmaceutical or veterinary composition may thus be used to deliver in some intestinal bacteria a nucleic acid of interest which can alter the intestinal microbiota composition or its metabolites (e.g. by inducing expression, overexpression or secretion of some molecules by said bacteria, for example molecules having a beneficial role on metabolic inflammation).
- the disclosure also concerns the use of a pharmaceutical or veterinary composition of the invention for the manufacture of a medicament for the treatment of a metabolic disorder including, for example, obesity, type 2 diabetes and nonalcoholic fatty liver disease. It also relates to a method for treating a metabolic disorder including, for example, obesity, type 2 diabetes and nonalcoholic fatty liver disease, comprising administering to a subject having a metabolic disorder in need of treatment the provided pharmaceutical or veterinary composition, in particular a therapeutically effective amount of the provided pharmaceutical or veterinary composition.
- the invention concerns a pharmaceutical or veterinary composition for use in the treatment of pathologies involving bacteria of the human microbiome, such as inflammatory and auto-immune diseases, cancers, infections or brain disorders. Indeed, some bacteria of the microbiome, without triggering any infection, can secrete molecules that will induce and/or enhance inflammatory or auto-immune diseases or cancer development. More specifically, the present invention relates also to modulating microbiome composition to improve the efficacy of immunotherapies based, for example, on CAR-T (Chimeric Antigen Receptor T) cells, TIL (Tumor Infiltrating Lymphocytes) and Tregs (Regulatory T cells) also known as suppressor T cells.
- CAR-T Chimeric Antigen Receptor T
- TIL Tuor Infiltrating Lymphocytes
- Tregs Regulatory T cells
- Modulation of the microbiome composition to improve the efficacy of immunotherapies may also include the use of immune checkpoint inhibitors well known in the art such as, without limitation, PD-1 (programmed cell death protein 1 ) inhibitor, PD-L1 (programmed death ligand 1 ) inhibitor and CTLA-4 (cytotoxic T lymphocyte associated protein 4).
- immune checkpoint inhibitors well known in the art such as, without limitation, PD-1 (programmed cell death protein 1 ) inhibitor, PD-L1 (programmed death ligand 1 ) inhibitor and CTLA-4 (cytotoxic T lymphocyte associated protein 4).
- said disease is not caused by bacteria.
- the disease to be treated is cancer or a proliferative disorder, including but not limited to, breast cancer (e.g., triple negative breast cancer, ER+ breast cancer, or ER- breast cancer), basal cell carcinoma, skin cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, brain cancer, medulloblastoma, glioma (including glioblastoma, oligodendroglioma, astrocytoma, ependymoma), neuroblastoma, colorectal cancer, ovarian cancer, liver cancer, pancreatic cancer (e.g., carcinoma, angiosarcoma, adenosarcoma), gastric cancer, gastroesophageal junction cancer, prostate cancer, cervical cancer, bladder cancer, head and neck cancer, lymphoma (e.g., mantle cell lymphoma, diffuse large B-cell lymphoma), solid tumors that cannot be removed by surgery, locally advanced solid tumors, meta
- the diseases to be treated include, but are not limited to, inflammatory or allergic diseases, including systemic anaphylaxis and hypersensitivity disorders, atopic dermatitis, urticaria, drug allergies, insect sting allergies, food allergies (including celiac disease and the like), and mastocytosis; inflammatory bowel diseases, including Crohn's disease, ulcerative colitis, ileitis, and enteritis; vasculitis, and Behcet's syndrome; psoriasis and inflammatory dermatoses, including dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria, viral cutaneous pathologies including those derived from human papillomavirus, HIV or RLV infection, bacterial, flugal, and other parasital cutaneous pathologies, and cutaneous lupus erythematosus; asthma and respiratory allergic diseases, including allergic asthma, exercise induced asthma, allergic rhinitis, otitis
- the disease to be treated may be an autoimmune disease such as autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmunocytopenia, antiphospholipid syndrome, dermatitis, gluten-sensitive enteropathy, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis, Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendo- crinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, myocarditis, IgA glomerulonephritis, dense deposit disease, rheumatic heart disease, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism, systemic lupus erythematosus, discoid lup
- the subject to be treated may have been diagnosed with, or may be at risk of developing an infection, a disorder and/or a disease preferably due to a bacterium. Diagnostic method of such infection, disorder and/or disease are well known by the man skilled in the art.
- the infection, disorder and/or disease presents a resistance to treatment, preferably the infection, disorder or disease presents an antibiotic resistance.
- the subject has never received any treatment prior to the administration of the vectors according to the invention, particularly a vector packaged into a delivery vehicle according to the invention, preferably a packaged plasmid or phagemid into a bacterial virus particle according to the invention, or of a pharmaceutical or veterinary composition according to the invention.
