WO2021126344A2 - Compositions and methods comprising engineered bacteriophage - Google Patents

Compositions and methods comprising engineered bacteriophage Download PDF

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WO2021126344A2
WO2021126344A2 PCT/US2020/053933 US2020053933W WO2021126344A2 WO 2021126344 A2 WO2021126344 A2 WO 2021126344A2 US 2020053933 W US2020053933 W US 2020053933W WO 2021126344 A2 WO2021126344 A2 WO 2021126344A2
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bacteriophage
phage
engineered
seq
nucleic acid
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WO2021126344A3 (en
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Bryan Boen HSU
Isaac Nathan PLANT
Jeffrey Charles Way
Pamela A. Silver
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President And Fellows Of Harvard College
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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 RNAses, DNAses
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10321—Viruses as such, e.g. new isolates, mutants or their genomic sequences

Definitions

  • compositions and methods comprising engineered bacteriophage.
  • the human gut microbiota is a collection of microbes colonizing the gastrointestinal tract and has been associated with various aspects of human health. While this community typically works concert with the human body, substantial perturbations such as antibiotics or infections can disrupt the microbial balance and lead to long lasting dysbiosis. In some instances, pathogenic bacteria do so by transmitting virulence factors encoded by these pathogens to commensal bacteria through plasmid- based and phage-based horizontal gene transfer (HOT). Remediating diseases associated with these pathogens while minimizing unintended and disruptive effects to the surrounding microbiota remains challenging especially with the limited tools available for targeting particular species.
  • HAT horizontal gene transfer
  • An alternative strategy is to modify bacterial function within its native environment.
  • one approach has been to develop drugs that target the virulence factors of anti-biotic resistant pathogens to specifically neutralize their deleterious effects and minimizing selection for resistance.
  • the targets for inhibition are generally limited to those accessible by small molecules and biologies (i.e., surface-bound and secreted proteins), may require multiple drugs targeting multiple virulence factors, and could have off target effects on other microbes and the host (see e.g., Dickey et al. Nature Reviews Drag Discover ' 16, 457 (2017)). While the principle of anti -virulence is encompasse, it remains challenging application.
  • Shigatoxin (Stx)-producing E. coli is one example of a pathogenic infection that is challenging to treat.
  • Anti-virulence drugs targeting the toxin have been investigated but failed clinical trial (see e.g., Trachtman et al, JAMA 290, 1337-1344 (2003)).
  • Antibiotics are contraindicated because of their potential to exacerbate virulence.
  • Stx is significantly associated with disease severity and can lead to hemolytic uremic syndrome.
  • Stxl and Stx2 the latter is -1000-fold more toxic.
  • Stx is not expressed while the phage is in a lysogenic state, i.e. stably integrated into the bacterial genome. It is not until induction, whether occurring spontaneously or from stimuli such as antibiotics, that the lytic life cycle is activated to produce Stx2 and progeny phage that can spread virulence genes to commensal E. coli species.
  • compositions and methods comprising engineered bacteriophage as alternative to an antimicrobial strategy for killing pathogens.
  • Described herein is a genetic-based anti-virulence strategy that can neutralize virulence before expression and minimize resistance until, e.g., the bacteria have been completely shed from the gastrointestinal tract.
  • Temperate phages offer a solution as they are genetically engineerable and can integrate into the bacterial chromosome as prophages for long-lasting effect as they confer fitness advantages to the bacterial host. Instead of relying on a non-native constituent of the gut that could face practical barriers for efficacy, temperate phages are abundantly found in human gut bacteria and can constitute large portions of the bacterial chromosome.
  • bacteriophage wherein the bacteriophage genome is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium.
  • the bacteriophage is an engineered lambda (l) phage.
  • the inhibitor comprises an inhibitor protein encoded by a phage endogenous to the bacterium.
  • the inhibitor comprises cl protein from enterobacteria phage 933 W.
  • the inhibitor comprises one of SEQ ID NOs: 9-10 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 9-10 that maintains the same function.
  • the inhibitor is engineered to be non- degradable.
  • the cl protein from enterobacteria phage 933W comprises a K178N mutation that causes the protein to be non-degradable.
  • the inhibitor comprises a Cas protein and at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor.
  • the Cas protein comprises S. aureus Cas9.
  • the Cas protein comprises SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 that maintains the same function.
  • the CRISPR guide RNA comprises a trans activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA).
  • tracrRNA trans activating CRISPR RNA
  • crRNA CRISPR RNA
  • the tracrRNA comprises SEQ ID NO: 25 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 25 that maintains the same function.
  • the crRNA comprises a variable targeting sequence and a region that is substantially complementary to a region of the tracrRNA.
  • variable targeting sequence of the crRNA is substantially complementary to a nucleic acid encoding a bacterial virulence factor.
  • the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 29 that maintains the same function.
  • the bacterium is Escherichia coli ( E . coli).
  • the bacterium is enterohemorrhagic E. coli
  • the virulence factor is Shiga Toxin (Stx).
  • the engineered bacteriophage genome further comprises a heterologous bacteriophage immunity region.
  • the heterologous bacteriophage immunity region is a lambdoid phage immunity region.
  • the lambdoid phage is selected from the group consisting of lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22.
  • the heterologous bacteriophage immunity region comprises one of SEQ ID NOs: 13-17 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 13-17 that maintains the same function.
  • the heterologous bacteriophage immunity region comprises SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 that maintains the same function.
  • the engineered bacteriophage genome further comprises a nucleic acid encoding a selectable marker.
  • bacteriophage wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • bacteriophage wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and (b) a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • bacteriophage wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • bacteriophage wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, and (b) a heterologous bacteriophage immunity region wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • a bacteriophage wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and (b) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • a bacteriophage wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, (b) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, and (c) a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • composition comprising the engineered bacteriophage as described herein and an acceptable carrier.
  • a method of treating a bacterial infection comprising administering an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein to a patient in need thereof.
  • the patient is infected with E. coli or EHEC.
  • a method of inhibiting bacterial growth or activity on a surface comprising contacting a surface with an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein.
  • compositions for use in a method of treating a bacterial infection comprising administering an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein to a patient in need thereof.
  • the patient is infected with E. coli or EHEC.
  • compositions for use in a method of inhibiting bacterial growth or activity on a surface comprising contacting a surface with an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein.
  • Fig. 1A-1E is a series of schematics and graphs showing that temperate phage robustly transduces E. coli colonizing the mouse gut.
  • the upper panel of Fig. 1A is a schematic showing that lytic phages decimate a bacterial population, selecting for resistant mutants that can repopulate over time i) In lytic phage infection, the phage particle injects its genetic material into a bacterium and ii) directs the cell to produce phage components, iii) which are released upon cell lysis to continue infection.
  • FIG. 1A is a schematic showing that temperate phages can infect a bacterial population to lysogenize phage-susceptible bacteria which persists overtime iv) Temperate phages can also participate in the lysogenic life cycle where they integrate their DNA into the bacterial genome and v) remain as a prophage during bacterial proliferation with the possibility of entering lytic replication in the future.
  • Fig. IB is a schematic showing an experimental timeline examining the impact of lBHI phage on pre-colonized E. coli in mice.
  • Fig. 1C is a dot plot showing fecal concentrations of free lBHI phage.
  • Fig. ID is a dot plot showing fecal concentrations of E. coli.
  • Fig. 2A-2D is a series of schematics and graphs showing that hybrid l phages overcome superinfection exclusion by prophage 933W.
  • Fig. 2A is a schematic showing a depiction of superinfection exclusion by the 933W prophage that inhibits infection by l phage but is ineffective against a hybrid phage that contains the immunity region from another lambdoid phage in a l phage background.
  • FIG. 2B shows a schematic representation of a portion of the l phage genome containing the l immunity region and its hybrids containing immunity regions from other lambdoid phages (e.g., 933W, 21, 434, and P22) in a l phage background.
  • Efficiency of plating (EOP) for l phage and its hybrids on E. coli 933W are shown in the right panel of Fig. 2B.
  • Fig. 2C is a schematic showing genetic schemes of E.
  • coli 933W showing: i) the 933W prophage expressing cl to maintain a lysogenic state in which stx2 is not expressed, ii) induction that causes degradation of the Cl protein leading to expression of the lytic genes including cro and stx2. This leads to cell lysis, releasing phage progeny and Stx2 protein iii) Expression of a non-degradable cl for the 933W prophage, 933W-cT nd ⁇ , from a genomically -integrated engineered temperate phage (anti -virulence prophage) can enforce the 933 W prophage to remain lysogenic despite induction and degradation of endogenous Cl protein.
  • FIG. 2D shows a schematic representation of lBHI phage, which is a l phage with a kanamycin resistance cassette ( KanR ) and 933W-c nd ⁇ inserted into the non-essential b2 region of the phage genome.
  • lBH2 phage is a product of a phage cross between lBHI and ' / .immPlldis resulting in a phage containing KanR and 933W-cF nd ⁇ genes with a P22 immunity region in a l phage background.
  • Their respective EOP against E. coli 933W are shown in the right panel of Fig. 2D. Symbols represent biological replicates with bars representing the geometric mean (e.g., in Fig. 2B, Fig. 2D.
  • Fig. 3A-3H is a series of schematics and graphs showing that engineered l phage neutralizes shigatoxin production from E. coli 933W in vitro.
  • Fig. 3B is a bar graph showing the concentration of Stx2 measured after 8 hours (h) of in vitro culture under non-induced conditions.
  • Fig. 3A-3H is a series of schematics and graphs showing that engineered l phage neutralizes shigatoxin production from E. coli 933W in vitro.
  • Fig. 3A is a schematic showing that E. coli 933 w was mixed with buffer, / «? «?P22dis or lBH2 free
  • FIG. 3C is a bar graph showing the concentration of Stx2 measured after 8 h of in vitro culture under induced conditions (e.g., 0.5 pg/mL of mitomycin c). Significance was calculated by one-way ANOVA with post-hoc Tukey test (e.g., in Fig. 3B-3C).
  • Fig. 3D is a bar graph showing total E. coli 933W measured over 8 h under non-induced and mitomycin c induced conditions.
  • Fig. 3E is a bar graph showing the percentage of bacteria lysogenized by lBH2 measured over 8 h under non-induced and mitomycin c induced conditions.
  • Fig. 3F is a schematic showing that E.
  • coli 933W lysogenized with lBHI or lBH2 was cultured in vitro and analyzed for Stx2 produced in Fig. 3G and Fig. 3H.
  • Fig. 3G is a bar graph showing Stx2 production under non-induced conditions.
  • Fig. 3H is a bar graph showing Stx2 production under induced conditions. Symbols represent biological replicates with bars or lines representing the geometric mean (e.g., in Fig. 3B-3E, Fig. 3G-3H).
  • Fig. 4A-4F is a series of schematics and graphs showing that lBH2 phage lysogenizes E. coli 933W in the murine gut and reduces fecal shigatoxin concentrations.
  • Fig. 4A is a schematic showing that streptomycin-treated mice pre-colonized with E. coli 933W received one dose of 5 x 10 9 pfu of lBH2 phage orally. Mitomycin c was administered thrice at 3 h intervals by intraperitoneal injection to induce Stx2 expression in the gut.
  • Fig. 4B is a bar graph showing concentrations of fecal Stx2 after induction with mitomycin c.
  • Fig. 4C is a bar graph showing concentrations of total fecal E. coli 933W
  • Fig. 4D is a dot plot showing the percentage of fecal E. coli 933W found to be lysogenized by lBH2 phage
  • Fig. 4E is a dot plot showing the concentration of fecal Stx2 as a function of fraction of fecal E. coli 933W lysogenic for lBH2 phage on day 3.
  • Fig. 4C is a bar graph showing concentrations of total fecal E. coli 933W
  • Fig. 4D is a dot plot showing the percentage of fecal E. coli 933W found to be lysogenized by lBH2 phage
  • Fig. 4E is a dot plot showing the concentration of fecal Stx2 as a function of fraction of fecal E. coli 933W lysogenic for lBH2 phage on
  • 4F is a dot plot showing the concentration of fecal Stx2 as a function of fraction of fecal E. coli 933W lysogenic for lBH2 phage on day 4.
  • Line and dashed lines represent mean and 95% confidence intervals of linear regression, respectively (e.g., in Fig. 4E-4F).
  • P value describes significance of slope being non-zero (e.g., in Fig. 4E-4F).
  • On Day 4, one buffer- treated mouse was unable to produce stool for analysis (e.g., in Fig. 4B-4C).
  • Bars or lines represent geometric means (e.g., in Fig. 4B-4D).
  • Fig. 5 is an image showing spot testing of l and Hmm933W phage against lawns of their lysogens in E. coli.
  • the zone of lysis (dark circle) on a lawn of E. coli indicates successful phage infection.
  • Both phages are capable of infecting non-lysogens and lysogens of the other phage (i.e. l phage infecting a imm933W lysogen and vise-versa) indicating the 933W cI and l-cl are not inhibitory to l phage and 933W phage infection, respectively.
  • Fig. 6A-6C is a series of schematics showing genetic map of immunity regions within l phage and its hybrids.
  • Fig. 6A is a schematic showing a genetic map of portions of the l phage genome with brackets indicating the regions within l that have been replaced with immunity regions from other lambdoid phages.
  • Fig. 6A-6C is a series of schematics showing genetic map of immunity regions within l phage and its hybrids.
  • Fig. 6A is a schematic showing a genetic map of portions of the l phage genome with brackets indicating the regions within l that have been replaced with immunity regions from other lambdoid phages.
  • Fig. 6A-6C is a series of schematics showing genetic map of immunity regions within l phage and its hybrids.
  • Fig. 6A is a schematic showing a genetic map of portions of the l phage genome with brackets indicating the regions within l that have been replaced with immunity regions from other lambdoid phage
  • 6B is a schematic; within Hmni933W phage, the genes homologous to 933W (see e.g., NCBI Accession ID NC.000924) are labeled in grey in or above the genetic map (e.g., cIII, ssB, L0079, N, L0081, stk, L0083, L0084, cl, cro, ell, O, P) and those homologous to l (see e.g., SEQ ID NO: 32 or NCBI Accession ID NC.001416.1) are labeled in black below the genetic map (e.g., kil, cIII, ealO, ren). Percent identities for genes less then identical are indicated in parenthesis.
