WO2019067621A1 - Compositions et procédés d'un système génétique crispr pour sensibiliser et/ou éliminer des bactéries cibles - Google Patents

Compositions et procédés d'un système génétique crispr pour sensibiliser et/ou éliminer des bactéries cibles Download PDF

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WO2019067621A1
WO2019067621A1 PCT/US2018/052962 US2018052962W WO2019067621A1 WO 2019067621 A1 WO2019067621 A1 WO 2019067621A1 US 2018052962 W US2018052962 W US 2018052962W WO 2019067621 A1 WO2019067621 A1 WO 2019067621A1
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Prior art keywords
bacteria
sequence
nucleotide sequence
composition
food
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English (en)
Inventor
Jorge BARDALES
Maricel SAENZ
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Nextbiotics Inc
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Nextbiotics Inc
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the disclosure is directed to the fields of molecular biology, genomic engineering, CRISPR, bacterial infection, antibacterial resistance and therapeutics.
  • the compositions and methods of the disclosure provide a CRISPR-based method for selectively sensitizing and/or eliminating target bacteria, which may be pathogenic.
  • the disclosure provides a nucleotide sequence comprising (a) a sequence encoding a DNA binding domain and (b) a sequence encoding a nuclease or nuclease domain, wherein the DNA binding domain specifically targets a sequence of a bacterial genome or a sequence of a bacterial plasmid and wherein the nuclease or nuclease domain induces at least one break in the sequence of the bacterial genome.
  • the nucleotide sequence comprises (a) a sequence encoding a DNA binding domain and (b) a sequence encoding a nuclease or nuclease domain, wherein the DNA binding domain specifically targets a sequence of a bacterial genome or a sequence of a bacterial plasmid and wherein the nuclease or nuclease domain induces at least one double-strand break in the sequence of the bacterial genome.
  • the nucleotide sequence encodes a Type V CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) construct.
  • the nuclease or nuclease domain is isolated or derived from a Cpfl polypeptide or an ortholog protein of the Cpfl polypeptide.
  • the DNA binding domain comprises an RNA sequence.
  • the RNA sequence comprises at least one guide RNA sequence.
  • the RNA sequence comprises at least two guide RNA sequences.
  • the RNA sequence comprises between 2 and 10 guide RNA sequences, inclusive of the endpoints.
  • the RNA sequence comprises 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 guide RNA sequences.
  • the RNA sequence comprises at least 10 guide RNA sequences.
  • each of the guide RNA sequences specifically targets a distinct sequence of the bacterial genome or a distinct sequence of a bacterial plasmid.
  • the sequence of the bacterial genome targeted by the DNA binding domain comprises a gene or at least one sequence essential for antibiotic resistance.
  • the gene comprises at least one sequence essential for antibiotic resistance.
  • the nuclease or nuclease domain induces at least one double strand break within the at least one sequence essential for antibiotic resistance.
  • the sequence of the bacterial plasmid targeted by the DNA binding domain comprises a gene or at least one sequence essential for antibiotic resistance.
  • the gene comprises at least one sequence essential for antibiotic resistance.
  • the nuclease or nuclease domain induces at least one double strand break within the at least one sequence essential for antibiotic resistance.
  • the DNA binding domain targets a gene or a sequence therein, which is essential for the bacteria's resistance to an antibiotic composition of the disclosure.
  • the gene or sequence therein essential for bacterial resistance encodes a TEM-1, TEM-2, SHV-3, SHV-4 ,SHV-5, OXA-1, OXA-2, OXA-48, OXA-24, OXA-58 CTXM-1, CTXM-2, CTXM-3, New Delhi Metallo-beta-lactamase-1 (NDM-1), New Delhi Metallo-beta-lactamase-2 (NDM-2), New Delhi Metallo-beta-lactamase-5 (NDM-5), GES-5, GES-18, IMI-l,IMI-2, SME-2, SME-3, KPC-7, KPC-8, VIM-2, VIM-7, IMP-6, IMP-7, DNA gyrase subunit A (Gyr)
  • ppxl Protoporphyrinogen oxidase
  • ppx2 Exopolyphosphatase 2
  • ppk Polyphosphate kinase
  • the gene or sequence therein is essential for bacterial resistance encodes a DNA gyrase subunit A (GyrA).
  • the sequence encoding a DNA binding domain is complementary to a sequence of the gene encoding a GyrA, a sequence variant, or a regulatory sequence thereof. In certain embodiments, the sequence encoding a DNA binding domain is complementary to one or more sequences flanking the gene encoding a GyrA, a sequence variant, or a regulatory sequence thereof.
  • the DNA binding domain targets a gene or a sequence therein that is essential for bacterial cell survival, viability or biofilm formation.
  • the nuclease or nuclease domain induces at least one double strand break within the gene or sequence therein that is essential for bacterial cell survival or viability.
  • the DNA binding domain targets a gene or a sequence therein that is essential for quorum sensing. In certain embodiments, the nuclease or nuclease domain induces at least one double strand break within the gene or sequence therein that is sequence essential quorum sensing. [015] In certain embodiments of the nucleotide sequence of the disclosure, the DNA binding domain targets a gene or a sequence therein that is essential for transformation to a virulent phenotype. In certain embodiments, the nuclease or nuclease domain induces at least one double strand break within the gene or sequence therein that is essential for
  • the one or more double strand breaks result in one or more of genomic instability of a bacterial cell, loss of antibiotic resistance of a bacterial cell, loss of homeostasis of a bacterial cell, loss of quorum sensing or virulent transformation of a bacterial cell.