- the subject has already received at least one line of treatment, preferably several lines of treatment, prior to the administration of the vectors according to the invention, particularly a vector packaged into a delivery vehicle according to the invention, preferably a packaged plasmid or phagemid into a bacterial virus particle according to the invention, or of a pharmaceutical or veterinary composition according to the invention.
- the treatment is administered regularly, preferably between every day and every month, more preferably between every day and every two weeks, more preferably between every day and every week, even more preferably the treatment is administered every day.
- the treatment is administered several times a day, preferably 2 or 3 times a day, even more preferably 3 times a day.
- the duration of treatment with vectors according to the invention is preferably comprised between 1 day and 20 weeks, more preferably between 1 day and 10 weeks, still more preferably between 1 day and 4 weeks, even more preferably between 1 day and 2 weeks.
- the duration of the treatment is of about 1 week.
- the treatment may last as long as the infection, disorder and/or disease persists.
- the form of the pharmaceutical or veterinary compositions, the route of administration and the dose of administration of vectors according to the invention, particularly of a vector packaged into a delivery vehicle according to the invention, preferably of a packaged plasmid or phagemid into a bacterial virus particle according to the invention, or of a pharmaceutical or veterinary composition according to the invention can be adjusted by the man skilled in the art according to the type and severity of the infection (e.g. depending on the bacteria species involved in the disease, disorder and/or infection and its localization in the patient’s or subject’s body), and to the patient or subject, in particular its age, weight, sex, and general physical condition.
- the amount of vectors according to the invention, particularly a vector packaged into a delivery vehicle according to the invention, preferably a packaged plasmid or phagemid into a bacterial virus particle according to the invention, or of a pharmaceutical or veterinary composition according to the invention, to be administered has to be determined by standard procedure well known by those of ordinary skills in the art. Physiological data of the patient or subject (e.g. age, size, and weight) and the routes of administration have to be taken into account to determine the appropriate dosage, so as a therapeutically effective amount will be administered to the patient or subject.
- the total amount of vectors particularly a vector packaged into a delivery vehicle according to the invention, preferably a plasmid or phagemid packaged into a bacterial virus particle according to the invention, for each administration is comprised between 10 4 and 10 15 delivery vehicles.
- the modulating method of the invention is for the cosmetic treatment of said host subject.
- the host organism is a plant
- the modulating method of the invention is for the agronomical, prophylactic or phytotherapeutic treatment of said host plant.
- said modulating method is for improving the growth of said host plants, for preventing a disease or for treating diseases affecting said host plants.
- the present invention also concerns a method for ex vivo modulating a microbiome from an environment by collecting targeted receiver bacterial cell from said environment and by delivering a nucleic acid of interest into said targeted receiver bacterial cell of said microbiome, said nucleic acid of interest producing a given effect, as disclosed above, on said targeted receiver bacterial cell, wherein said method comprises contacting a nucleic acid vector comprising said nucleic acid of interest with said microbiome, wherein said vector further comprises a conditional origin of replication which is inactive in the targeted receiver bacterial cell but is active in a donor bacterial cell, and said vector is devoid of antibiotic resistance marker, thereby delivering said nucleic acid of interest into the targeted receiver bacterial cell, and wherein, once delivered into said targeted receiver bacterial cell, said nucleic acid of interest produces said given effect on said targeted receiver bacterial cell while said vector is not replicated in said targeted receiver bacterial cell.
- environment is meant herein all the elements which surround a species and among which some directly or indirectly contribute to the subsistence of said species.
- said environment is not an animal.
- said environment is an animal, in particular a human.
- said environment can be any medium wherein said microbiome lives, such as a solid or semi-solid surface or a liquid medium, such as water, in particular waste water.
- said ex vivo method is for protecting a surface against biofouling. In another particular embodiment, said ex vivo method is for decontaminating water.
- Figure 1 BLAST analysis of the PICI-CFT073 origin in Escherichia coli.
- Figure 2 BLAST analysis of modified p15a origin of replication of sequence SEQ ID NO: 4 in Escherichia coli.
- Figure 3 BLAST analysis of the PICI-CFT073 origin in the domain Bacteria.
- Figure 4 BLAST analysis of the modified p15a origin in the domain Bacteria.
- Figure 5 Transformation of a 2.3kb payload containing the primase-origin of replication (p1319) in a production strain harboring an inducible primase RBS library in trans.
- Figure 6 Comparison of packaged phagemids titers obtained with a plasmid containing the primase-ori in production strains (p1319) tested against 7 different primase RBS. Right column, in black, control plasmid with a p15a-derived origin of replication (p1220). Titers shown are after a 10X concentration.
- Figure 7 Comparison of cells transduced with a primase-ori plasmid (top row, p1319) and a p15a-based packaged phagemids (p1220) on LB agar plus 25 pg/mL chloramphenicol.