  • NCBI Accession ID NC.000924 the genes homologous to 933W (see e.g., NCBI Accession ID NC.000924) are labeled in grey in or above the genetic map (e.g., cIII, ss
  • Fig. 6C is a schematic; within l m/i? P22dis phage, the genes homologous to P22 (see e.g., GenBank Accession ID AF217253) are labeled in grey in or above the genetic map (e.g., orf59, mnt, arc, ant, 9, orf485, gtrB, gtrA, abc2, abcl, erf, arf, kil, cIII, 17, orf67, orf78, ral, sieB, 24, c2, cro, cl, orf48, 18, 12) and those homologous to l (see e.g., NCBI Accession ID NC.001416.1) are labeled in black in or below the genetic map (e.g., J, ninB).
  • the genes homologous to P22 see e.g., GenBank Accession ID AF217253
  • the genes homologous to P22 are labeled in grey in or
  • Fig. 7 is a series of schematics showing genetic maps of lBHI and lBH2 phage.
  • Fig. 8 is a series of pictures showing spot assays of 3 pL of ⁇ 10 7 pfu/mL of l, imm933W, imm434, and 7/mmP22dis phages against non-lysogenic E. coli, its l, lBHI, or lBH2 lysogen.
  • Fig. 9 is a series of line graphs showing Shigatoxin producing E. coli mixed with various phages (e.g., WT phage, engineered phage) in liquid culture with shigatoxin measured overtime.
  • various phages e.g., WT phage, engineered phage
  • lambda temperate
  • lambda-immP22dis temperate
  • the lytic T4 phage shows an initial suppression of shigatoxin production, but the expansion of T4-resistant E. coli leads to the eventual increase in shigatoxin production.
  • Fig. 10 is a series of dot plots showing in vivo testing by colonizing mice with shigatoxin- producing E. coli, then administering no phage, vl or v2. Comparison of the shigatoxin production in stool between phage treatments (left panel) shows that the v2 engineered phage successfully represses shigatoxin in the gut. It was confirmed that a large fraction of the shigatoxin-producing bacteria harbor the v2 engineered phage using a selection marker (right panel).
  • Fig. 11 is a series of images showing lambda phage that were engineered to include a deactivated Cas9 (dCas9) that binds to targeted regions using a CRISPR RNA guide sequence.
  • dCas9 deactivated Cas9
  • Fig. 12 is a plot showing flow cytometry data for mCherry fluorescence in E. coli.
  • E. coli was mixed with buffer or engineered dCas9 phage with and without crRNA for 1 day and analyzed by flow cytometry to quantitate intracellular mCherry fluorescence.
  • E. coli treated with ::dCas9-crRNA showed similar fluorescence levels to those receiving buffer whereas E. coli treated with ::dCas9+crRNA.
  • engineered phage comprising crRNA targeting the mCherry gene for repression showed a marked shift in fluorescence towards that of the E. coli control that lacks mCherry.
  • Fig. 13A-13E is a series of schematics and graphs showing in vitro gene repression by /.::dCas9 rlp
  • Fig. 13A is a scheme of in vitro experiment examining repression by engineered l phage.
  • Fig. 13B-13C is a series of graphs showing E. coli cultures mixed with phage buffer, l::dCas9 phage or l::dCas9 Ifp phage tracked for RFP fluorescence (Fig. 13B) and bacterial density (Fig. 13C).
  • Fig. 13A-13E is a series of schematics and graphs showing in vitro gene repression by /.::dCas9 rlp
  • Fig. 13A is a scheme of in vitro experiment examining repression by engineered l phage.
  • Fig. 13B-13C is a series of graphs showing E. coli cultures mixed with phage buffer, l::
  • 13D-13E are a series of graphs showing non-lysogenic, l: :dCas9 lysogenic or /.::dCas9 rlp lysogenic E. coli cultures tracked for RFP fluorescence (Fig. 13D) and bacterial density (Fig. 13E). Lines represent means and shaded regions represent standard errors.
  • Fig. 14A-14F is a series of schematics and graphs showing phage delivered genetic repression in vivo.
  • Fig. 14A is a schematic showing that engineered phage or vehicle was orally administered in bicarbonate solution to mice pre-colonized with E. coli expressing RFP and GFP.
  • Fig. 14B-14D is a series of graphs showing that after oral administration the following measured were quantified: fecal phage (Fig. 14B); total E. coli (Fig. 14C); and percentage of E. coli lysogenized by phage (Fig. 14D).
  • Fig. 14E is a series of representative fluorescence images of colonies of fecal lysogens shown compared to in vitro cultured controls.
  • Fig. 15A-15E is a series of schematics and graphs showing that encapsulation protects oral phage but does not impair function.
  • Fig. 15A is a schematic showing that free l::dCas9 Gfp phage in bicarbonate buffer (to ensure survival) or encapsulated l::dCas9 Ifp phage in water were orally administered to mice pre-colonized with E. coli expressing RFP and GFP.
  • Fig. 15B-15D is a series of graphs showing that after oral administration the following measured were quantified: fecal phage (Fig. 15B); total E. coli (Fig. 15C); and percentage of E. coli lysogenized by phage (Fig.
  • Fig. 15E shows an ensemble view of relative fluorescence from lysogens with in vitro cultured controls. Symbols represent individual colonies ( ⁇ 50 colonies per mouse sample or culture) with lines indicating the median.
  • Fig. 16A-16C is a series of schematics and graphs showing plasmid based in vitro gene repression.
  • Fig. 15A is a schematic showing E. coli transformed with ate inducible expression of dCas9 with and without crRNA targeting rfp.
  • Fig. 16B is a graph showing relative fluorescence of E. coli over time after induction with 25 ng ate.
  • Fig. 16C is a graph showing relative fluorescence as a function of ate concentration after 6 h incubation.
  • compositions and methods comprising engineered bacteriophage as alternative to an antimicrobial strategy for killing pathogens.
  • bacteriophage refers to a virus that infects and replicates within bacteria (e.g., prokaryotes) and/or archaea. In some embodiments of any of the aspects, the bacteriophage does not infect and replicate within eukaryotes. In some embodiments of any of the aspects, the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium.
  • lysogenic refers to a specific viral life cycle characterized by integration of the bacteriophage nucleic acid into the host bacterium's genome or formation of a circular replicon in the bacterial cytoplasm; lysogenic phage can also be referred to herein as temperate phage.
  • the engineered bacteriophage is not lytic, which is to say that it does not kill or lyse the bacterium.
  • the target bacterium is the natural host of the engineered bacteriophage.
  • the target bacterium is not the natural host of the engineered bacteriophage, and the bacteriophage has been further engineered to infect a bacterium (e.g., by insertion or modulation of specific bacterial binding proteins in the phage, as well known in the art).
  • the bacteriophage is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor. In some embodiments of any of the aspects, the bacteriophage is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, or a heterologous bacteriophage immunity region. In some embodiments of any of the aspects, the bacteriophage is engineered to comprise a heterologous bacteriophage immunity region. In some embodiments of any of the aspects, the bacteriophage is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, and a heterologous bacteriophage immunity region.
  • the bacteriophage is an engineered lambda (l) phage. In some embodiments of any of the aspects, the bacteriophage is an engineered phage that infects and/or replicates in E. coli. In some embodiments of any of the aspects, the bacteriophage is an engineered phage that infects and/or replicates in at least one of the bacteria described herein.
  • the un-engineered wild-type lambda (l) phage comprises genome SEQ ID NO: 32 (Escherichia phage Lambda, complete genome GenBank: J02459.1 or NCBI Accession ID NC.001416.1) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 32 which retains the same activity as SEQ ID NO: 32.
  • the un-engineered lambda (l) phage genome comprises SEQ ID NO: 32 or a sequence that is at least 95% identical to SEQ ID NO: 32 which retains the same activity as SEQ ID NO: 32.
  • Any of the foregoing un-engineered lambda (l) phage genomes can be engineered as described herein.
  • the lambda (l) phage genome is engineered through homologous recombination with at least one heterologous nucleic acid sequence comprising two homology arms (also referred to herein as homology regions).
  • the nucleic acid sequence in between the homology arms in the lambda (l) phage genome e.g., SEQ ID NO: 32
  • the nucleic acid sequence in between the homology arms in the heterologous nucleic acid sequence e.g., at least one of SEQ ID NOs: 1-6 or 20-21.
  • the at least one heterologous nucleic acid sequence is inserted (e.g., through homologous recombination) in place of or within at least one non-essential viral gene in the phage genome.
  • the engineered lambda (l) phage genome comprises at least one heterologous nucleic acid sequence selected from at least one of SEQ ID NOs: 1-6 or 20-21 or the reverse complement of at least one of SEQ ID NOs: 1-6 or 20-21.
  • the heterologous nucleic acid sequence comprises a nucleic acid encoding an inhibitor (e.g., SEQ ID NO: 1 or one of SEQ ID NOs: 20-21).
  • the heterologous nucleic acid sequence comprises an immunity region of a heterologous bacteriophage (e.g., one SEQ ID NO: 2-6).
  • the heterologous nucleic acid comprises one of SEQ ID NOs: 1-6 or 20-21 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 1-6 or 20-21 which retains the same activity as one of SEQ ID NOs: 1-6 or 20-21.
  • the heterologous nucleic acid comprises one of SEQ ID NOs: 1-6 or 20-21 or a sequence that is at least 95% identical to one of SEQ ID NOs: 1-6 or 20-21 which retains the same activity as one of SEQ ID NOs: 1-6 or 20-21. See e.g., Fig. 6A-6C, Fig. 7, or Fig.
  • FIG. 13A for exemplary schematics of lambda (l) phage engineered with a heterologous nucleic acid through recombination.
  • the engineered lambda (l) phage genome comprises an immunity region of a heterologous bacteriophage (e.g., one of SEQ ID NO: 2-6). In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises a nucleic acid encoding an inhibitor (e.g., SEQ ID NO: 1 or one of SEQ ID NOs: 20-21).
  • the engineered lambda (l) phage genome comprises an immunity region of a heterologous bacteriophage (e.g., one SEQ ID NO: 2-6) and a nucleic acid encoding an inhibitor (e.g., SEQ ID NO: 1 or one of SEQ ID NOs: 20-21).
  • a heterologous bacteriophage e.g., one SEQ ID NO: 2-6
  • a nucleic acid encoding an inhibitor e.g., SEQ ID NO: 1 or one of SEQ ID NOs: 20-21.
  • the engineered lambda (l) phage genome comprises an engineered SEQ ID NO: 32, wherein at least one internal region of SEQ ID NO: 32 is replaced through homologous recombination with at least one of SEQ ID NOs: 1-6 or 20-21, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the engineered SEQ ID NO: 32 which retains the same activity as the engineered SEQ ID NO: 32.
  • the engineered lambda (l) phage genome comprises an engineered SEQ ID NO: 32, wherein at least one internal region of SEQ ID NO: 32 is replaced through homologous recombination with at least one of SEQ ID NOs: 1-6 or 20-21, or a sequence that is at least 95%, or at least 99.9% identical to the engineered SEQ ID NO: 32 which retains the same activity as the engineered SEQ ID NO: 32.
  • the engineered lambda (l) phage genome comprises SEQ ID NO: 34 (e.g., lBH2), wherein SEQ ID NO: 1 and SEQ ID NO: 6 have been recombined into the wild-type l phage (e.g., SEQ ID NO: 32).
  • the engineered lambda (l) phage genome comprises SEQ ID NO: 35 (e.g., lBHI), wherein SEQ ID NO: 1 has been recombined into the wild-type l phage (e.g., SEQ ID NO: 32).
  • the engineered lambda (l) phage genome comprises SEQ ID NO: 36 (e.g., /xlCas9). wherein SEQ ID NO: 20 has been recombined into the wild-type l phage (e.g.,
  • the engineered lambda (l) phage genome comprises SEQ ID NO: 37 (e.g., dCas9rfp), wherein SEQ ID NO: 21 has been recombined into the wild-type l phage (e.g., SEQ ID NO: 32).
  • the engineered lambda (l) phage genome comprises one of SEQ ID NOs: 34-37 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 34-37 which retains the same activity as one of SEQ ID NOs: 34-37.
  • the engineered lambda (l) phage genome comprises one of SEQ ID NOs: 34-37 or a sequence that is at least 95% identical to one of SEQ ID NOs: 34-37 which retains the same activity as one of SEQ ID NOs: 34-37.
  • the inhibitor comprises an inhibitor protein encoded by a phage endogenous to the bacterium.
  • endogenous phage also referred to herein as a “prophage” refers to a phage genome that has been integrated into the bacterial host genome.
  • phage 933 W can be an endogenous phage of specific E. coli strains.
  • the inhibitor comprises an inhibitor protein encoded by phage 933 W.
  • the inhibitor comprises cl protein from enterobacteria phage 933 W.
  • the 933 W cl protein comprises SEQ ID NO: 9.
  • the inhibitor comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 9, which retains the same activity or function as SEQ ID NO: 9 or 933W cl protein.
  • the inhibitor comprises a sequence at least 95% identical to SEQ ID NO: 9, which retains the same activity or function as SEQ ID NO: 9 or 933W cl protein. In some embodiments of any of the aspects, the inhibitor comprises SEQ ID NO: 9 with a K178N mutation.
  • SEQ ID NO: 9 repressor protein Cl [Enterobacteria phage 933W]; NCBI Reference Sequence: NP_049485.1; 235 amino acids; residue 178 is shown bold and double-underlined.
  • the engineered bacteriophage comprises SEQ ID NO: 1.
  • the engineered bacteriophage comprises the unformatted black text of SEQ ID NO: 1 (see e.g., Table 3 and SEQ ID NO: 11). In some embodiments of any of the aspects, the engineered bacteriophage comprises SEQ ID NO: 10.
  • the inhibitor comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 10, which retains the same activity or function as SEQ ID NO: 10 or 933W cl protein. In some embodiments of any of the aspects, the inhibitor comprises a sequence that is at least 95% identical to SEQ ID NO: 10, which retains the same activity or function as SEQ ID NO: 10 or 933W cl protein. In some embodiments of any of the aspects, the inhibitor comprises SEQ ID NO: 10 with a N178K mutation.
  • SEQ ID NO: 10 933W cl protein, 235 amino acids; residue 178 is shown bold and double-underlined
  • the inhibitor is engineered to be non- degradable or less degradable.