  • the sequence of a bacterial genome or the sequence of a bacterial plasmid is isolated or derived from a sequence of a pathogenic bacteria.
  • the pathogenic bacteria is from the family Enterobacteriaceae.
  • the pathogenic bacteria is from the family Lactobacillaceae.
  • the pathogenic bacteria is from the family Staphylococcaceae spp.
  • the pathogenic bacteria is from the family Streptococcaceae spp.
  • the pathogenic bacteria is from the family Peptoniphilyus spp.
  • the pathogenic bacteria is from the family Anaerococcus spp.
  • the pathogenic bacteria is from the family Corynebacterium spp.
  • the disclosure provides a composition comprising a nucleotide sequence of the disclosure.
  • the disclosure provides a vector comprising a nucleotide sequence of the disclosure.
  • the vector is a viral vector.
  • the viral vector is a replication-deficient virus.
  • the viral vector is a bacteriophage vector.
  • the viral vector is a plasmid.
  • the plasmid is a phagemid.
  • the disclosure provides a composition comprising a vector of the disclosure.
  • the disclosure provides a phagemid comprising: (a) a nucleotide of the disclosure, and (b) one or more sequences encoding a protein having a predetermined three-dimensional structure, wherein the predetermined three-dimensional structure confers stability, increases bioavailability and/or reduces immunogenicity to the protein when compared to a protein lacking the predetermined three-dimensional structure.
  • the one or more sequences encoding a protein having a predetermined three-dimensional structure comprise at least one sequence isolated or derived from a bacteriophage.
  • the at least one sequence isolated or derived from a bacteriophage has 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or any percentage in between of complementarity or identity with a sequence of a bacteriophage.
  • the one or more sequences encoding a protein having a predetermined three-dimensional structure improves a polarity towards a species and/or a strain of bacteria.
  • the one or more sequences encoding a protein having a predetermined three-dimensional comprise a sequence encoding a viral capsid protein.
  • the one or more sequences encoding a protein having a predetermined three-dimensional comprise a sequence encoding a viral filament protein.
  • the disclosure provides a composition comprising a phagemid of the disclosure.
  • the composition further comprises an antibacterial composition.
  • the antibiotic composition comprises one or more of a penicillin, a tetracycline, a cephalosporin, a quinolone, a lincomycin, a macrolide, a sulfonamide, a glycopeptide, an aminoglycoside, and a carbapenem.
  • the penicillin comprises benzylpenicillin (penicillin G), penicillin V (phenoxymethylpenicillin), penicillin V potassium, amoxicillin, amoxicillin/clavulanate (Augmentin), ampicillin, cl oxacillin, flucloxacillin, piperacillin or ticarcillin.
  • the tetracycline comprises doxycycline, doxycycline/salicylic acid, tetracycline, minocycline, demeclocycline, or oxytetracycline.
  • the cephalosporin comprises cefuroxime, ceftriaxone, or Cefdinir.
  • the quinolone comprises ciprofloxacin, levofloxacin or moxifloxacin.
  • the lincomycin comprises clindamycin or lincomycin.
  • the macrolide comprises azithromycin,
  • the sulfonamide comprises sulfamethoxazole-trimethoprim, sulfasalazine or sulfisoxazole.
  • the glycopeptide comprises dalbavancin, oritavancin, telavancin or vancomycin.
  • the aminoglycoside comprises gentamicin, tobramycin or amikacin.
  • the carbapenem comprises imipenem/cilastatin, meropenem, doripenem or ertapenem. [024] In some embodiments of the compositions of the disclosure, the compositions further comprise a pharmaceutically acceptable carrier.
  • the composition is formulated as a liquid. In some embodiments, the composition is formulated as a gel, ointment, paste, suspension, semi-solid, a spreadable format or a dispersible format. In some embodiments, the composition is formulated as a solid.
  • the composition comprises a substrate.
  • the substrate is semi-solid or solid.
  • the substrate comprises a scaffold or a surface.
  • the disclosure provides a method of modifying a genomic sequence of a bacteria comprising contacting the bacteria and an effective amount of a nucleotide sequence of the disclosure.
  • the contacting step comprises introducing the nucleotide sequence into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the nucleotide sequence into the bacteria.
  • the disclosure provides a method of selectively eliminating a bacteria comprising contacting the bacteria and an effective amount of a nucleotide sequence of the disclosure.
  • the contacting step comprises introducing the nucleotide sequence into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the nucleotide sequence into the bacteria.
  • the disclosure provides a method of modifying a genomic sequence of a bacteria comprising contacting the bacteria with an effective amount of a vector of the disclosure.
  • the contacting step comprises introducing the vector into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the vector into the bacteria.
  • the disclosure provides a method of selectively eliminating a bacteria comprising contacting the bacteria with an effective amount of a vector of the disclosure.
  • the contacting step comprises introducing the vector into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the vector into the bacteria.
  • the disclosure provides a method of modifying a genomic sequence of a bacteria comprising contacting the bacteria with an effective amount of a phagemid of the disclosure.