- RBS 3 is SEQ ID NO: 20.
- Figure 8 Comparison of killing activity in the absence of antibiotic selection of E. coli MG1655 transduced with a nuclease circuit targeting the lacZ gene. Black line, primase-ori (conditional replication, p1322); grey line, modified p15a-ori, replicative (p780).
- Figure 9 Packaged phagemids titers obtained with ⁇ 12kb plasmids harboring the mutated primase-ori in production strains containing primase RBS 3. Titers shown are after a 10X concentration. Left bar, lacZ target (p1326); right bar, 4stx target (p1327).
- Figure 10 Nuclease-mediated killing of different 0157 strains mediated by targeting lacZ by transduction of packaged phagemids harboring a conditional origin of replication, payload p1326 (grey line c, an 0157 strain lacking the lacZ gene serves as a non-killing control).
- Figure 11 Nuclease-mediated killing of four 0157 strains mediated by stx targeting after transduction of packaged phagemids harboring a conditional origin of replication (payload p1327).
- Figure 12 Change in colonization in colonized mice orally administered with either a neutralizing buffer alone (‘+ buffer’) or 10 12 particles of packaged phagemids in a neutralizing buffer (‘+ EB’). Change in colonization between TO and T8h is reported for each animal, and the median and quartiles of each experimental group were graphed. **** p ⁇ 0.0001 by unpaired t test.
- Figure 13 Adenine base editing of p-lactamase on the E. coli genome after phagemid transduction in vitro using a payload comprising a conditional origin of replication of sequence SEQ ID NO: 7. 96 individual colonies for each MOI were spotted on LB and LB (carbenicillin) plates and base editing efficiencies were calculated.
- Packaged phagemids are being used to deliver a DNA payload to target bacteria with high efficiency.
- Features required for phagemid packaging are the presence of a packaging site and an origin of replication that is functional in the producer cell line.
- the residence time is increased.
- phages have a precise tropism towards the same or closely related species in which they are produced, the packaged phagemids derived from this phage, once their payloads delivered in the target bacteria, will keep replicating, unless the phage has been engineered to infect/inject in a new group of bacteria.
- the payload Since the payload is replicative, some events of injection will cause the plasmid to spread. • Moreover, when the payload is based on a common origin of replication present in many Enterobacteria (for example, a ColE-type origin), the risk of recombination with already- existing plasmids in target bacterial strains may be high. For regulatory purposes, this poses a problem since the transduced cells are considered as GMOs and are then replicative GMOs, which poses a containment risk that has to be evaluated accordingly.
- the inventors show for the first time that phagemids can be packaged at high titers with a conditional ORI with ori and protein required for replication in trans, • The inventors show the additional advantage of using a ORI system that can be found in PICI genomes as opposed to other systems based on plasmid derived ORI (from a bacterial origin), which significantly limits the risk of spread. Furthermore, even if the ORI system is actually present in the transduced bacteria, meaning that a natural PICI harboring the same ORI system is found in the bacteria, it has to be active (in a lytic cycle) for the introduced phagemid to be replicated, since the primase gene in a PICI is inactive unless found in the induced (lytic) state. This is totally different for a bacterial ORI, since it would mean that it would be active naturally and constitutively.
- the inventors sought to develop a system in which the payload contains the 282- bp primase origin and the primase protein is supplied in trans (SEQ ID NO: 8 and SEQ ID NO: 9).
- the PICI primase gene was extracted from the genome of E. coli CFT073, cloned into a plasmid under the control of an inducible system and an RBS (ribosome-binding site) library generated. This series of plasmids were cloned in the lambda production strain s1965.
- the inventors constructed a small payload harboring the primase- ori instead of the p15a-based origin of replication to yield the 2.3 kb payload p1319 (SEQ ID NO: 16). Since this plasmid is, in principle, non-replicative, competent cells of s1965 harboring the RBS library of inducible primase constructs were made, the p1319 plasmid transformed in them and plated in LB agar + kanamycin and chloramphenicol in the presence of the inducer DAPG (to induce the expression of the primase in trans). Next day, the inventors observed that the plates contained hundreds of colonies, suggesting that the primase-origin system in trans works (Figure 5).
- the plasmid encoding the inducible primase in the 7 clones tested was miniprepped and sequenced (SEQ ID NO: 18 to 24). They were also transformed into MG1655 cells (s003): these strains were used to verify the titers obtained, since the payloads should not be replicative in the absence of the primase protein supplied in trans.
- PICI primase and origin can be stably maintained in production strains, are compatible with lambda-based phagemids packaging judging by the titers obtained and the payloads are dependent on the presence of its cognate primase for active replication and maintenance in target strains.