  • Such engineering can include mutation of enzymatic action sites, e.g., protease recognition sequences, protease cleavage sites, and the like or the use of non-natural amino acids or peptide backbone structures.
  • the 933W cl protein comprises a K178N mutation that causes the protein to be non-degradable or degraded less than the wild-type 933W cl protein.
  • the 933W cl protein comprises an asparagine at residue 178 that causes the protein to be non-degradable or degraded less than the wild-type 933W cl protein.
  • the at least one nucleic acid of the engineered bacteriophage encoding an inhibitor comprises the unformatted black text of SEQ ID NO: 1 (see e.g., Table 3). In some embodiments of any of the aspects, the at least one nucleic acid of the engineered bacteriophage encoding an inhibitor comprises SEQ ID NO: 11 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 11, which retains the same activity as SEQ ID NO: 11, or a codon- optimized version thereof.
  • the at least one nucleic acid of the engineered bacteriophage encoding an inhibitor comprises SEQ ID NO: 11 or a sequence that is at least 95% identical to SEQ ID NO: 11, which retains the same activity or function as SEQ ID NO: 11, or a codon-optimized version thereof.
  • the engineered bacteriophage genome further comprises a promoter for the nucleic acid encoding the inhibitor.
  • the promoter for the inhibitor is engineered to be non-inducible.
  • the promoter for the inhibitor is the LacUV5 promoter.
  • the promoter for the inhibitor is mutated for constitutive expression.
  • the promoter of the inhibitor comprises SEQ ID NO: 12 (see e.g., Table 3) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 12 which retains the same activity as SEQ ID NO: 12.
  • the promoter of the inhibitor comprises SEQ ID NO: 12 (see e.g., Table 3) or a sequence that is at least 95% identical to SEQ ID NO: 12 which retains the same activity as SEQ ID NO: 12.
  • SEQ ID NO: 12 LacUV5 promoter with mutated nucleotides (uppercase; e.g., nucleotides 76, 77, 90, 101 of SEQ ID NO: 12) for constitutive expression gcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatAAtgtgtggaattaTgagcggat aaTaatttcacacaggaaacagct
  • the inhibitor comprises a Cas9 protein and at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, the inhibitor comprises a Cas9 protein or at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor.
  • the inhibitor comprises a Cas9 protein. In some embodiments of any of the aspects, the inhibitor comprises at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, Cas9 protein is administered or supplied with the engineered bacteriophage comprising at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, the CRISPR guide RNA (also referred to herein as crRNA) can be designed according to methods as known in the art. In some embodiments of any of the aspects, the Cas9 is a deactivated Cas9 (dCas9), in other words the Cas9 is catalytically inactive or lacks nuclease activity.
  • dCas9 deactivated Cas9
  • the inhibitor comprises a CRISPR-Cas protein selected from the group consisting of C2cl, C2c3, Casl, CaslOO, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, Casl3c, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl, CaslB, CaslO, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csa5, Csa5, CsaX, Csbl, Csb2, Csb3, Cscl, Csc2, Csel, Cse2, Csfl, Csf2, Csf3, Csf4, Csm2, Csm3, Csmm2, Csm3, Csmm2, Cs
  • the CRISPR-Cas protein can be from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Leptospira, Desulfovibrio,
  • the CRISPR-Cas protein is Cas9 from Staphylococcus aureus.
  • the Cas9 protein is modified with nuclease -inactivating mutations in the HNH and/or RuvC catalytic regions.
  • the Cas9 protein comprises a D10A mutation and/or aN580A mutation (see e.g., Friedland et al. Genome Biol. 16, 257 (2015); Nishimasu et al. Cell 162, 1113-1126 (2015)).
  • the engineered bacteriophage genome further comprises a promoter for the nucleic acid encoding the inhibitor (e.g., Cas9).
  • the promoter for the inhibitor is inducible.
  • the promoter for the inhibitor is inducible by anhydrotetracy cline (ATc).
  • the promoter for the inhibitor is the ProC promoter.
  • the promoter of the inhibitor comprises SEQ ID NO: 22 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 22 which retains the same activity as SEQ ID NO: 22.
  • the promoter of the inhibitor comprises SEQ ID NO: 22 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 22 which retains the same activity as SEQ ID NO: 22.
  • the inhibitor is encoded by a nucleotide sequence comprising SEQ ID NO: 23, a functional fragment of SEQ ID NO: 23, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 22, which retains the same activity as SEQ ID NO: 22, or a codon-optimized version thereof.
  • the inhibitor is encoded by a nucleotide sequence comprising SEQ ID NO: 23, a functional fragment of SEQ ID NO: 23, or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 22, which retains the same activity as SEQ ID NO: 22, or a codon-optimized version thereof.
  • SEQ ID NO: 23 dSaCas9; bold double -underlined text indicates D10A mutation (see e.g., nt 28-30 of SEQ ID NO: 23); italicized double-underlined text indicates N580A mutation (see e.g., nt 1738-1740 of SEQ ID NO: 23).
  • the inhibitor comprises SEQ ID NO: 19, a functional fragment of SEQ ID NO: 19, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 19, which retains the same activity as SEQ ID NO: 19.
  • the inhibitor comprises SEQ ID NO: 19, a functional fragment of SEQ ID NO: 19, or an amino acid sequence that is at least 95 identical to SEQ ID NO: 19, which retains the same activity as SEQ ID NO: 19.
  • SEQ ID NO: 19 SEQ ID NO: 19, dSaCas9; bold double-underlined text indicates D10A mutation; italicized double-underlined text indicates N580A mutation.
  • the inhibitor comprises a Cas protein in combination with a CRISPR guide RNA (gRNA).
  • gRNA CRISPR guide RNA
  • the CRISPR gRNA comprises a tracrRNA and/or crRNA.
  • the engineered bacteriophage genome further comprises at least one tracrRNA and/or at least one crRNA.
  • the tracrRNA and crRNA are separate RNA molecules.
  • the tracrRNA and crRNA are comprised by the same RNA molecule.
  • the full-length guide nucleic acid strand can be any length.
  • the guide nucleic acid strand can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • a nucleic acid strand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the guide nucleic acid sequence is 10-30 nucleotides long.
  • the guide nucleic acid is designed using a guide design tool (e.g., BenchlingTM; Broad Institute GPPTM; CasOFFinderTM; CHOPCHOPTM; CRISPORTM; DeskgenTM; E-CRISPTM; GeneiousTM; GenHubTM; GUIDESTM (e.g., for library design); Horizon DiscoveryTM; IDTTM; Off- SpotterTM; and SynthegoTM; which are available on the world wide web).
  • a guide design tool e.g., BenchlingTM; Broad Institute GPPTM; CasOFFinderTM; CHOPCHOPTM; CRISPORTM; DeskgenTM; E-CRISPTM; GeneiousTM; GenHubTM; GUIDESTM (e.g., for library design); Horizon DiscoveryTM; IDTTM; Off- SpotterTM; and SynthegoTM; which are available on the world wide web).
  • the engineered bacteriophage genome further comprises a promoter for the at least one tracrRNA and/or at least one crRNA.
  • the promoter for the tracrRNA is constitutive.
  • the promoter for the crRNA is constitutive.
  • the promoter (e.g., for the tracrRNA) comprises SEQ ID NO: 24 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 24 which retains the same activity as SEQ ID NO: 24.
  • the promoter (e.g., for the tracrRNA) comprises SEQ ID NO: 24 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 24 which retains the same activity as SEQ ID NO: 24.
  • the promoter (e.g., for the crRNA) comprises the J23100 promoter.
  • the promoter (e.g., for the crRNA) comprises SEQ ID NO: 26 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 26 which retains the same activity as SEQ ID NO: 26.
  • the promoter (e.g., for the crRNA) comprises SEQ ID NO: 24 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 26 which retains the same activity as SEQ ID NO: 26.
  • the trans-activating CRISPR RNA specifically binds with the CRISPR-Cas protein (e.g., Cas9). In some embodiments of any of the aspects, the tracrRNA hybridizes with the crRNA. In some embodiments of any of the aspects, the tracrRNA specifically binds with the CRISPR-Cas protein (e.g., Cas9) and hybridizes with the crRNA.
  • the tracrRNA comprises SEQ ID NO: 25 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 25 which retains the same activity as SEQ ID NO: 25.
  • the tracrRNA comprises SEQ ID NO: 25 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 25 which retains the same activity as SEQ ID NO: 25.
  • the CRISPR RNA comprises a variable targeting sequence and a region that is substantially complementary to a region of the tracrRNA. In some embodiments of any of the aspects, the CRISPR RNA (crRNA) comprises a variable targeting sequence. In some embodiments of any of the aspects, the variable targeting sequence of the crRNA is substantially complementary to a target nucleic acid. In some embodiments of any of the aspects, the variable targeting sequence of the crRNA is substantially complementary to a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, the variable targeting sequence of the crRNA is substantially complementary to RFP (see e.g., SEQ ID NO: 28).
  • variable targeting sequence of a negative control crRNA is substantially complementary to no known nucleic acid sequence (see e.g., SEQ ID NO: 27).
  • the variable targeting sequence comprises one of SEQ ID NOs: 27-28 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 27-28 which retains the same activity as one of SEQ ID NOs: 27-28.
  • variable targeting sequence comprises one of SEQ ID NOs: 27-28 (see e.g., Table 4) or a sequence that is at least 95% identical to one of SEQ ID NOs: 27-28 which retains the same activity as one of SEQ ID NOs: 27-28.
  • the CRISPR RNA comprises a region that is substantially complementary to a region of the tracrRNA.
  • the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 29 which retains the same activity as SEQ ID NO: 29.
  • the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 29 which retains the same activity as SEQ ID NO: 29.
  • SEQ ID NO: 26 J23100 promoter (e.g., promoter for crRNA)
  • SEQ ID NO: 27 Bsal spacer (e.g, control gRNA) GAGACCGACTGAGGTCTCA [00109] SEQ ID NO: 28, rip l gRNA TGGTAACTTTCAGTTTAGCGGT [00110] SEQ ID NO: 29, SaCas9 repeat (e.g., for hybridization with tracrRNA) GTTTTAGTACTCTGTAATTTTAGGTATGAGGTAGAC
  • a vector comprises at least one of the following: l phage homology arms, a promoter for the CRISPR-Cas protein (e.g., Cas9), the CRISPR- Cas protein (e.g., S. aureus dCas9), the tracrRNA under a constitutive promoter, the crRNA under a constitutive promoter, and a resistance cassette (e.g., pACYCDuet chloramphenicol resistance cassette).
  • a promoter for the CRISPR-Cas protein e.g., Cas9
  • the CRISPR- Cas protein e.g., S. aureus dCas9
  • the tracrRNA under a constitutive promoter
  • the crRNA under a constitutive promoter
  • a resistance cassette e.g., pACYCDuet chloramphenicol resistance cassette
  • the vector comprises one of SEQ ID NOs: 30- 31 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 30-31 which retains the same activity as one of SEQ ID NOs: 30-31.
  • the vector comprises one of SEQ ID NOs: 30-31 or a sequence that is at least 95% identical to one of SEQ ID NOs: 30-31 which retains the same activity as one of SEQ ID NOs: 30-31.
  • the bacterium (e.g., targeted by the engineered bacteriophage) is Escherichia coli ( E . coli). In some embodiments of any of the aspects, the bacterium is enterohemorrhagic E. coli (EHEC). In some embodiments of any of the aspects, the bacterium is found within the human intestinal microbiome.
  • the following microbes that causes diseases and/or associated microbial matter can be amendable to the compositions and methods of various aspects described herein (e.g., can be targeted by the engineered phage described herein): Bartonella henselae, Borrelia burgdorferi, Campylobacter jejuni, Campylobacterfetus, Chlamydia trachomatis, Chlamydia pneumoniae, Chylamydia psittaci, Simkania negevensis, Escherichia coli (e.g., 0157:H7 and K88), Ehrlichia chafeensis, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Enterococcus faecalis, Haemophilius influenzae, Haemophilius ducreyi, Coccidioides immitis, Bordetella pertussis
  • the virulence factor is Shiga Toxin (Stx).
  • Stx Shiga Toxin
  • Non-limiting examples of virulence factors include bacterial hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases, and enterotoxins.
  • the virulence factor is encoded by an endogenous phage, including but not limited to phage 933 W.
  • the engineered phage further comprises a heterologous bacteriophage immunity region.
  • heterologous refers to that which is not endogenous to, or naturally occurring in, a referenced sequence, molecule (including e.g., a protein), virus, cell, tissue, or organism.
  • a heterologous sequence of the present disclosure can be derived from a different species, or from the same species but substantially modified from an original form.
  • a nucleic acid sequence that is not normally expressed in a virus or a cell is a heterologous nucleic acid sequence.
  • heterologous can refer to DNA, RNA, or protein that does not occur naturally as part of the organism (which is used herein to comprise viruses) in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature. It is DNA, RNA, or protein that is not endogenous to the virus or cell and has been artificially introduced into the virus or cell.
  • immuno region refers to a nucleic acid locus in a temperate phage that is responsible for maintaining the lysogenic state and, as a consequence, effecting immunity against superinfecting phages. In some embodiments of any of the aspects, the immunity region inhibits infection of the bacteria by other phage, i.e., immunity against superinfection.
  • superinfection refers to bacteria infected with at least bacteriophage being infected by at least one other different bacteriophage.
  • the heterologous bacteriophage immunity region is a lambdoid phage immunity region.
  • the lambdoid phage is selected from the group consisting of lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22.
  • the lambdoid phage is selected from the group consisting of 933W phage, lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22.
  • the heterologous bacteriophage immunity region is an immunity region from 933W phage (e.g., SEQ ID NO: 13).
  • the heterologous bacteriophage immunity region is an immunity region from lambdoid phage 21 (e.g., SEQ ID NO: 15). In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is an immunity region from lambdoid phage 434 (e.g., SEQ ID NO: 16). In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is an immunity region from lambdoid phage P22 (e.g., SEQ ID NO: 14 or 17).
  • the heterologous bacteriophage immunity region comprises any one of SEQ ID NOs: 2-6, a portion of any one of SEQ ID NOs: 2-6 (e.g., as indicated in Table 3), or a sequence that is at least 95% identical to any one of SEQ ID NOs: 2-6 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 2-6 or a portion thereof.