  • the contacting step comprises introducing the phagemid into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the phagemid into the bacteria.
  • the disclosure provides a method of selectively eliminating a bacteria comprising contacting the bacteria with an effective amount of a phagemid of the disclosure.
  • the contacting step comprises introducing the phagemid into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the phagemid into the bacteria.
  • the disclosure provides a method of modifying a genomic sequence of a bacteria comprising contacting the bacteria with an effective amount of a composition of the disclosure.
  • the contacting step comprises introducing the composition into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the composition into the bacteria.
  • the disclosure provides a method of selectively eliminating a bacteria comprising contacting the bacteria with an effective amount of a composition of the disclosure.
  • the contacting step comprises introducing the composition into the bacteria.
  • the contacting step comprises a transformation of the bacteria to introduce the composition into the bacteria.
  • the contacting step occurs under conditions suitable for expression of the nucleotide sequence. In certain embodiments of the methods of the disclosure, including methods of modifying a genomic sequence of a bacteria and methods of selectively eliminating a bacteria of the disclosure, the contacting step occurs under conditions suitable for expression of the nucleotide sequence comprised a vector or a phagemid.
  • the pathogenic bacteria is from the family Enterobacteriaceae. In certain embodiments, the pathogenic bacteria is from the family Lactobacillaceae. In certain embodiments, the pathogenic bacteria is from the family Staphylococcaceae spp. In certain embodiments, the pathogenic bacteria is from the family Streptococcaceae spp. In certain embodiments, the pathogenic bacteria is from the family Peptoniphilyus spp. In certain embodiments, the pathogenic bacteria is from the family Anaerococcus spp. In certain embodiments, the pathogenic bacteria is from the family Corynebacterium spp.
  • the disclosure provides a method of treating an infection in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a nucleotide sequence of the disclosure.
  • the disclosure provides a method of treating an infection in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a vector of the disclosure.
  • the disclosure provides a method of treating an infection in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a phagemid of the disclosure.
  • the disclosure provides a method of treating an infection in a subject in need thereof comprising administering to the subject a therapeutically-effective amount of a composition of the disclosure.
  • the composition further comprises an antibiotic composition.
  • the method further comprises administering an antibiotic composition.
  • the antibiotic composition comprises one or more of a penicillin, a tetracycline, a cephalosporin, a quinolone, a lincomycin, a macrolide, a sulfonamide, a glycopeptide, an aminoglycoside, and a carbapenem.
  • the penicillin comprises benzylpenicillin (penicillin G), penicillin V (phenoxymethylpenicillin), penicillin V potassium, amoxicillin,
  • the tetracycline comprises doxycycline
  • the cephalosporin comprises cefuroxime, ceftriaxone, or Cefdinir.
  • the quinolone comprises ciprofloxacin, levofloxacin or moxifloxacin.
  • the lincomycin comprises clindamycin or lincomycin.
  • the macrolide comprises azithromycin, clarithromycin or erythromycin.
  • the sulfonamide comprises sulfamethoxazole-trimethoprim, sulfasalazine or sulfisoxazole.
  • the glycopeptide comprises dalbavancin, oritavancin, telavancin or vancomycin. In certain embodiments, the
  • aminoglycoside comprises gentamicin, tobramycin or amikacin.
  • the carbapenem comprises imipenem/cilastatin, meropenem, doripenem or ertapenem.
  • the nucleotide sequence is administered by a local route or by a systemic route.
  • the vector is administered by a local route or by a systemic route.
  • the phagemid is administered by a local route or by a systemic route.
  • the composition comprising a nucleotide sequence, vector or phagemid of the disclosure is administered by a local route or by a systemic route.
  • the antibiotic composition is administered by a local route or by a systemic route.
  • the local route comprises topical administration.
  • the local route comprises administration directly to the cerebral spinal fluid (CSF).
  • the local route comprises an intraspinal, intrathecal or intracortical route.
  • the local route comprises administration directly to the eye.
  • the local route comprises an intraocular, intravitreous or subretinal route.
  • the local route comprises administration directly to the bone.
  • the local route comprises an intraosseus route.
  • the systemic route comprises an intravenous, a subcutaneous, an oral, an intranasal, an inhaled, an intramuscular or an intraperitoneal route.
  • the systemic route comprises an intravenous route.
  • the systemic route comprises an intravenous infusion over a period of time from between about one minute to about one hour.
  • the intravenous infusion occurs once. In certain embodiments of the methods of the disclosure, including methods of treating an infection in a subject in need thereof, the intravenous infusion occurs once per day, once per week or once per month.
  • the subject is a mammal.
  • the subject is a human.
  • the subject is a neonate, an infant, a child, a young adult, an adult or an elderly adult.
  • the subject is exposed to one or more bacteria.
  • the subject is at risk of infection or at risk of infecting another person.
  • the subject is immune-compromised.
  • the target bacteria prior to contacting a nucleotide sequence, a vector, a phagemid or a composition of the disclosure, is resistant to an antibiotic composition and, after contacting a nucleotide sequence, a vector, a phagemid or a composition of the disclosure the target bacteria is sensitive to an antibiotic composition.
  • the antibiotic composition comprises one or more of a penicillin, a tetracycline, a cephalosporin, a quinolone, a lincomycin, a macrolide, a sulfonamide, a glycopeptide, an aminoglycoside, and a carbapenem.