- the inventors constructed a large plasmid ( ⁇ 12kb) exchanging the p15a-based origin of replication by the primase origin.
- This plasmid targets the lacZ gene (p1322, SEQ ID NO: 25) and also contains a chloramphenicol marker. Since it was ignored if the RBS strength would need to be modified to replicate a large plasmid, the inventors transformed this plasmid into the production strain s1965 harboring an inducible primase RBS library in trans, as done for the initial, smaller payload. Next day, the inventors observed that the plates contained hundreds of colonies.
- the packaged phagemids targeting lacZ and containing the p15a-based origin (control) or the primase origin were serially diluted 3X; this approach allowed for testing different MOIs.
- 90 pL of cells were added to each well containing a packaged phagemid dilution. After 30 min- incubation at 37 e C, 10X dilutions of each reaction were performed, 10 pL plated on LB agar plates and incubated overnight at 37 e C.
- conditional origins of replication based on PICIs allow for production at high titers of large payloads ( ⁇ 12kb) and nuclease-mediated killing of a target strain in the absence of selection and primase protein.
- the inventors analyzed the 282-bp PICI origin and found that it contains the 0157 restriction site GAAABCC (GAAAGCC). The inventors modified this site within the origin and obtained the sequence GAAAGCa (small cap represents the mutation introduced) which should not be recognized by 0157 strains.
- the modified PICI origin (SEQ ID NO: 6) was then cloned into ⁇ 12kb payloads containing a Cpf1 nuclease circuit targeting the lacZ gene as mentioned in Example 3 (p1326, SEQ ID NO: 28) and also a quadruplex crRNA guide targeting stx1 and stx2 genes (p1327, SEQ ID NO: 29).
- the inventors previously designed a bacterial cell line producing an engineered lambdabased capsid, comprising a chimeric 1 A2 gpJ protein and a chimeric STF-V10[Helix], able to inject efficiently in 0157 strains (s15816), so these two plasmids were transformed in this production strain containing the primase RBS 3 in trans.
- the non-targeted strain shows a similar profile as that seen for MG1655: dense spots at high MOIs and low dilutions (the cells cannot actively divide due to cell density and cannot lose the plasmid) and weaker density spots, translucid, at lower MOIs and higher dilutions, indicative of cell death due to exposure to the antibiotics.
- the present example demonstrates efficient decolonization in vivo by specifically killing bacteria bearing six genes using a packaged phagemid with a conditional origin of replication.
- mice Streptomycin-treated mice were orally administered with either a target bacterial strain (hereafter referred to as ‘Target strain’) or a mutant of the same bacterial strain deleted for a specific gene of interest, namely a stx gene (hereafter referred to as ‘Non-Target strain’) to establish a durable intestinal colonization with these bacterial strains.
- Target strain a target bacterial strain
- Non-Target strain a mutant of the same bacterial strain deleted for a specific gene of interest
- mice colonized with either strain were given 100 pl of packaged phagemids (approximately 10 12 particles) along with 100 pl of a buffer (sucrose and bicarbonate in water) aimed at temporarily neutralizing the gastric pH.
- a buffer saccharide and bicarbonate in water
- This example presents a method for the base editing of the nucleic acid sequence encoding ⁇ -lactamase (SEQ ID NO: 30) on the E. coli MG1655 genome after phagemid transduction in vitro using a payload comprising a conditional origin of replication of sequence SEQ ID NO: 7, based on a primase-helicase.
- the non-replicative payload comprises an adenine base editor (ABE8e), a transcribed guideRNA targeting the active site of the p-lactamase gene (K71 E) on the genome, a lambda packaging sequence, a chloramphenicol resistance marker, and the conditional origin of replication of sequence SEQ ID NO: 7.
- Transduced cells were plated on LB plates 2 hours post transduction at different multiplicity of infections (MOI). The next day, 96 individual colonies for each MOI were spotted on LB and LB (carbenicillin) plates in order to analyse the base editing efficiency.
- MOI multiplicity of infections
Abstract
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CN202180094940.XA CN116940677A (en) | 2020-12-30 | 2021-12-29 | Microbiome modulation of a host by delivery of DNA payloads with minimal transmission |
EP21847963.2A EP4271808A1 (en) | 2020-12-30 | 2021-12-29 | Microbiome modulation of a host by delivery of dna payloads with minimized spread |
JP2023540163A JP2024501870A (en) | 2020-12-30 | 2021-12-29 | Host microbiome modulation by delivery of DNA payloads with minimal diffusion |
CA3205876A CA3205876A1 (en) | 2020-12-30 | 2021-12-29 | Microbiome modulation of a host by delivery of dna payloads with minimized spread |
KR1020237025323A KR20230127265A (en) | 2020-12-30 | 2021-12-29 | Regulation of the host microbiome through the delivery of DNA payloads with minimal spread |
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