  • the heterologous bacteriophage immunity region comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to any one of SEQ ID NOs: 2-6 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 2-6 or a portion thereof.
  • the heterologous bacteriophage immunity region comprises any one of SEQ ID NOs: 13-17, a portion of any one of SEQ ID NOs: 13-17, or a sequence that is at least 95% identical to any one of SEQ ID NOs: 13-17 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 13-17 or a portion thereof.
  • the heterologous bacteriophage immunity region comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to any one of SEQ ID NOs: 13-17 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 13-17 or a portion thereof.
  • SEQ ID NO: 13 immunity region of 933W phage (e.g., in limm933W phage; see e.g., SEQ ID NO: 2 in Table 3)
  • SEQ ID NO: 14 immunity region of P22 phage (e.g., in l ⁇ hihR22 ⁇ 8 phage; see e.g.,
  • SEQ ID NO: 15 immunity region of 21 phage (e.g., in l ⁇ ihih21 phage; see e.g., SEQ ID NO: 4 in Table 3)
  • SEQ ID NO: 16 immunity region of 434 phage (e.g., in l ⁇ hih434 phage; see e.g., SEQ ID NO: 5 in Table 3)
  • SEQ ID NO: 17 immunity region of P22 phage (e.g., in lBH2 phage; see e.g., SEQ ID NO: 6 in Table 3; SEQ ID NO: 17 comprises nucleotides 11,060-16,195 of SEQ ID NO: 14)
  • the engineered bacteriophage genome further comprises a nucleic acid encoding a selectable marker.
  • selectable markers include a positive selection marker; a negative selection marker; a positive and negative selection marker; resistance to at least one of chloramphenicol, ampicillin, kanamycin, and/or triclosan; or an auxotrophy marker.
  • the selectable marker is selected from the group consisting of CmR (a chloramphenicol resistance gene; e.g., the pACYCDuet chloramphenicol resistance cassette) beta-lactamase, Neo gene (e.g., Kanamycin resistance cassette) from Tn5, mutant Fabl gene, and an auxotrophic mutation.
  • CmR chloramphenicol resistance gene
  • Neo gene e.g., Kanamycin resistance cassette
  • the selectable marker is encoded by a nucleotide sequence comprising one of SEQ ID NOs: 18 or 33 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 18 or 33, which retains the same activity or function as one of SEQ ID NOs: 18 or 33, or a codon-optimized version thereof.
  • SEQ ID NO: 18 kanamycin resistance cassette (Tn5) gccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcg catgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctc tgatgccgcgtgttccggctgtcagcgcaggggcgcccggtttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggc aggc agc agcgc agcgcggctatcgtggccacgac
  • described herein is a method of treating a bacterial infection, comprising administering an effective amount of an engineered bacteriophage as described or a pharmaceutical composition as described herein to a patient in need thereof.
  • the patient is infected with E. coli or EHEC.
  • levels of at least one virulence factor can be increased in a bacterial infection and/or in subjects with a bacterial infection. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising administering at least one engineered bacteriophage as described herein to a subject determined to have a level of at least one virulence factor that is increased relative to a reference.
  • a method of treating a bacterial infection in a subject in need thereof comprising: a) determining the level of at least one virulence factor in a sample obtained from a subject; and b) administering at least one engineered bacteriophage as described herein to the subject if the level of at least one virulence factor is increased relative to a reference.
  • the method comprises administering at least one engineered bacteriophage as described herein to a subject previously determined to have a level of at least one virulence factor that is increased relative to a reference.
  • described herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising: a) first determining the level of at least one virulence factor in a sample obtained from a subject; and b) then administering at least one engineered bacteriophage as described herein to the subject if the level of at least one virulence factor is increased relative to a reference.
  • a method of treating a bacterial infection in a subject in need thereof comprising: a) determining if the subject has an increased level of at least one virulence factor; and b) administering at least one engineered bacteriophage as described herein to the subject if the level of at least one virulence factor is increased relative to a reference.
  • the step of determining if the subject has an increased level of at least one virulence factor can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise performing or having performed an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject.
  • the step of determining if the subject has an increased level of at least one virulence factor can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise receiving the results of an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject.
  • the step of determining if the subject has an increased level of at least one virulence factor can comprise receiving a report, results, or other means of identifying the subject as a subject with an increased level of at least one virulence factor.
  • a method of treating a bacterial infection in a subject in need thereof comprising: a) determining if the subject has an increased level of at least one virulence factor; and b) instructing or directing that the subject be administered at least one engineered bacteriophage as described herein if the level of at least one virulence factor is increased relative to a reference.
  • the step of determining if the subject has an increased level of at least one virulence factor can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise performing or having performed an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject.
  • the step of determining if the subject has an increased level of at least one virulence factor can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject.
  • the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results.
  • the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results.
  • described herein is a method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of at least one engineered bacteriophage as described herein or an effective amount of at least one pharmaceutical composition as described herein.
  • described herein are methods of inhibiting or delaying the formation of biofdms, comprising administering to a subject in need thereof or contacting a surface with an effective amount of at least one engineered bacteriophage as described herein or an effective amount of a pharmaceutical composition as described herein.
  • a “biofilm” refers to mass of microorganisms attached to a surface, such as a surface of a medical device, and the associated extracellular substances produced by one or more of the attached microorganisms.
  • the extracellular substances are typically polymeric substances that commonly include a matrix of complex polysaccharides, proteinaceous substances and glycopeptides.
  • the microorganisms can include, but are not limited to, bacteria, fungi and protozoa.
  • the microorganisms include one or more species of bacteria. The nature of a biofilm, such as its structure and composition, can depend on the particular species of bacteria present in the biofilm.
  • Biofilms Bacteria present in a biofilm are commonly genetically or phenotypically different than corresponding bacteria not in a biofilm, such as isolated bacteria or bacteria in a colony. "Polymicrobic biofilms" are biofilms that include a plurality of bacterial species.
  • the terms and phrases "delaying”, “delay of formation”, and “delaying formation of' have their ordinary and customary meanings, and are generally directed to increasing the period of time prior to the formation of biofilm, or a slow growing bacterial infection in a subject or on a surface.
  • the delay may be, for example, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more.
  • Inhibiting formation of a biofilm refers to avoiding the partial or full development or progression of a biofilm, for example, on a surface, such as a surface of an indwelling medical device.
  • biofilms can be practiced wherever bacteria, such as persistent, slow-growing, stationary-phase, or biofilm forming bacteria, can be encountered.
  • the methods described herein can be practiced on the surface of or inside of an animal, such as a human; on an inert surface, such as a counter or bench top; on a surface of a piece of medical or laboratory equipment; on a surface of a medical or laboratory tool; or on a surface of an in-dwelling medical device.
  • a biofilm on a surface or on a porous material comprising applying to or contacting a surface or a porous material upon which a biofdm can form at least one engineered bacteriophage or a pharmaceutical composition as described herein in an amount sufficient to inhibit the formation of a biofilm.
  • the surface is an inert surface, such as the surface of an in-dwelling medical device.
  • a biofilm on a surface or on a porous material comprising applying to or contacting a surface or a porous material upon which a biofilm can form at least one engineered bacteriophage or a pharmaceutical composition as described herein in an amount sufficient to delay the formation of a biofilm.
  • the surface is an inert surface, such as the surface of an in-dwelling medical device.
  • kits for preventing the colonization of a surface by persistent bacteria comprising applying to or contacting a surface with at least one engineered bacteriophage or a pharmaceutical composition as described herein in an amount sufficient to prevent colonization of the surface by persistent bacteria.
  • the material comprising the surface or the porous material can be any material that can be used to form a surface or a porous material.
  • the material is selected from: polyethylene, polytetrafluoroethylene, polypropylene, polystyrene, polyacrylamide, polyacrylonitrile, poly(methyl methacrylate), polyamide, polyester, polyurethane, polycarbonate, silicone, polyvinyl chloride, polyvinyl alcohol, polyethylene terephthalate, cobalt, a cobalt-base alloy, titanium, a titanium base alloy, steel, silver, gold, lead, aluminum, silica, alumina, yttria stabilized zirconia polycrystal, calcium phosphate, calcium carbonate, calcium fluoride, carbon, cotton, wool and paper.
  • an antibiotic in embodiments of the methods described herein of inhibiting or delaying the formation of a biofdm, or preventing the colonization of a surface by persistent bacteria, can be applied concurrently with at least one engineered bacteriophage or a pharmaceutical composition as described herein.
  • Suitable antibiotics include, for example, aminoglycosides (e.g., gentamicin, streptomycin, kanamycin), b-lactams (e.g, penicillins and cephalosporins), vancomycins, bacitracins, macrolides (e.g., erythromycins), lincosamides (e.g., clindamycin), chloramphenicols, tetracyclines, amphotericins, cefazolins, clindamycins, mupirocins, sulfonamides and trimethoprim, rifampicins, metronidazoles, quinolones, novobiocins, polymyxins, gramicidins, or any salts or variants thereof.
  • aminoglycosides e.g., gentamicin, streptomycin, kanamycin
  • b-lactams e.g,
  • the persistent, slow growing, stationary-phase or biofdm bacteria is any bacterial species or population that comprises persistent cells, can exist in a slow growing or stationary-phase, and/or that can form a biofilm.
  • the bacteria is E. coli, Staphylococcus aureus, Staphylococcus epidermidis, a vancomycin-susceptible enterococci, a vancomycin-resistant enterococci, a Staphylococcus species or a Streptococcus species.
  • the bacteria is selected from vancomycin (VAN)-susceptible Enterococcus faecalis (VSE), VAN-resistant E. faecalis (VRE), and Staph epidermidis .
  • VAN vancomycin
  • VSE vancomycin-susceptible Enterococcus faecalis
  • VRE VAN-resistant E. faecalis
  • Staph epidermidis Staph epidermidis .
  • the term "contacting” is meant to broadly refer to bringing a bacterial cell and at least one engineered bacteriophage or a pharmaceutical composition as described herein into sufficient proximity that the at least one engineered bacteriophage or a pharmaceutical composition as described herein can exert their effects on any bacterial cell present.
  • the skilled artisan will understand that the term “contacting” includes physical interaction between at least one engineered bacteriophage or a pharmaceutical composition as described herein and a bacterial cell, as well as interactions that do not require physical interaction.
  • the methods described herein further encompass surfaces coated by at least one engineered bacteriophage or a pharmaceutical composition as described herein, and/or impregnated with at least one engineered bacteriophage or a pharmaceutical composition as described herein.
  • Such surfaces include any that can come into contact with a persistent, slow growing, stationary-phase, biofilm bacteria.
  • such surfaces include any surface made of an inert material (although surfaces of a living animal are encompassed within the scope of the methods described herein), including the surface of a counter or bench top, the surface of a piece of medical or laboratory equipment or a tool, the surface of a medical device such as a respirator, and the surface of an in-dwelling medical device.
  • such surfaces include those of an in dwelling medical device, such as surgical implants, orthopedic devices, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time.
  • Such devices include, but are not limited to, artificial joints, artificial hearts and implants; valves, such as heart valves; pacemakers; vascular grafts; catheters, such as vascular, urinary and continuous ambulatory peritoneal dialysis (CAPD) catheters; shunts, such as cerebrospinal fluid shunts; hoses and tubing; plates; bolts; valves; patches; wound closures, including sutures and staples; dressings; and bone cement.
  • an in dwelling medical device such as surgical implants, orthopedic devices, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time.
  • Such devices include, but are not limited to, artificial joints, artificial hearts and implants; valves, such as heart valves; pacemakers; vascular
  • intravascular catheters for example, intravenous and intra-arterial
  • right heart flow-directed catheters Hickman catheters
  • arteriovenous fistulae catheters used in hemodialysis and peritoneal dialysis
  • vascular access ports for example, silastic, central venous, Tenckhoff, and Teflon catheters
  • indwelling urinary catheters urinary catheters
  • silicone catheters silicone catheters
  • ventricular catheters synthetic vascular prostheses (for example, aortofemoral and femoropopliteal)
  • prosthetic heart valves prosthetic joints
  • orthopedic implants penile implants
  • shunts for example, Scribner, Torkildsen, central nervous system, portasystemic, ventricular, ventriculoperitoneal
  • intrauterine devices for example, tampons, dental implants, stents (for example, ureter
  • a subcategory of indwelling medical devices refer to implantable devices that are typically more deeply and/or permanently introduced into the body.
  • Indwelling medical devices can be introduced by any suitable means, for example, by percutaneous, intravascular, intraurethral, intraorbital, intratracheal, intraesophageal, stomal, or other route, or by surgical implantation, for example intraarticular placement of a prosthetic joint.
  • the in-dwelling medical device is coated by a solution, such as through bathing or spraying, containing a concentration of about 10 pg/ml to about 500 mg/ml of at least one engineered bacteriophage or a pharmaceutical composition as described herein.
  • more specific ranges of concentrations of the at least one engineered bacteriophage or a pharmaceutical composition as described herein can be used, including: about 10 pg/ml to about 1 mg/ml, about 1 mg/ml to about 100 mg/ml, about 10 mg/ml to about 500 mg/ml, about 50 mg/ml to about 200 mg/ml, about 10 mg/ml to about 100 mg/ml, about 100 mg/ml to about 500 mg/ml.
  • specific concentrations of at least one engineered bacteriophage or a pharmaceutical composition as described herein can be used, including: about 10 pg/ml, about 50 pg/ml, about 100 pg/ml, about 250 pg/ml, about 500 pg/ml, about 750 pg/ml, about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 250 mg/ml, about 500 mg/ml, about 600 mg/ml, about 750 mg/ml, and about 900 mg/ml.
  • the in-dwelling medical device can be coated by the solution comprising at least one engineered bacteriophage or a pharmaceutical composition as described herein before its insertion in the body.
  • the methods described herein relate to treating a subject having or diagnosed as having a bacterial infection.
  • Subjects having a bacterial infection can be identified by a physician using current methods of diagnosing bacterial infections.
  • Symptoms and/or complications of a bacterial infection which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to fever; fatigue; swollen lymph nodes in the neck, armpits, or groin; headache; and/or nausea or vomiting.
  • Tests that may aid in a diagnosis of, e.g. a bacterial infection include, but are not limited to, a blood test, a urine test, a culture test, rt-qPCR, MALDI- TOF MS, or other methods as known in the art.