  • the penicillin comprises benzylpenicillin (penicillin G), penicillin V (phenoxymethylpenicillin), penicillin V potassium, amoxicillin,
  • the tetracycline comprises doxycycline
  • the cephalosporin comprises cefuroxime, ceftriaxone, or Cefdinir.
  • the quinolone comprises ciprofloxacin, levofloxacin or moxifloxacin.
  • the lincomycin comprises clindamycin or lincomycin.
  • the macrolide comprises azithromycin, clarithromycin or erythromycin.
  • the sulfonamide comprises sulfamethoxazole-trimethoprim, sulfasalazine or sulfisoxazole.
  • the glycopeptide comprises dalbavancin, oritavancin, telavancin or vancomycin. In certain embodiments, the
  • aminoglycoside comprises gentamicin, tobramycin or amikacin.
  • the carbapenem comprises imipenem/cilastatin, meropenem, doripenem or ertapenem.
  • the target bacteria prior to contacting a nucleotide sequence, a vector, a phagemid or a composition of the disclosure, is resistant to an antibiotic composition and, after contacting a nucleotide sequence, a vector, a phagemid or a composition of the disclosure the target bacteria is no longer viable or dies.
  • the bacteria or the nucleotide sequence further contacts a surface.
  • the bacteria or the vector further contacts a surface.
  • the bacteria or the phagemid furthers contact a surface.
  • the bacteria or the composition further contacts a surface.
  • a bacteria of the disclosure including those comprising a contacting step wherein the bacteria and one or more of a nucleotide sequence, vector, phagemid or composition of the disclosure further contacts a surface
  • the surface comprises a plastic, a composite, a metal, a glass, a ceramic, a textile, a wood, a mineral, a stone, a leather, a liquid or any combination thereof.
  • the surface comprises a coating and wherein the coating comprises a paint, a polyurethane, a wax, a laminate, a powder coatings, a metallic electroplating, a glass, a ceramic, a plastic, a rubber or any combination thereof.
  • the surface comprises the composition.
  • the composition covers at least a portion of the surface.
  • a bacteria of the disclosure including those comprising a contacting step wherein the bacteria and one or more of a nucleotide sequence, vector, phagemid or composition of the disclosure further contacts a surface, the surface is exposed to pathogenic bacteria.
  • the surface is located in a hospital, an operating room, an emergency room, a trauma center, a doctor's office, a pharmacy, a blood donation or infusion center, a dialysis center, a physical rehabilitation facility, a dentistry office, a nursing home, a residential care facility, an assisted living facility, a psychiatric office, a mental health facility, a school nursing office, a medical device store, a shopping center, a restaurant, a grocery store, a farm, a food processing or packaging facility, an industrial kitchen, a home, an office, a school, a university, a method of public transportation, an airport, a train station, a bus station, a gymnasium, an exercise facility, a swimming pool, a spa, a nail salon, a hair salon, a body piercing store, a tattoo parlor.
  • the disclosure provides a method of modulating a microbiome of a surface comprising contacting the surface and an effective amount of the nucleotide sequence of the disclosure, wherein the nucleotide selectively targets and removes at least one pathogenic bacteria from the microbiome of the surface.
  • the disclosure provides a method of modulating a microbiome of a surface comprising contacting the surface and an effective amount of the vector of the disclosure, wherein the vector selectively targets and removes at least one pathogenic bacteria from the microbiome of the surface.
  • the disclosure provides a method of modulating a microbiome of a surface comprising contacting the surface and an effective amount of the phagemid of the disclosure, wherein the phagemid selectively targets and removes at least one pathogenic bacteria from the microbiome of the surface.
  • the disclosure provides a method of modulating a microbiome of a surface comprising contacting the surface and an effective amount of the composition of the disclosure wherein the composition selectively targets and removes at least one pathogenic bacteria from the microbiome of the surface.
  • the at least one pathogenic bacteria is resistant to at least one antibiotic composition.
  • the method maintains a diverse population of non-pathogenic bacteria within the microbiome of the surface.
  • the diverse population of nonpathogenic bacteria does not contain a bacteria or strain of bacteria resistant to at least one antibiotic composition.
  • the contacting is systemic.
  • the systemic contact comprises an aerosolized delivery of the nucleotide sequence.
  • the systemic contact comprises an aerosolized delivery of the vector.
  • the systemic contact comprises an aerosolized delivery of the phagemid.
  • the systemic contact comprises an aerosolized delivery of the composition.
  • the aerosolized delivery diffuses through a volume of an entry way, a room, a hallway, a ventilation system, or an air supply of a building.
  • the contacting is local.
  • the local contact comprises a concentrated delivery of the nucleotide sequence.
  • a liquid, semi-solid, or solid formulation comprises the nucleotide sequence.
  • the local contact comprises a concentrated delivery of the vector.
  • a liquid, semi-solid, or solid formulation comprises the vector.
  • the local contact comprises a concentrated delivery of the phagemid.
  • a liquid, semisolid, or solid formulation comprises the phagemid.
  • the local contact comprises a concentrated delivery of the composition.
  • a liquid, semisolid, or solid formulation comprises the composition.
  • the local delivery encompasses a volume or a portion of a surface of an object.
  • the object has a volume of less than 10 cubic meters.