  • compositions and methods described herein can be administered to a subject having or diagnosed as having a bacterial infection.
  • the methods described herein comprise administering an effective amount of compositions described herein, e.g. at least one engineered bacteriophage as described herein, to a subject in order to alleviate a symptom of a bacterial infection.
  • “alleviating a symptom of a bacterial infection” is ameliorating any condition or symptom associated with the bacterial infection. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • a variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
  • the term “effective amount” as used herein refers to the amount of an engineered bacteriophage as described herein or a pharmaceutical composition comprising at least one engineered bacteriophage as described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmaceutical composition to provide the desired effect.
  • the term “therapeutically effective amount” therefore refers to an amount of an engineered bacteriophage that is sufficient to provide a particular anti-bacterial effect (e.g., especially with regard to decreasing the level of at least one virulence factor as described herein) when administered to a typical subject.
  • an effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e..
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for bacteriophage such as a plaque assay as described herein, among others.
  • the dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the technology described herein relates to a pharmaceutical composition comprising at least one engineered bacteriophage as described herein, and optionally a pharmaceutically acceptable carrier.
  • the active ingredients of the pharmaceutical composition comprise at least one engineered bacteriophage as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of at least one engineered bacteriophage as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of at least one engineered bacteriophage as described herein.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include:
  • sugars such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol;
  • polyols such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) other non toxic compatible substances employed in pharmaceutical formulations.
  • PEG polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • agar such as magnesium hydro
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent, e.g. at least one engineered bacteriophage as described herein.
  • the pharmaceutical composition comprising at least one engineered bacteriophage as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.
  • parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • controlled-re lease parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS ® -type dosage forms and dose-dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms of at least one engineered bacteriophage as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non- aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lac
  • compositions comprising at least one engineered bacteriophage as described herein can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non- aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion.
  • Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally,
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • the pharmaceutical composition comprising at least one engineered bacteriophage as described herein can be administered in a sustained release formulation.
  • Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Kim Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
  • Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated herein by reference.
  • dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
  • active ingredients for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS ® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
  • OROS ® Alza Corporation, Mountain View, Calif. USA
  • the pharmaceutical composition comprising at least one engineered bacteriophage as described herein is administered as a monotherapy, e.g., another treatment for the bacterial infection is not administered to the subject.
  • the methods described herein can further comprise administering a second antimicrobial agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
  • the antimicrobial agent can be selected from aminoglycosides, ansamycins, beta- lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron-sequestering glycoproteins, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, tetracyclines, and any combinations thereof.
  • the antimicrobial agent can comprise an antibiotic.
  • Some exemplary specific antimicrobial agents include broad penicillins, amoxicillin (e.g., Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin), Penicillins and Beta Lactamase Inhibitors (e.g., Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam, Benzylpenicillin, Cloxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin), Cephalosporins (e.g., Cephalosporin I Generation, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine),
  • amoxicillin e.g., Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ti
  • Cephalosporin II Generation e.g., Cefaclor, Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Ceftnetazole, Cefuroxime, Loracarbef
  • Cephalosporin III Generation e.g., Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone
  • Cephalosporin IV Generation e.g., Cefepime
  • Macrolides and Lincosamides e.g., Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin, Troleandomycin
  • Quinolones and Fluoroquinolones e.g., Cinoxacin, Ciprofloxacin, Enoxacin, Gat
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy.
  • a second agent and/or treatment known to be beneficial for subjects suffering from pain or inflammation.
  • agents and/or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g.
  • opiates e.g. endorphins, enkephalins, and dynorphin
  • an effective dose of a composition comprising at least one engineered bacteriophage as described herein can be administered to a patient once.
  • an effective dose of a composition comprising at least one engineered bacteriophage as described herein can be administered to a patient repeatedly.
  • subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, at least 10 PFU/dose (plaque-forming units per dose), at least 10 1 PFU/dose, at least 10 2 PFU/dose, at least 10 3 PFU/dose, at least 10 4 PFU/dose, at least 10 5 PFU/dose, at least 10 6 PFU/dose, at least 10 7 PFU/dose, at least 10 8 PFU/dose, at least 10 9 PFU/dose, or at least 10 10 PFU/dose.
  • PFU/dose plaque-forming units per dose
  • subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, about 10 7 PFU/dose. In some embodiments of any of the aspects, subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, about 5xl0 3 PFU/dose.
  • a composition (e.g., for administration to a patient in need thereof) comprises 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, or at least 10 engineered phage as described herein.
  • At least one of the engineered phage as described herein is administered unencapsulated, e.g., in a saline solution or buffer (e.g., bicarbonate buffer).
  • at least one of the engineered phage as described herein is administered encapsulated or as part of an encapsulation composition, e.g., to protect it from stomach acid.
  • the phage can be administered on a substrate or in/on a composition, e.g., beads, nanoparticles, hydrogel.
  • the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
  • Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. a bacterial infection by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to at least one engineered bacteriophage as described herein.
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
  • dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
  • a composition comprising at least one engineered bacteriophage as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the dosage ranges for the administration of a composition comprising at least one engineered bacteriophage as described herein, according to the methods described herein depend upon, for example, the form of the engineered bacteriophage, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for at least one virulence factor.
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • compositions comprising at least one engineered bacteriophage as described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. a bacterial infection) can be determined by the skilled clinician.
  • a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. at least one virulence factor as described herein. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. fever, levels of bacteria in a sample, levels of at least one virulence factor). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of a bacterial infection. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. at least one virulence factor as described herein.
  • compositions comprising at least one engineered bacteriophage as described herein.
  • effects of a dose of composition comprising at least one engineered bacteriophage as described herein can be assessed by measurement of at least one virulence factor in cell culture or in an animal model of infection (see e.g., Examples 1 and 2).
  • one or more of the genes described herein is expressed in a recombinant expression vector or plasmid.
  • the term “vector” refers to a polynucleotide sequence suitable for transferring transgenes into a host cell.
  • the term “vector” includes plasmids, mini-chromosomes, phage, naked DNA and the like. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989).
  • vectors refers to a circular double stranded DNA loop into which additional DNA segments are ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors" . In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” is used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses
  • a cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence can be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis.
  • replication can occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an RNA transcript.
  • Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., b-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • the vectors used herein are capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
  • the promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene.
  • the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene.
  • a variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule.
  • the promoter is the LacUV5 promoter (e.g., SEQ ID NO: 12).
  • the precise nature of the regulatory sequences needed for gene expression can vary between species or cell types, but in general can include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
  • the vector is pJETl .2.
  • the genes (e.g., of the engineered bacteriophage) described herein can be included in one vector or separate vectors.
  • the virulence factor inhibitor gene or the immunity region gene can be included in the same vector; or the virulence factor inhibitor gene and the immunity region gene can be included in the same vector.
  • the virulence factor inhibitor gene can be included in a first vector, and the immunity region gene can be included in a second vector.
  • one or more of the recombinantly expressed gene can be integrated into the genome of the cell.
  • a nucleic acid molecule that encodes the enzyme of the claimed invention can be introduced into a cell or cells using methods and techniques that are standard in the art.
  • nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc.
  • Expressing the nucleic acid molecule encoding the enzymes of the claimed invention also may be accomplished by integrating the nucleic acid molecule into the genome.
  • “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.
  • “Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount.
  • the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a “increase” is a statistically significant increase in such
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms, “individual,” “patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a bacterial infection.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. a bacterial infection) or one or more complications related to such a condition, and optionally, have already undergone treatment for a bacterial infection or the one or more complications related to a bacterial infection.
  • a subject can also be one who has not been previously diagnosed as having a bacterial infection or one or more complications related to a bacterial infection.
  • a subject can be one who exhibits one or more risk factors for a bacterial infection or one or more complications related to a bacterial infection or a subject who does not exhibit risk factors.
  • a “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • protein and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • modified amino acids e.g., phosphorylated, glycated, glycosylated, etc.
  • amino acid analogs regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • variants naturally occurring or otherwise
  • alleles homologs
  • conservatively modified variants conservative substitution variants of any of the particular polypeptides described are encompassed.
  • amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide.
  • conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
  • a given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn).
  • Other such conservative substitutions e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known.
  • Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. activity and specificity of a native or reference polypeptide is retained.
  • Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).
  • Naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
  • Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
  • Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into He or into Leu.
  • the polypeptide described herein can be a functional fragment of one of the amino acid sequences described herein.
  • a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wild-type reference polypeptide’s activity according to the assays described below herein.
  • a functional fragment can comprise conservative substitutions of the sequences disclosed herein.
  • the polypeptide described herein can be a variant of a sequence described herein.
  • the variant is a conservatively modified variant.
  • Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example.
  • a “variant,” as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions.
  • Variant polypeptide encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity.
  • a wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan.
  • a variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence.
  • the degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).
  • Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide -directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required.
  • nucleic acid or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA, cDNA, bacterial DNA, or viral DNA.
  • Suitable RNA can include, e.g., mRNA, bacterial RNA, viral RNA, crRNA, tracrRNA, or gRNA.
  • substantially complementary refers both to complete complementarity of the two nucleic acids, in some cases referred to as an identical sequence, as well as complementarity sufficient to achieve the desired binding of the two nucleic acids.
  • the substantially complementary region can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments of any of the aspects, substantially complementary region is less than about 75,
  • the substantially complementary region is 10-30 nucleotides long. In some embodiments of any of the aspects, the substantially complementary region is 15-25 nucleotides long. In some embodiments of any of the aspects, the substantially complementary region is 18-22 nucleotides long. In some embodiments of any of the aspects, the substantially complementary region is about 20 nucleotides long.
  • expression refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing.
  • Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
  • the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are global. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is systemic.
  • “Expression products” include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene.
  • the term “gene” means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences.
  • the gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • Marker in the context of the present invention refers to an expression product, e.g., nucleic acid or polypeptide which is differentially present in a sample taken from subjects having a bacterial infection, as compared to a comparable sample taken from control subjects (e.g., a healthy subject).
  • biomarker is used interchangeably with the term “marker.”
  • the methods described herein relate to measuring, detecting, or determining the level of at least one marker.
  • detecting or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.
  • a polypeptide, nucleic acid, or cell as described herein can be engineered.
  • engineered refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be “engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.
  • progeny of an engineered cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.
  • the engineered bacteriophage described herein is exogenous. In some embodiments of any of the aspects, the engineered bacteriophage described herein is ectopic. In some embodiments of any of the aspects, the engineered bacteriophage described herein is not endogenous.
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • exogenous can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • endogenous refers to a substance that is native to the biological system or cell.
  • ectopic refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.
  • a nucleic acid encoding a polypeptide as described herein is comprised by a vector.
  • a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof is operably linked to a vector.
  • the term "vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • a vector can be viral or non-viral.
  • the term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells.
  • a vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
  • the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).
  • non-native e.g., heterologous
  • the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system.
  • the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism).
  • the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector.
  • sequences expressed will often, but not necessarily, be heterologous to the cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes.
  • the vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies.
  • the vector is episomal.
  • the use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. a bacterial infection.
  • the term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a bacterial infection.
  • Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
  • contacting refers to any suitable means for delivering, or exposing, an agent to at least one cell.
  • exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art.
  • contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
  • compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid.
  • Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.
  • specific binding refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third non-target entity.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • a pharmacological composition comprising the engineered bacteriophage of any of paragraphs 1-13 and an acceptable carrier.
  • a method of treating a bacterial infection comprising administering an effective amount of an engineered bacteriophage of any of paragraphs 1-13 or a pharmaceutical composition of paragraph 14 to a patient in need thereof.
  • a method of inhibiting bacterial growth or activity on a surface comprising contacting a surface with an effective amount of an engineered bacteriophage of any of paragraphs 1-13 or a pharmaceutical composition of paragraph 14.
  • a bacteriophage wherein the bacteriophage genome is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium.
  • the engineered bacteriophage of paragraph 1 wherein the bacteriophage is an engineered lambda (l) phage.
  • the CRISPR guide RNA comprises a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA).
  • tracrRNA trans-activating CRISPR RNA
  • crRNA CRISPR RNA
  • the crRNA comprises a variable targeting sequence and a region that is substantially complementary to a region of the tracrRNA.
  • variable targeting sequence of the crRNA is substantially complementary to a nucleic acid encoding a bacterial virulence factor.
  • the engineered bacteriophage of any of paragraphs 1-14 wherein the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 29 that maintains the same function.
  • the engineered bacteriophage of any of paragraphs 1-15 wherein the bacterium is Escherichia coli ( E . coli).
  • EHEC EHEC
  • a bacteriophage wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • cteriophage wherein the bacteriophage genome is engineered to comprise: a.
  • the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, b. a heterologous bacteriophage immunity region wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
  • cteriophage wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and b. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i.
  • cteriophage wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, b. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises i. a Cas protein and ii.
  • a pharmaceutical composition comprising the engineered bacteriophage of any of paragraphs 1-30 and an acceptable carrier.
  • a method of treating a bacterial infection comprising administering an effective amount of an engineered bacteriophage of any of paragraphs 1-30 or a pharmaceutical composition of paragraph 31 to a patient in need thereof.
  • a method of inhibiting bacterial growth or activity on a surface comprising contacting a surface with an effective amount of an engineered bacteriophage of any of paragraphs 1-30 or a pharmaceutical composition of paragraph 31.
  • Phages typically replicate by a lytic mechanism in which a bacterium infected by a phage turns its cellular machinery towards producing phage components that assemble into viral particles and are released upon cell lysis. This produces hundreds of progeny phage to infect new bacterial hosts (see e.g., Fig. 1A sections i-iii). Lytic phages replicate solely by this life cycle and the decimation of phage -susceptible bacteria selects for phage-resistant mutants that can repopulate over time (see e.g., Fig. 1A upper panel).
  • temperate phages can also integrate their genetic material into the host chromosome as a prophage to co-replicate with the bacterial genome during cell proliferation (see e.g., Fig. 1A sections iv-v). In a bacterial population, this leads to the lysogenic conversion of phage -susceptible species that coexist with phage -resistant species (see e.g., Fig. 1A lower panel). Because anti-bacterial approaches can enrich for resistance, including phage therapy which typically utilizes lytic phages, temperate phages were engineered to deliver an anti-virulence payload that neutralizes virulence in a manner that minimizes the selection for resistance.
  • bacteriophage l were shown to transduce a substantial fraction of targeted bacteria in the mammalian gut.
  • Fig. IB a streptomycin-treated mouse model was used to quantitate temperate phage lysogeny on E. coli colonizing the mammalian gastrointestinal tract.
  • E. coli MG1655 One day after colonization with E. coli MG1655, lBHI phage was introduced by oral gavage and daily stool samples were collected for analysis of bacterial and phage titers.
  • lBHI were constructed from l phage by inserting an antibiotic resistance cassette for quantification of lysogens (see e.g., Fig. 2D).
  • fecal phage levels reached equilibrium approximately two days later and persisted at substantial concentrations (> 10 6 plaque forming units per gram of stool (pfu/g stool)) for the duration of the experiment (see e.g., Fig. 1C).
  • phage in the absence of its cognate bacterial host is undetectable in the stool of mice ⁇ 2 days after administration, these results indicate that lBHI phage is capable of continuous replication in the gut, permitting its expansion throughout the bacterial population from a single dose.
  • introduction of lBHI phage did not significantly alter fecal E. coli concentrations (see e.g., Fig.
  • coli 933W excludes l phage infection but genetic hybrids of l with other lambdoid phages restore infectivity.
  • the 933W prophage inhibits infection from l phage by recognition of its immunity region, e. indispensable genes responsible for the lysis- lysogeny decision in the phage life cycle.
  • lambdoid phages have similarities in genetic function and organization despite dissimilar sequences, it is feasible to replace the l immunity region with orthologous immunity regions from other lambdoid phages to overcome the superinfection exclusion.
  • the efficiency of plating (EOP) of l phage against E The efficiency of plating (EOP) of l phage against E.
  • coli 933W was ⁇ 10 6 -fold lower than that of the non-lysogen (see e.g., Fig. 2B), confirming its superinfection exclusion. This effect was not due to a cl-based immunity (see e.g., Fig. 5).
  • the EOP was then determined for genetic hybrids of l phage in which the l immunity region was swapped with that of other lambdoid phages (e.g. 21, 434, and P22; see e.g., Fig. 6A-6C and Table 3). As shown in Fig. 2B, these hybrid phages had substantially improved EOPs against E. coli 933W with 6.0% for Ximmll and 6.7% for imm434 phages.
  • Stx2 is dependent upon induction of the 933 W prophage.
  • the 933W prophage in E. coli 933W maintains a dormant state by expression of its repressor protein, cl, which blocks expression of cro and consequently lytic genes including Stx2.
  • Induction which occurs spontaneously and by stimuli such as antibiotics, causes activation of the bacterial SOS response and RecA-mediated degradation of Cl (see e.g., Fig. 2C panel ii). This ultimately leads to expression of the lytic genes that produce phage progeny and Stx2.
  • phage encoded repressor for the 933W prophage, cl is key to blocking lytic induction and maintaining the dormant lysogenic state, constitutive expression of a non-degradable mutant of this repressor ( 933W-cT nd ⁇ ) that contains a Lysl78Asn mutation blocks induction of the 933W prophage and consequently neutralizes production of progeny phage and Stx2 (see e.g., Fig. 2C panel Hi), ultimately demonstrating anti-virulence at a genetic level.
  • Efficient gene transduction permits the delivery of anti-virulence genes.
  • Genes for 933W-cF nd ⁇ and a kanamycin resistant cassette (to quantitate lysogeny) were inserted into the non- essential b2 region of l, 30 producing lBHI (see e.g., Fig. 2D and Fig. 7).
  • lBHI see e.g., Fig. 2D and Fig. 7
  • 933W-cF nd ⁇ expressed from lBHI was functional (see e.g., Fig. 8).
  • a P22 immunity region was utilized instead of a l immunity region. A phage cross between lBHI and l/m/i?
  • P22dis resulted in the replacement of ⁇ 6 kb of the immunity region of lBHI with a ⁇ 5 kb portion of that from / «? «?P22dis while retaining 933W-cE nd ⁇ and KarP genes (see e.g., Fig. 2D, Fig. 7, and Table 3).
  • This new phage, lBH2 showed improved EOP to 90% (see e.g., Fig. 2D) and demonstrated a functional loss of l immunity and gain of P22 immunity, as well as expression of functional 933W-cF nd ⁇ (see e.g., Fig. 8).
  • Stx2 repression is maintained under inducing conditions.
  • DNA damaging agents such as antibiotics can induce lambdoid prophages towards lysis (see e.g., Fig. 1A, lower panel) by activating the bacterial SOS response leading to RecA-mediated degradation of Cl (see e.g., Fig. 2C).
  • Stx2 produced in cultures of lBH2 phage mixed with E. coli 933W was measured in the presence of an inducing agent, mitomycin c. As shown in Fig. 3 C, E.
  • coli 933 w receiving buffer alone produced substantially more Stx2 when incubated with mitomycin c due to induction of the 933 W prophage.
  • This induction also directed other phages towards primarily lytic replication and so the introduction of /. mm P22dis phage significantly reduced E. coli 933W concentrations (see e.g., Fig. 3D induced) and consequently Stx2 concentrations (see e.g., Fig. 3C).
  • lBH2 phage treatment achieved significantly lower Stx2 concentrations than those measured for buffer and /J «? «?P22dis phage conditions (see e.g., Fig. 3C) because it was capable of repressing six 2 expression from a large fraction of E. coli 933W as shown by the substantial lysogenic conversion (see e.g., Fig. 3E induced).
  • lBH2 lysogens of E. coli 933W were cultured for up to 8 h in the presence of mitomycin c (see e.g., Fig. 3F).
  • the toxin was undetectable, indicating repression was maintained under inducing conditions (see e.g., Fig. 3H).
  • lBH2 reduced fecal Stx2 concentrations in mice.
  • a mouse model of enteric Stx2 intoxication from Stx- producing E. coli was used. While it is challenging to model the effect of enteric pathogens including Stx-producing E. coli in mice, mitomycin c injections can induce substantial quantities of Stx that is otherwise too low to be detected in stool. Mice pre-colonized by E. coli 933W were orally treated with lBH2 phage and then received three doses of mitomycin c by intraperitoneal injection to induce stx2 expression (see e.g., Fig. 4A).
  • coli lacking the 933W prophage see e.g., Fig. IE.
  • a high level of induction can disfavor lysogeny, and the use of a mitomycin c mouse model may underestimate the achievable degree of lysogeny in more typical, less-inducing conditions such as those shown in Fig. 1B-1E.
  • Temperate phage l were genetically engineered to express a repressor that neutralizes Stx production in E. coli and take advantage of the genetic mosaicism of lambdoid phages to create a hybrid phage that is capable of overcoming phage resistance mechanisms.
  • the anti virulence phage not only efficiently infected, lysogenized, and inhibited Stx2 production from E. coli in vitro, but it also was effective at propagating in the murine gut from a single dose to significantly reduce Stx2 production in vivo.
  • mice Female BALB/c mice (Charles River LaboratoriesTM) 6-7 week old were acclimated for one week prior to experiments.
  • mice received 5 g/L of streptomycin sulfate (Gold BioTM) in their drinking water which was replaced every 2-3 days. On day 0, 100 pL of streptomycin-resistant A. coli MG 1655 was administered to mice by oral gavage.
  • the bacterial gavage solution was prepared from an overnight culture in LB, washed twice with PBS, and then diluted 100-fold into PBS, yielding ⁇ 10 7 cfu/mL (colony forming units per milliliter).
  • mice received 100 pL of lBHI phage which consisted of a 5 x 10 7 pfu/rnL solution diluted 1:10 into 100 mM sodium bicarbonate immediately prior to gavage.
  • Daily stool samples were collected for microbial quantification.
  • To quantify fecal phage fresh non-frozen samples were gently suspended into 1 mL of phage buffer, incubated at 4°C for ⁇ 10 min with a few drops of chloroform, and then pelleted at 4000 rpm at 4°C. Phage concentration was determined using a double-agar overlay plaque assay in which serially diluted phage solutions were incubated for 20 min at r.t. (room temperature) with a hardened overlay of E.
  • coli MG1655 in 0.3% agar in TNT media over a 1.5% agar in TNT base. After aspiration, plates were incubated at 37°C overnight after which plaques were counted.
  • frozen stool was thawed from -80°C and suspended into 1 mL of PBS by vortexing for 10 min at 4°C followed by low-speed centrifugation at 200 rpm for 20 min to settle fecal debris. The fecal suspension was then serially diluted into PBS and 100 pL was plated onto MacConkey agar (RemelTM) plates supplemented with 100 pg/mL streptomycin sulfate to quantify total E. coli or supplemented with 100 pg/mL streptomycin and 50 pg/mL kanamycin to quantify lBHI lysogens of E. coli.
  • RemelTM MacConkey agar
  • mice were treated with similar conditions as described above with the following modifications.
  • mice received 100 pL of similarly prepared streptomycin-resistant E. coli 933W in PBS by oral gavage.
  • mice received 100 pL of lBH2 phage, which was a 5 x 10 10 pfu/mL solution diluted 1:10 into 100 mM sodium bicarbonate immediately prior to gavage.
  • mice received three intraperitoneal injections of 0.25 mg/kg of mitomycin c at 3 h intervals.
  • Fecal E. coli was quantified by plating as described above, and fecal Stx2 was quantified from the same suspension of stool in PBS by mixing 10: 1 with 20 mg/mL of polymyxin B, incubating at 37°C for ⁇ 20 min and then storing at -20°C until analysis by ELISA as described below.
  • E. coli 933W was generated by a previously described method (see e.g., O’Brien et al. Science 226, 694 (1984))., in which 933W phage was produced from the supernatant of a log phase culture of E. coli 0157:H7 strain edl933 in a modified LB media (10 g/L tryptone, 5 g/L yeast extract, 5 mM sodium chloride, 10 mM calcium chloride, and 0.001% thiamine) and then stored at 4°C. Molten top agar containing 100 pL of E.
  • E. coli lysogens of l and Hmni933W were generated similarly using TNT media instead of modified LB media and screened for successful lysogeny by resistance to l or Hmni933W, respectively, and ability to produce phage progeny.
  • Phage strains A table of phage used herein is listed in Table 2.
  • lBHI phage was generated using the inherent ' /.red recombination system of l phage expressed during lytic replication.
  • a crude phage lysate containing recombinant phage was produced by mixing 100 pL of E. coli C600, containing a plasmid vector with Tn5-933W-cI md flanked by 400 bp homology to ea59 and ea47 in a pJET1.2 backbone (see e.g., Table 3), with 100 pL of serially-diluted l phage, incubated for 20 min at r.t.
  • top agar molten top agar
  • TNT media with 0.3% top agar at 45 °C
  • molten top agar TNT media with 0.3% top agar at 45 °C
  • top agar from the plate with the greatest plaque density was resuspended into 5 mL of phage buffer (50 mM tris, 100 mM sodium chloride, 10 mM magnesium sulfate, and 0.01% gelatin, pH 7.5), sterile filtered, and stored at 4°C.
  • 50 pL of crude phage lysate was mixed with 50 pL of E.
  • coli C600 grown to log-phase in LB and incubated for ⁇ 3 h at 37°C. After incubation, 100 pL was plated onto LB containing 50 pg/mL of kanamycin and grown overnight at 37°C with individual colonies re streaked twice. To additionally purify by plaque purification, colonies were grown overnight in LB, and their sterile filtered supernatants were spotted onto TNT top agar of E. coli C600. Individual plaques were streaked onto LB containing 50 pg/mL of kanamycin and sequenced to confirm insertion in the correct locus of l phage.
  • lBH2 phage was generated by a phage cross between lBHI phage and /./m «?P22dis phage.
  • 200 pL of a log phase E. coli C600 culture (7 x 10 7 cfii/mL) in T-broth (l%tryptone and 0.5% sodium chloride) with 0.4% maltose was mixed with a 200 pL solution of lBHI phage (1.5 x 10 8 pfu/mL) and /./m«?P22dis (1.5 x 10 8 pfu/mL) in phage buffer.
  • Phage lysates were prepared by culturing colonies overnight in TNT media, pelleting and sterile filtering the supernatant, and then incubating 100 pL of this phage mixture with 100 pL of E. coli C600 (MOI-0.1) at 37°C for 20 min and then plating onto LB with 50 mg/mL of kanamycin. After overnight incubation at 37°C, phage was plaque purified by preparing phage lysates from individual colonies as described above and streaking 10 pL onto hardened top agar containing E. coli C600. After overnight incubation at 37°C, individual plaques were picked and restreaked onto LB with kanamycin. The resultant lBH2 lysogen of E.
  • coli was confirmed susceptible to l and ' kimmA3A as well as resistant to Hmni933W and ' /.immPlldis (see e.g., Fig. 8). Sequencing confirmed the presence of the P22 immunity region and 933Wc nd ⁇ gene (see e.g., Fig. 7 and Table 3).
  • E. coli suspension was mixed with 1 mL of 4 x 10 8 pfii/mL of lBH2 or / «? «P22dis phage solution.
  • E. coli concentrations in solution aliquots were collected, serially-diluted into PBS and the spotted (10 pL) onto LB or LB with 50 pg/mL kanamycin plates to quantify total E. coli and lBH2 lysogens, respectively. After 8 h, aliquots were mixed 10: 1 with 20 mg/mL of polymyxin B, incubated at 37°C for ⁇ 20 min and stored at -20°C for quantification of Stx2 by ELISA.
  • temperate phages can be engineered to modulate bacterial gene expression. Key is that this approach does not kill the targeted bacterium and instead induces targeted gene knockdown. This approach can be seen as a modular anti-virulence approach that reduces the encouragement of resistance development typically seen with bacteriolytic approaches like lytic phages and antibiotics. In some cases, antibiotics are not prescribed because they can make the bacterial infection worse (e.g., shigatoxin producing bacteria, enterohemorrhagic E. coli).
  • Temperate phages can infect a bacterium and predominantly integrate their genomes into the bacterial genome (e.g., as a prophage), replicating along with the bacterium.
  • an engineered phage was used to intracellularly produce a repressor of shigatoxin (e.g., cl protein of 933 W prophage) and demonstrate that this neutralizes shigatoxin expression in vitro and in enteric mouse models of infection.