  • the object has a surface having an area of less than 10 square meters.
  • the object is a medical device or a portion thereof.
  • the object comprises a plastic tubing, a catheter, a needle, a central line, a scalpel, a surgical instrument or any combination thereof.
  • the liquid or semi-solid formulation comprises a viscosity or a volatility effective to form a coating of a surface.
  • the method restores or generates a microbiome on the surface comprising a diversity of bacterial strains equal to or greater than a diversity of bacterial strains found in the microbiome of a gut of a healthy human subject.
  • the restored or generated microbiome and the microbiome of the gut of a healthy human subject do not comprise a pathogenic bacteria or a pathogenic strain of bacteria.
  • the restored or generated microbiome and the microbiome of the gut of a healthy human subject comprise a pathogenic bacteria or a pathogenic strain of bacteria and wherein the method reduces or prevents the pathogenic bacteria or the pathogenic strain of bacteria from representing greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% ,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or any percentage in between of the total number of bacteria or bacterial strains in the microbiome.
  • the pathogenic bacteria or the pathogenic strain of bacteria are not resistant to at least one antibiotic composition.
  • the pathogenic bacteria or the pathogenic strain of bacteria are resistant to at least one antibiotic composition.
  • the effective amount of the nucleotide sequence inhibits at least one pathogenic bacteria from replicating, initiating horizontal gene transfer, and/or initiating or participating in quorum sensing.
  • the effective amount of the vector inhibits at least one pathogenic bacteria from replicating, initiating horizontal gene transfer, and/or initiating or participating in quorum sensing.
  • the effective amount of the phagemid inhibits at least one pathogenic bacteria from replicating, initiating horizontal gene transfer, and/or initiating or participating in quorum sensing.
  • the effective amount of the composition inhibits at least one pathogenic bacteria from replicating, initiating horizontal gene transfer, and/or initiating or participating in quorum sensing.
  • the method does not target, kill, or decrease the viability of a non-pathogenic bacteria or non-pathogenic bacterial strain of the microbiome of the surface. In some embodiments of the methods of modulating a microbiome of a surface of the disclosure, the method does not alter one or more of a diversity, a proportional representation or a spatial distribution of a non-pathogenic bacteria or a non-pathogenic bacterial strain of the microbiome of the surface.
  • the disclosure provides a method of selectively eliminating a target bacteria in a subject comprising administering to the subject an effective amount of a composition of the disclosure, wherein the subject is a livestock animal.
  • the livestock animal or a portion thereof enters a food supply chain.
  • the livestock is a cow, pig, goat, elk, deer, turkey, chicken, duck, pigeon, shrimp, bass, oyster, clam, mussel, salmon, or tilapia.
  • the composition further comprises animal feed.
  • the composition is administered simultaneously with animal feed.
  • the composition is administered sequentially with the animal feed.
  • the disclosure provides a method of selectively eliminating a target bacteria comprising contacting the target bacteria with an effective amount of a composition of the disclosure, wherein one or more of a raw food, a food supply, a food product or a food production surface comprises the target bacteria.
  • the raw food, the food supply, the food product comprises an animal tissue.
  • the animal tissue is isolated or derived from a chicken, fish, turkey, pork, beef, or a combination thereof.
  • the animal tissue comprises meat or a meat product and wherein the meat or the meat product comprises whole muscle meat, ground meat, soft tissue or a combination thereof.
  • the meat product is selected from deli turkey, ground beef, or a combination thereof.
  • the raw food, the food supply, the food product comprises a plant tissue.
  • the plant tissue is isolated or derived from one or more of a fruit, a vegetable, a seed, a grain, a grass, a bean, and a nut.
  • the fruit is an intact or fragmented apple, orange, banana, berry, lemon, or a combination thereof.
  • the food supply or the food product comprises wheat, rice, oats, barley, bread, pasta, or a combination thereof.
  • the food supply or the food product comprises spinach, carrots, onions, peppers, avocado, broccoli, or a combination thereof.
  • the food supply or the food product comprises guacamole.
  • the raw food, the food supply, the food product comprises a dairy product.
  • the dairy product comprises milk, butter, yogurt, cheese, ice cream, queso fresco, or a combinations thereof.
  • the food production surface directly or indirectly contacts a raw food, a food supply, or a food product.
  • the food production surface directly contacts the raw food, the food supply, or the food product and wherein the direct contact comprises physical contact of the surface and the raw food, the food supply, or the food product and communication of the surface and the raw food, the food supply, or the food product through air or fluid.
  • the food production surface indirectly contacts the raw food, the food supply, or the food product and wherein the indirect contact comprises an agent communicating between the surface and the raw food, the food supply, or the food product.
  • the agent is a subject or a second surface that simultaneously or sequentially contacts the food production surface and the raw food, the food supply, or the food product.
  • the contacting step occurs in a food production facility.
  • the food production facility is a farm, a boat, a food distribution facility, a food processing plant, a food retail location, a home, or a restaurant.
  • the contacting occurs on or within a machine or equipment within the food production facility.
  • the target bacteria is Escherichia coli, Escherichia coli 0157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Infantis, Salmonella Heidelberg,
  • the target bacteria is Escherichia coli. In some embodiments, the target bacteria is Escherichia coli 0157:H7. In some embodiments, the target bacteria is Salmonella Enteritidis. In some embodiments, the target bacteria is Salmonella Typhimurium. In some embodiments, the target bacteria is Campylobacter jejuni.
  • the target bacteria is Staphylococcus aureus. In some embodiments, the target bacteria is Listeria monocytogenes. In some embodiments, the target bacteria is Pseudomonas aeuriginosa. In some embodiments, the target bacteria is Klebisella pneumonia.
  • the target bacteria is a pathogenic bacteria.
  • Figure 1A is a schematic diagram depicting an exemplary vector comprising an exemplary nucleotide sequence of the disclosure (left) and a corresponding target bacterial cell (right).
  • the vector is a phagemid.
  • Figure IB is a schematic diagram depicting an exemplary vector comprising an exemplary nucleotide sequence of the disclosure (left) and a corresponding target bacterial cell (right).
  • the vector is a phagemid.
  • the phagemid shown in this figure comprises a protein having a predetermined three-dimensional structure that improves the targeting or polarity of the phagemid for a target bacterial cell (on the right).
  • the phagemid may be designed to match a polarity or to selectively or specifically bind one species or one strain of bacteria within a mixed bacterial population.
  • the specificity of the vector or phagemid for the target bacteria is depicted here as each of the vector or phagemid and the bacteria having matching boundaries.
  • Figure 1C is a schematic diagram depicting a further embodiment of the phagemid and bacteria shown in Figure IB.
  • Figure ID is a schematic diagram depicting a further embodiment of the phagemid and bacteria shown in Figure IB.
  • Figure IE is a schematic diagram depicting a further embodiment of the phagemid and bacteria shown in Figure IB.
  • Figure IF is a schematic diagram depicting a further embodiment of the phagemid and bacteria shown in Figure IB.
  • Figure 2A is a schematic diagram depicting an exemplary vector comprising an exemplary nucleotide sequence of the disclosure (left) and a corresponding target bacterial cell (right).
  • the vector is a bacteriophage.
  • Figure 2B is a schematic diagram depicting an exemplary nucleotide sequence of the disclosure (for example, as shown in Figure 2A), comprising a sequence encoding a DNA binding domain, wherein the DNA binding domain comprises between one and ten guide RNAs (gRNAs) that target a sequence of a bacterial genome or a bacterial plasmid and a sequence encoding a nuclease or nuclease domain, wherein the nuclease comprises a Cpfl polypeptide or an ortholog thereof.
  • the gRNA depicted herein may each specifically bind to a distinct sequence of a bacterial genome or a bacterial plasmid.
  • Figure 2C is a schematic diagram depicting contact between a bacteriophage comprising a nucleotide sequence of the disclosure, wherein the bacteriophage inserts the nucleotide sequence into the bacteria to be expressed, and wherein the expressed Type V CRISPR construct comprising at least one gRNA and a Cpfl nuclease or ortholog thereof scans the bacteria's genomic and plasmid DNA, making double-stranded breaks at sites of complementarity between the at least one gRNA and a target sequence.
  • Preferred target sequences reduce or eliminate the bacterial cell's resistance to an antibacterial composition or the bacterial cell's viability, ultimately leading to cell death.
  • Figure 2D is a schematic diagram depicting contacting a plurality of bacteria and a plurality of bacteriophage (e.g. as shown in Figures 2A, 2B, and 2C), the result of which is the selective elimination of bacteria targeted by the bacteriophage.
  • Figure 2E is a schematic diagram depicting contacting a plurality of bacteria, a plurality of bacteriophage (e.g. as shown in Figures 2A, 2B, and 2C), and an antibacterial composition, the result of which is the synergistic selective elimination of bacteria targeted by the bacteriophage.
  • the plurality of bacteriophage e.g. as shown in Figures 2A, 2B, and 2C
  • Figure 3 is a photograph showing antibiotic resistant bacteria plated on media in the presence of control (left panel), a composition comprising at least one antibiotic to which the bacteria have acquired resistance (middle panel), or the combination of a composition comprising at least one antibiotic to which the bacteria have acquired resistance and a composition comprising a nucleotide sequence of the present disclosure that induces at least one break in a sequence of the genome of the bacteria present on the plates shown in the right-hand panel.
  • Figure 4 is a schematic outlining the bioinformatic platform for identifying
  • Figure 5 is a map of the plasmid encoding a Cpfl phagemid and an antibiotic resistance gene targeting gRNA.
  • Figure 6 is a graph showing viability of antibiotic resistant E. coli in the presence of control, buffer, a CRISPR nuclease of the present disclosure, and a composition comprising a nucleotide sequence of the present disclosure comprising a CRISPR nuclease and a guide RNA (gRNA) targeting GES gene that induces at least one break in a sequence of the genome of the bacteria.
  • gRNA guide RNA
  • Figure 7 is a graph showing viability of antibiotic resistant E. coli in the presence of control, buffer, a CRISPR nuclease of the present disclosure, and a composition comprising a nucleotide sequence of the present disclosure comprising a CRISPR nuclease and a guide RNA (gRNA) targeting NDM gene that induces at least one break in a sequence of the genome of the bacteria.
  • gRNA guide RNA
  • Figure 8A is a graph showing the viability of HEK cells in the presence of bacteriophages of the disclosure after 48 and 72 hrs of exposure. Relative cell number is presented on the Y-axis and bacteriophage concentration is presented on the X-axis.