  • engineered phage using a deactivated Cas9 were used to allow the customization of the target gene for repression. As shown herein, this phage can repress expression of a fluorescent protein in E. coli.
  • lambda phage were engineered to include a deactivated Cas9 (dCas9) that binds to targeted regions using a crispr RNA guide sequence.
  • dCas9 deactivated Cas9
  • the engineered phage with crRNA targeting RFP neutralize RFP fluorescence (see e.g., Fig. 11 panel iii) whereas the same construct without the crRNA has no effect (see e.g., Fig. 11 panel iv).
  • FIG. 12 An example of flow cytometry for mCherry fluorescence in E. coli is shown in Fig. 12.
  • E. coli was mixed with buffer or engineered dCas9 phage with and without crRNA for 1 d and analyzed by flow cytometry to quantitate intracellular mCherry fluorescence.
  • E. coli treated with ::dCas9- crRNA showed similar fluorescence levels to those receiving buffer whereas E. coli treated with ::dCas9+crRNA.
  • engineered phage comprising crRNA targeting the mCherry gene for repression showed a marked shift in fluorescence towards that of the E. coli control that lacks mCherry.
  • This strategy can be used to selectively repress any number of genes.
  • the selectivity of phage allows the specific targeting of a particular species within a complex mixture, such as the gut microbiome, as demonstrated herein.
  • This method is also adaptable using an activating Cas9 construct to increase gene expression as well.
  • the targeted anti-virulence of pathogens can be used as an alternative to antibiotics that minimizes the development of resistance.
  • the gut microbiome has numerous associations with human health.
  • This bacterial community contains hundreds of densely colonizing species with a composition that varies along the gastrointestinal tract, between individuals, and overtime.
  • the complexity of this ecosystem makes it challenging to precisely target specific bacteria without unintended impacts to the microbiota.
  • generalizable tools are needed, especially ones capable of modifying specific bacterial functions while minimizing disruption to non-targeted genes, microbes, and host physiology.
  • Phages are viewed as possible therapeutics due to their capability of targeting specific bacteria, even when the bacteria are part of complex consortia. Lytic phages, which kill their cognate bacteria during phage propagation, have been of particular interest due to the prevalence of antibiotic resistant infections. Temperate phages have been of less interest therapeutically because they do not primarily pursue lysis and can integrate themselves into the bacterial genomes as prophages. While temperate phages may have some utility as antibacterial agents on their own or as adjuvants, their specificity and lysogenic conversion of bacteria offers an alternative non-lytic strategy: introducing new genes or reprogramming endogenous gene expression in precisely targeted bacteria within their natural ecosystems such as the gut microbiota. As described in Example 1, bacterial virulence can be repressed in vitro and in the mammalian gut using a lysogenic phage engineered to specifically repress shigatoxin expression.
  • Described herein is a non-invasive strategy to modify gene expression of specific bacteria in the mammalian gut via oral delivery.
  • temperate phage l were engineered to express a nuclease-deactivated Cas9 (dCas9) that specifically represses gene expression in bacteria both in vitro and when colonizing the mouse gut.
  • dCas9 nuclease-deactivated Cas9
  • an encapsulation formulation was developed to protect phage during oral delivery.
  • described herein is a non-invasive and minimally-disruptive in situ modification of bacteria in the mammalian gut.
  • Engineered phage containing dCas9 represses target gene function.
  • the engineered construct was inserted into the l genome by replacing a portion of the non- essential b2 region.
  • the system was tested in E. coli containing genomically-integrated rfp and gfp genes.
  • the plasmid-based S. aureus dCas9 was effective in repressing fluorescence (see e.g., Fig. 16A-16C).
  • phage containing crRNA targeting rfp (7::dCas9 rlp was added to E.
  • the efficacy of the engineered phage was tested in vivo, by administration of /.::dCas9 /.::dCas9 rlp or vehicle (phage buffer) to mice pre-colonized with RFP-expressing E. coli.
  • the fecal phage and E. coli concentrations were then tracked overtime (see e.g., Fig. 14A).
  • phage solutions and vehicle were diluted 10-fold into a sodium bicarbonate solution immediately prior to oral gavage.
  • Figure 14B fecal phage were detectable at high concentrations soon after administration.
  • Lysogenized E. coli have reduced fluorescence. Functional gene repression by the engineered phage was assessed by measuring the relative RFP fluorescence of l: ⁇ Oh89 and /.::dCas9 rlp lysogens isolated from mouse stool after oral phage administration. As shown in the representative culture plates in Figure 14E, fecal E. coli colonies from mice receiving /.::dCas9 rlp phage demonstrated a maintained GFP and reduced RFP fluorescence compared to fecal colonies from mice receiving /.::dCas9 phage.
  • Non-invasive and minimally disruptive modification of gut bacteria modifies gut bacteria in situ without compromising the gastric barrier.
  • /.::dCas9 rlp phage significantly repressed RFP fluorescence in gut bacteria compared to l: :dCas9 phage, which lacks targeting to the rfp gene of E. coli.
  • Phage administered in water is readily inactivated during oral administration unless the stomach acid is neutralized, phage is given at very high concentration, or phage is encapsulated (data not shown).
  • a low concentration (5 xlO 3 pfu) of this phage was administered either as encapsulated phage suspended in water or as free phage mixed with bicarbonate buffer to neutralize the stomach acid (see e.g., Fig. 15A).
  • Examination of mouse stool after oral phage administration revealed similar results in the following measurements for the bicarbonate-buffered phage compared to the encapsulated phage: fecal phage concentration (see e.g., Fig. 15B); total fecal E. coli concentration (see e.g., Fig.
  • Manipulating bacterial processes in situ is a powerful tool for dissecting the causal relationship between microbial and host physiology.
  • Current approaches, such as the use of small molecules to interrogate the impact of microbial metabolites are limited by off target effects.
  • the approach described herein allows for a fine tuned genetic control of a single gene within a single microbe, which makes it possible to systematically screen diverse genetic targets, characterize cause- effect relationships, and identify cascading effects to the surrounding microbiota and host.
  • the phage described herein can be used as a therapeutic, with durable modification of gut bacteria using a single, self-propagating dose.
  • microbiomes of other bodily regions such as the mouth, skin, bladder, nose, ears, eyes, and lungs can also be targeted by the engineered phage described herein.
  • microbiomes in animals, plants, insects, soils, and bodies of water are all be targets for the modification of specific microbes using the engineered phage described herein.
  • Golden Gate Assembly was used for cloning plasmids (see e.g., SEQ ID NOs: 30-31).
  • Q5 Hot StartTM polymerase was used to amplify the proC promoter, l phage homology arms, pACYCDuet origin of replication, and pACYCDuet chloramphenicol resistance cassette. Restriction sites were also added during PCR.
  • Chloramphenicol acetyltransferase was used as the antibiotic resistance and Cas9 from Staphylococcus aureus because of their minimal size. Cas9 was modified with nuclease -inactivating mutations in the HNH and RuvC catalytic regions (see e.g., Friedland et al. Genome Biol. 16, 257 (2015); Nishimasu et al. Cell 162, 1113-1126 (2015)).
  • Golden Gate reactions were run using 10xT4 Ligase Buffer (PromegaTM), T4 Ligase (2,000,000 units/mL, NEB), and bovine serum albumin (BSA) (10 mg/mL, NEB) as well as the appropriate restriction enzyme, either Eco31I, Esp3I, or Sapl (Thermo FastDigestTM). Golden Gate reactions were desalinated using drop dialysis (for a minimum of 10 minutes) and electroporated in DH 10b Electrocompetent Cells (Thermo FischerTM). Plasmids were verified by sequencing all junctions as well as the entirety of the gBlocks.
  • the gRNA spacers were synthesized as complementary oligos then added to plasmids by Golden Gate after being annealed and phosphorylated. This was done by incubating the oligos with T4 Ligase Buffer (NEB) and T4 Poly Nucleotide Kinase (NEB), heating to boiling, and then slowly cooled to room temperature. Annealed oligos were added to plasmids using Eco31I. The gRNA spacers were verified by sequencing.
  • a double agar layer plaque assay method 100 pL of this culture was mixed with 100 pL of serially diluted l phage in phage buffer (e.g., 50 mM tris, 100 mM sodium chloride, 10 mM magnesium chloride, and 0.01% gelatin at pH 7.5) and then mixed with 3 mL of molten top agar (e.g., TNT with 0.3% agar) and immediately poured onto TNT agar plates to harden. After overnight culture at 37°C, the top agar from plates containing the highest density of individual plaques were resuspended into 5mL of phage buffer with gentle rocking at 4°C for 2h. These suspensions were then pelleted with the supernatant filtered through 0.45 um syringe filters, yielding crude phage lysates.
  • phage buffer e.g., 50 mM tris, 100 mM sodium chloride, 10 mM magnesium chloride, and 0.0
  • E. coli C600 grown to late log in TNT with 0.4% maltose was concentrated by pelleting and resuspension to ⁇ 10 10 cfti/mL. 200 pL of this bacterial suspension was mixed with 200 pL of crude phage lysate and incubated at 37°C for 2.5 h, statically. Cultures were then plated onto LB with 34 ug/mL chloramphenicol and incubated overnight at 37°C. For plaque purification, phage was isolated by culturing streak-purified colonies in TNT overnight at 37°C, then treated with chloroform and pelleted. Plaques were then generated from serial dilutions of the supernatant by double overlay plaque assay with E.
  • GFP fluorescence (ex 485nm/em 528nm), and RFP fluorescence (ex 555nm/ em 584 nm) at 5 min intervals (BioTek Synergy H1MFTM).
  • Studies of non-lysogens, l:AOh89 lysogens or /.::dCas9 rlp lysogens were setup similarly except that 200 pL of log-phase cultures were used.
  • coli was prepared by inoculating an overnight culture (-16-20 h) in LB with 100 pg/mL streptomycin, then washing twice by pelleting and then re-suspending in an equal volume of PBS, and then diluting 10-fold into PBS to yield -10 7 - 10 8 cfii/mL.
  • 100 pL of phage solution was administered by oral gavage.
  • E. coli was quantified by plating lOOuL of serial 10-fold dilutions in PBS onto MacConkey agar (RemelTM) supplemented with (100 pg/mL) streptomycin to quantify total E.
  • mice were allowed free access to food and water, then sacrificed under CO2 and cervical dislocation, dissected, and a pH probe was immediately inserted into the gastric contents for measurement.
  • SEQ ID NO: 20 4865 nt, 991-5855 of IP 567 (SEQ ID NO: 30); l phage homology arms, proC promoter, S. aureus dCas9, the dCas9’s tracrRNA under a constitutive promoter, a control crRNA (Bsal spacer) under a constitutive promoter, and pACYCDuet chloramphenicol resistance cassette for insertion into l. See e.g., Table 4 for region descriptions.
  • SEQ ID NO: 21, 4868 nt, 991-5858 of IP 568 SEQ ID NO: 31
  • l phage homology arms proC promoter, S. aureus dCas9, the dCas9’s tracrRNA under a constitutive promoter, the dCas9’s crRNA under a constitutive promoter, and pACYCDuet chloramphenicol resistance cassette for insertion into l. See e.g., Table 4 for region descriptions.
  • a TCTA GTA AAA GA A GGGA AAA TGGGGTGTTTA TGTGGTGA A GTTGTTGGGGA GA TTT

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Abstract

The technology described herein is directed to compositions and methods comprising engineered bacteriophage. In one aspect, described herein is an engineered bacteriophage comprising at least one nucleic acid encoding an inhibitor of a virulence factor encoded by a bacterium; and an immunity region from a second bacteriophage; wherein the engineered bacteriophage is lysogenic and infects the bacterium without killing the bacterium. In one aspect, described herein is a method of treating a bacterial infection comprising administering an engineered bacteriophage. In one aspect described herein is a method of inhibiting bacterial growth or activity on a surface comprising contacting a surface with an engineered bacteriophage.

Description

COMPOSITIONS AND METHODS COMPRISING ENGINEERED BACTERIOPHAGE
CROSS-REFERENCE TO REUATED APPUICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/909,988 filed October 3, 2019, the contents of which are incorporated herein by reference in their entirety.
GOVERNMENT SUPPORT
[0002] This invention was made with government support under Grant No. HR0011-15-C-0094 awarded by the Defense Advanced Research Projects Agency. The government has certain rights in the invention.
SEQUENCE UISTING
[0003] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 1, 2020, is named 002806_094760_SL.txt and is 463,392 bytes in size.
TECHNICAL FIELD
[0004] The technology described herein relates to compositions and methods comprising engineered bacteriophage.
BACKGROUND
[0005] The human gut microbiota is a collection of microbes colonizing the gastrointestinal tract and has been associated with various aspects of human health. While this community typically works concert with the human body, substantial perturbations such as antibiotics or infections can disrupt the microbial balance and lead to long lasting dysbiosis. In some instances, pathogenic bacteria do so by transmitting virulence factors encoded by these pathogens to commensal bacteria through plasmid- based and phage-based horizontal gene transfer (HOT). Remediating diseases associated with these pathogens while minimizing unintended and disruptive effects to the surrounding microbiota remains challenging especially with the limited tools available for targeting particular species.
[0006] The ability to manipulate the composition and function of the gut microbiota is presently limited in terms of precision and durability. Antibiotics non-specifically decimate swaths of gut species, dietary changes affect both the overall microbiota and mammalian host, probiotics poorly engraft due to colonization resistance, and even highly-specific lytic phages can cause cascading effects in the bacterial community despite targeting specific species. While in some cases these strategies may show transient efficacy, the emergence of resistant mutants can impact therapeutic effect. Broadly resetting the gut microbiota through fecal microbiota transplants (FMTs) has been promising especially for treating Clostridium difficile infections, but they are difficult to characterize and may transmit unintended traits such as obesity.
[0007] An alternative strategy is to modify bacterial function within its native environment. For example, one approach has been to develop drugs that target the virulence factors of anti-biotic resistant pathogens to specifically neutralize their deleterious effects and minimizing selection for resistance. While a number of anti-virulence drugs are under investigation the targets for inhibition are generally limited to those accessible by small molecules and biologies (i.e., surface-bound and secreted proteins), may require multiple drugs targeting multiple virulence factors, and could have off target effects on other microbes and the host (see e.g., Dickey et al. Nature Reviews Drag Discover ' 16, 457 (2017)). While the principle of anti -virulence is atractive, it remains challenging application.