  • Figure 8B is a graph showing the viability of HEKa cells in the presence of SDS after 48 and 72 hrs of exposure. Relative cell number is presented on the Y-axis and SDS concentration is presented on the X-axis.
  • antibiotic resistance has been identified as one of the greatest global health challenges, currently 2 million people are diagnosed with antibiotic resistance every year, furthermore the WHO estimates that by 2050 10 million people will die from antibiotic resistant infections.
  • Antibiotic resistance is caused by multiple factors, including but not limited to, meat consumption, exposure to bacteria in the environment and in food, among others. Having the ability to provide targeted and preventive care is extremely important, bacteria are quickly evolving and superbugs are becoming more prominent, aggressive action against this global health threat is necessary.
  • compositions and methods of the disclosure modify genomic and plasmid sequences of target bacteria to sensitize the target bacteria to antibiotic drugs. Alternatively, or in addition, the compositions and methods of the disclosure modify genomic and plasmid sequences of target bacteria to reduce or elimination the bacteria's viability, ultimately leading to the death of targeted bacterial cells, species and/or strains.
  • DNA binding domains of the disclosure may comprise or consist of one or more of a DNA, RNA or protein sequence.
  • DNA binding domains of the disclosure may comprise or consist of a single-stranded or a double-stranded nucleic acid sequence.
  • the DNA binding domain comprises an RNA sequence.
  • the RNA sequence comprises a guide RNA (gRNA).
  • gRNA guide RNA
  • the RNA sequence is single-stranded.
  • the single-stranded RNA comprises a guide RNA (sgRNA).
  • DNA binding domains of the disclosure may comprise or consist of one or more gRNA sequences. Each gRNA sequence may target a distinct sequence of a bacterial genome or bacterial plasmid.
  • DNA binding domains of the disclosure do not comprise a nuclear localization signal (NLS).
  • NLS nuclear localization signal
  • DNA binding domains of the disclosure comprise non-naturally occurring sequences.
  • the non-naturally occurring sequences may be recombinant or chimeric.
  • Guide RNAs of the disclosure comprise a target-specific sequence.
  • the target-specific sequence may comprise a spacer sequence, a protospacer sequence and/or a crispr RNA (crRNA) sequence.
  • the gRNA may further comprise a trans -activating crRNA (tracrRNA) sequence.
  • the guide RNA sequence comprises at least 15, 16, 17, 18, 19, 20, 25 nucleotides. In certain embodiments, the guide RNA sequence comprises between 10 and 30 nucleotides, inclusive of the endpoints. In certain embodiments, the guide RNA sequence comprises between 15 and 25, inclusive of the endpoints. In certain embodiments, the guide RNA sequence comprises between 15-20 nucleotides, inclusive of the endpoints.
  • the spacer sequence, the protospacer sequence and/or the crRNA sequence comprises at least 15, 16, 17, 18, 19, 20, 25 nucleotides. In certain embodiments, the spacer sequence, the protospacer sequence and/or the crRNA sequence comprises between 10 and 30 nucleotides, inclusive of the endpoints. In certain
  • the spacer sequence, the protospacer sequence and/or the crRNA sequence comprises between 15 and 25, inclusive of the endpoints. In certain embodiments, the spacer sequence, the protospacer sequence and/or the crRNA sequence comprises between 15-20 nucleotides, inclusive of the endpoints.
  • the tracrRNA sequence comprises at least 15, 16, 17, 18, 19, 20, 25 nucleotides. In certain embodiments, the tracrRNA sequence comprises between 10 and 30 nucleotides, inclusive of the endpoints. In certain embodiments, the tracrRNA sequence comprises between 15 and 25, inclusive of the endpoints. In certain embodiments, the tracrRNA sequence comprises between 15-20 nucleotides, inclusive of the endpoints.
  • the gRNA has no more than 1, 2, 5, 7, 10, 15, 20, or any number in between of off-target sequences.
  • the off-target sequences of the gRNA contain at least 1 , 2, 3, 4, or 5 mismatches that decrease binding efficacy or prevent off-target binding.
  • the off-target sequences of the gRNA are located in an area of a bacterial genome or a bacterial plasmid with minimal or no negative side effects.
  • the break at the off-target site does not interfere with the efficacy of the composition and methods of the disclosure.
  • Exemplary gRNAs of the disclosure contain a target-specific sequence that targets an antibiotic resistance gene.
  • Exemplary resistance genes include beta-lactamases, specifically the TEM family of beta-lactamases.
  • Exemplary gRNAs of the disclosure contain a target-specific sequence that targets an gene essential to the survival of a bacterial species. Exemplary gene sequences that can be targeted by a gRNA of the disclosure are listed in Table 4.
  • 931 ispH 5639 icdA 10350 - 15061 - 932 VC0686 5640 trmU/mnmA 10351 - 15062 -

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Abstract

L'invention concerne des compositions comprenant une séquence de nucléotides, un vecteur ou un phagémide comprenant (a) une séquence codant pour un domaine de liaison à l'ADN et (b) une séquence codant pour une nucléase ou un domaine nucléasique, où le domaine de liaison à l'ADN cible spécifiquement une séquence d'un génome bactérien ou une séquence d'un plasmide bactérien et où la nucléase ou le domaine nucléasique induit au moins une rupture dans la séquence du génome bactérien. Dans certains modes de réalisation, les compositions comprennent une construction CRISPR de type V. Des procédés de préparation et d'utilisation desdites compositions sont en outre décrits.
PCT/US2018/052962 2017-09-26 2018-09-26 Compositions et procédés d'un système génétique crispr pour sensibiliser et/ou éliminer des bactéries cibles Ceased WO2019067621A1 (fr)

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US12279989B2 (en) 2011-02-04 2025-04-22 Seed Health, Inc. Method and system for increasing beneficial bacteria and decreasing pathogenic bacteria in the oral cavity
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US12533312B2 (en) 2011-02-04 2026-01-27 Seed Health, Inc. Method and system for preventing sore throat in humans
US11951140B2 (en) 2011-02-04 2024-04-09 Seed Health, Inc. Modulation of an individual's gut microbiome to address osteoporosis and bone disease
US11998479B2 (en) 2011-02-04 2024-06-04 Seed Health, Inc. Method and system for addressing adverse effects on the oral microbiome and restoring gingival health caused by sodium lauryl sulphate exposure
US11844720B2 (en) 2011-02-04 2023-12-19 Seed Health, Inc. Method and system to reduce the likelihood of dental caries and halitosis
US12318411B2 (en) 2013-12-20 2025-06-03 Seed Health, Inc. Probiotic method and composition for maintaining a healthy vaginal microbiome
US12318414B2 (en) 2013-12-20 2025-06-03 Seed Health, Inc. Method and system to modify an individual's gut-brain axis to provide neurocognitive protection
US12246043B2 (en) 2013-12-20 2025-03-11 Seed Health, Inc. Topical application to treat acne vulgaris
US11826388B2 (en) 2013-12-20 2023-11-28 Seed Health, Inc. Topical application of Lactobacillus crispatus to ameliorate barrier damage and inflammation
US12005085B2 (en) 2013-12-20 2024-06-11 Seed Health, Inc. Probiotic method and composition for maintaining a healthy vaginal microbiome
US11833177B2 (en) 2013-12-20 2023-12-05 Seed Health, Inc. Probiotic to enhance an individual's skin microbiome
US11839632B2 (en) 2013-12-20 2023-12-12 Seed Health, Inc. Topical application of CRISPR-modified bacteria to treat acne vulgaris
US11980643B2 (en) 2013-12-20 2024-05-14 Seed Health, Inc. Method and system to modify an individual's gut-brain axis to provide neurocognitive protection
US11998574B2 (en) 2013-12-20 2024-06-04 Seed Health, Inc. Method and system for modulating an individual's skin microbiome
US12329783B2 (en) 2013-12-20 2025-06-17 Seed Health, Inc. Method and system to improve the health of a person's skin microbiome
US12357662B2 (en) 2013-12-20 2025-07-15 Seed Health, Inc. Modulation of an individual's gut microbiome to address osteoporosis and bone disease
US11969445B2 (en) 2013-12-20 2024-04-30 Seed Health, Inc. Probiotic composition and method for controlling excess weight, obesity, NAFLD and NASH
US11951139B2 (en) 2015-11-30 2024-04-09 Seed Health, Inc. Method and system for reducing the likelihood of osteoporosis
US12257272B2 (en) 2015-12-24 2025-03-25 Seed Health, Inc. Method and system for reducing the likelihood of developing depression in an individual
CN108998406A (zh) * 2018-08-03 2018-12-14 福州大学 一种人类原代培养细胞基因组编辑、定点基因敲入方法
EP4090678A4 (fr) * 2020-01-10 2024-01-10 Intron Biotechnology, Inc. Procédé de préparation de myoglobine porcine à l'aide d' escherichia coli
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CN113073099A (zh) * 2021-03-19 2021-07-06 深圳市第三人民医院 sgRNA库、敲低基因文库及敲低基因文库的构建方法和应用
CN113416739A (zh) * 2021-06-24 2021-09-21 黑龙江八一农垦大学 鲁氏酵母菌基因在提高微生物产hdmf的产量中的应用
EP4384620A4 (fr) * 2021-08-13 2025-09-17 1E Therapeutics Ltd Dnazymes ciblant des enzymes de synthèse de paroi cellulaire et leurs utilisations
CN114921463B (zh) * 2022-04-11 2023-12-01 中国农业科学院生物技术研究所 一种调控萜类化合物合成产量的人工非编码RNA分子CsiY及其应用
CN114921463A (zh) * 2022-04-11 2022-08-19 中国农业科学院生物技术研究所 一种调控萜类化合物合成产量的人工非编码RNA分子CsiY及其应用
WO2023205267A1 (fr) * 2022-04-20 2023-10-26 The Administrators Of The Tulane Educational Fund Synthèse de bactériophage acellulaire améliorée par modulation génétique de machinerie de transcription/traduction (txtl) bactérienne
CN116478953B (zh) * 2023-06-14 2023-09-12 四川大学华西医院 鲍曼不动杆菌DlaT重组蛋白、制备方法及应用
CN116478953A (zh) * 2023-06-14 2023-07-25 四川大学华西医院 鲍曼不动杆菌DlaT重组蛋白、制备方法及应用

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