[0008] Shigatoxin (Stx)-producing E. coli is one example of a pathogenic infection that is challenging to treat. Anti-virulence drugs targeting the toxin have been investigated but failed clinical trial (see e.g., Trachtman et al, JAMA 290, 1337-1344 (2003)). Antibiotics are contraindicated because of their potential to exacerbate virulence. While there are multiple virulence factors in the foodbome pathogen enterohemorrhagic E. coli (EHEC), Stx is significantly associated with disease severity and can lead to hemolytic uremic syndrome. Of the two main Stx variants — Stxl and Stx2 — the latter is -1000-fold more toxic. Similar to a number of other prophage-encoded virulence factors, Stx is not expressed while the phage is in a lysogenic state, i.e. stably integrated into the bacterial genome. It is not until induction, whether occurring spontaneously or from stimuli such as antibiotics, that the lytic life cycle is activated to produce Stx2 and progeny phage that can spread virulence genes to commensal E. coli species.
SUMMARY
[0009] The technology described herein is directed to compositions and methods comprising engineered bacteriophage as alternative to an antimicrobial strategy for killing pathogens. Described herein is a genetic-based anti-virulence strategy that can neutralize virulence before expression and minimize resistance until, e.g., the bacteria have been completely shed from the gastrointestinal tract. Temperate phages offer a solution as they are genetically engineerable and can integrate into the bacterial chromosome as prophages for long-lasting effect as they confer fitness advantages to the bacterial host. Instead of relying on a non-native constituent of the gut that could face practical barriers for efficacy, temperate phages are abundantly found in human gut bacteria and can constitute large portions of the bacterial chromosome. [0010] To modify the function of specific species within the complex community in the mammalian gut, in a manner that avoids the emergence of resistance, described herein is a genetically-engineered temperate phage to repress Six in an established E. coli infection. Genetic hybrids between lambdoid phages can overcome phage resistance mechanisms while maintaining function. A transcriptional repressor of Stx was then genetically encoded in the engineered phage, and this engineered phage substantially reduced Stx produced by E. coli in vitro. Finally, the engineered phage, when administered to mice pre-colonized by Stx -producing E. coli, can propagate throughout the murine gut from a single dose to significantly reduce fecal Stx concentrations. Thus, described herein is a therapeutic framew ork for the in situ modification of gut bacteria for genetic-based anti- virulence.
[0011] Accordingly, in one aspect described herein is a bacteriophage, wherein the bacteriophage genome is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium.
[0012] In some embodiments of any of the aspects, the bacteriophage is an engineered lambda (l) phage.
[0013] In some embodiments of any of the aspects, the inhibitor comprises an inhibitor protein encoded by a phage endogenous to the bacterium.
[0014] In some embodiments of any of the aspects, the inhibitor comprises cl protein from enterobacteria phage 933 W.
[0015] In some embodiments of any of the aspects, the inhibitor comprises one of SEQ ID NOs: 9-10 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 9-10 that maintains the same function.
[0016] In some embodiments of any of the aspects, the inhibitor is engineered to be non- degradable.
[0017] In some embodiments of any of the aspects, the cl protein from enterobacteria phage 933W comprises a K178N mutation that causes the protein to be non-degradable.
[0018] In some embodiments of any of the aspects, the inhibitor comprises a Cas protein and at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor.
[0019] In some embodiments of any of the aspects, the Cas protein comprises S. aureus Cas9.
[0020] In some embodiments of any of the aspects, the Cas protein comprises SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 that maintains the same function.
[0021] In some embodiments of any of the aspects, the CRISPR guide RNA comprises a trans activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA). [0022] In some embodiments of any of the aspects, the tracrRNA comprises SEQ ID NO: 25 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 25 that maintains the same function.
[0023] In some embodiments of any of the aspects, the crRNA comprises a variable targeting sequence and a region that is substantially complementary to a region of the tracrRNA.
[0024] In some embodiments of any of the aspects, variable targeting sequence of the crRNA is substantially complementary to a nucleic acid encoding a bacterial virulence factor.
[0025] In some embodiments of any of the aspects, the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 29 that maintains the same function.
[0026] In some embodiments of any of the aspects, the bacterium is Escherichia coli ( E . coli).
[0027] In some embodiments of any of the aspects, the bacterium is enterohemorrhagic E. coli
(EHEC).
[0028] In some embodiments of any of the aspects, the virulence factor is Shiga Toxin (Stx).
[0029] In some embodiments of any of the aspects, the engineered bacteriophage genome further comprises a heterologous bacteriophage immunity region.
[0030] In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is a lambdoid phage immunity region.
[0031] In some embodiments of any of the aspects, the lambdoid phage is selected from the group consisting of lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22.
[0032] In some embodiments of any of the aspects, the heterologous bacteriophage immunity region comprises one of SEQ ID NOs: 13-17 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 13-17 that maintains the same function.
[0033] In some embodiments of any of the aspects, the heterologous bacteriophage immunity region comprises SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 that maintains the same function.
[0034] In some embodiments of any of the aspects, the engineered bacteriophage genome further comprises a nucleic acid encoding a selectable marker.
[0035] In one aspect described herein is a bacteriophage, wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
[0036] In one aspect described herein is a bacteriophage, wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and (b) a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium. [0037] In one aspect described herein is a bacteriophage, wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
[0038] In one aspect described herein is a bacteriophage, wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, and (b) a heterologous bacteriophage immunity region wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
[0039] In one aspect described herein is a bacteriophage, wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and (b) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
[0040] In one aspect described herein is a bacteriophage, wherein the bacteriophage genome is engineered to comprise: (a) at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, (b) at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: (i) a Cas protein and (ii) at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, and (c) a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
[0041] In one aspect described herein is a pharmaceutical composition comprising the engineered bacteriophage as described herein and an acceptable carrier.
[0042] In one aspect described herein is a method of treating a bacterial infection, comprising administering an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein to a patient in need thereof.
[0043] In some embodiments of any of the aspects, the patient is infected with E. coli or EHEC.
[0044] In one aspect described herein is a method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein.
[0045] In one aspect described herein is a composition for use in a method of treating a bacterial infection, the method comprising administering an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein to a patient in need thereof. [0046] In some embodiments of any of the aspects, the patient is infected with E. coli or EHEC.
[0047] In one aspect described herein is a composition for use in a method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of an engineered bacteriophage as described herein or a pharmaceutical composition as described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] Fig. 1A-1E is a series of schematics and graphs showing that temperate phage robustly transduces E. coli colonizing the mouse gut. The upper panel of Fig. 1A is a schematic showing that lytic phages decimate a bacterial population, selecting for resistant mutants that can repopulate over time i) In lytic phage infection, the phage particle injects its genetic material into a bacterium and ii) directs the cell to produce phage components, iii) which are released upon cell lysis to continue infection. The lower panel of Fig. 1A is a schematic showing that temperate phages can infect a bacterial population to lysogenize phage-susceptible bacteria which persists overtime iv) Temperate phages can also participate in the lysogenic life cycle where they integrate their DNA into the bacterial genome and v) remain as a prophage during bacterial proliferation with the possibility of entering lytic replication in the future. Fig. IB is a schematic showing an experimental timeline examining the impact of lBHI phage on pre-colonized E. coli in mice. Fig. 1C is a dot plot showing fecal concentrations of free lBHI phage. Fig. ID is a dot plot showing fecal concentrations of E. coli. Fig. IE is a bar graph showing percentage of fecal E. coli lysogenized by lBHI. Symbols (e.g., in Fig. 1C-1E) represent individual mice (n = 3) with lines or bars representing the geometric mean. [0049] Fig. 2A-2D is a series of schematics and graphs showing that hybrid l phages overcome superinfection exclusion by prophage 933W. Fig. 2A is a schematic showing a depiction of superinfection exclusion by the 933W prophage that inhibits infection by l phage but is ineffective against a hybrid phage that contains the immunity region from another lambdoid phage in a l phage background. Fig. 2B shows a schematic representation of a portion of the l phage genome containing the l immunity region and its hybrids containing immunity regions from other lambdoid phages (e.g., 933W, 21, 434, and P22) in a l phage background. Efficiency of plating (EOP) for l phage and its hybrids on E. coli 933W are shown in the right panel of Fig. 2B. Fig. 2C is a schematic showing genetic schemes of E. coli 933W showing: i) the 933W prophage expressing cl to maintain a lysogenic state in which stx2 is not expressed, ii) induction that causes degradation of the Cl protein leading to expression of the lytic genes including cro and stx2. This leads to cell lysis, releasing phage progeny and Stx2 protein iii) Expression of a non-degradable cl for the 933W prophage, 933W-cTnd~, from a genomically -integrated engineered temperate phage (anti -virulence prophage) can enforce the 933 W prophage to remain lysogenic despite induction and degradation of endogenous Cl protein. Fig. 2D shows a schematic representation of lBHI phage, which is a l phage with a kanamycin resistance cassette ( KanR ) and 933W-c nd~ inserted into the non-essential b2 region of the phage genome. lBH2 phage is a product of a phage cross between lBHI and ' /.immPlldis resulting in a phage containing KanR and 933W-cFnd~ genes with a P22 immunity region in a l phage background. Their respective EOP against E. coli 933W are shown in the right panel of Fig. 2D. Symbols represent biological replicates with bars representing the geometric mean (e.g., in Fig. 2B, Fig. 2D.
[0050] Fig. 3A-3H is a series of schematics and graphs showing that engineered l phage neutralizes shigatoxin production from E. coli 933W in vitro. Fig. 3A is a schematic showing that E. coli 933 w was mixed with buffer, / «?«?P22dis or lBH2 free phage (MOI-l) at t = 0. from which the concentration of Stx2 was measured in Fig. 3B and Fig. 3C. (Fig. 3B is a bar graph showing the concentration of Stx2 measured after 8 hours (h) of in vitro culture under non-induced conditions. Fig. 3C is a bar graph showing the concentration of Stx2 measured after 8 h of in vitro culture under induced conditions (e.g., 0.5 pg/mL of mitomycin c). Significance was calculated by one-way ANOVA with post-hoc Tukey test (e.g., in Fig. 3B-3C). Fig. 3D is a bar graph showing total E. coli 933W measured over 8 h under non-induced and mitomycin c induced conditions. Fig. 3E is a bar graph showing the percentage of bacteria lysogenized by lBH2 measured over 8 h under non-induced and mitomycin c induced conditions. Fig. 3F is a schematic showing that E. coli 933W lysogenized with lBHI or lBH2 was cultured in vitro and analyzed for Stx2 produced in Fig. 3G and Fig. 3H. Fig. 3G is a bar graph showing Stx2 production under non-induced conditions. Fig. 3H is a bar graph showing Stx2 production under induced conditions. Symbols represent biological replicates with bars or lines representing the geometric mean (e.g., in Fig. 3B-3E, Fig. 3G-3H).
[0051] Fig. 4A-4F is a series of schematics and graphs showing that lBH2 phage lysogenizes E. coli 933W in the murine gut and reduces fecal shigatoxin concentrations. Fig. 4A is a schematic showing that streptomycin-treated mice pre-colonized with E. coli 933W received one dose of 5 x 109 pfu of lBH2 phage orally. Mitomycin c was administered thrice at 3 h intervals by intraperitoneal injection to induce Stx2 expression in the gut. Fig. 4B is a bar graph showing concentrations of fecal Stx2 after induction with mitomycin c. Mann-Whitney tests were used to compare Buffer and lBH2 conditions while Wilcoxon tests were used to compare between Day 3 and Day 4 within the same group. Fig. 4C is a bar graph showing concentrations of total fecal E. coli 933W, and Fig. 4D is a dot plot showing the percentage of fecal E. coli 933W found to be lysogenized by lBH2 phage. Fig. 4E is a dot plot showing the concentration of fecal Stx2 as a function of fraction of fecal E. coli 933W lysogenic for lBH2 phage on day 3. Fig. 4F is a dot plot showing the concentration of fecal Stx2 as a function of fraction of fecal E. coli 933W lysogenic for lBH2 phage on day 4. Line and dashed lines represent mean and 95% confidence intervals of linear regression, respectively (e.g., in Fig. 4E-4F). P value describes significance of slope being non-zero (e.g., in Fig. 4E-4F). Symbols represent individual mice for Buffer (n = 9) and lBH2 conditions (n = 10) (e.g., in Fig. 4B-4F). On Day 4, one buffer- treated mouse was unable to produce stool for analysis (e.g., in Fig. 4B-4C). Bars or lines represent geometric means (e.g., in Fig. 4B-4D).
[0052] Fig. 5 is an image showing spot testing of l and Hmm933W phage against lawns of their lysogens in E. coli. The zone of lysis (dark circle) on a lawn of E. coli indicates successful phage infection. Both phages are capable of infecting non-lysogens and lysogens of the other phage (i.e. l phage infecting a imm933W lysogen and vise-versa) indicating the 933W cI and l-cl are not inhibitory to l phage and 933W phage infection, respectively.
[0053] Fig. 6A-6C is a series of schematics showing genetic map of immunity regions within l phage and its hybrids. Fig. 6A is a schematic showing a genetic map of portions of the l phage genome with brackets indicating the regions within l that have been replaced with immunity regions from other lambdoid phages. Fig. 6B is a schematic; within Hmni933W phage, the genes homologous to 933W (see e.g., NCBI Accession ID NC.000924) are labeled in grey in or above the genetic map (e.g., cIII, ssB, L0079, N, L0081, stk, L0083, L0084, cl, cro, ell, O, P) and those homologous to l (see e.g., SEQ ID NO: 32 or NCBI Accession ID NC.001416.1) are labeled in black below the genetic map (e.g., kil, cIII, ealO, ren). Percent identities for genes less then identical are indicated in parenthesis. Fig. 6C is a schematic; within l m/i? P22dis phage, the genes homologous to P22 (see e.g., GenBank Accession ID AF217253) are labeled in grey in or above the genetic map (e.g., orf59, mnt, arc, ant, 9, orf485, gtrB, gtrA, abc2, abcl, erf, arf, kil, cIII, 17, orf67, orf78, ral, sieB, 24, c2, cro, cl, orf48, 18, 12) and those homologous to l (see e.g., NCBI Accession ID NC.001416.1) are labeled in black in or below the genetic map (e.g., J, ninB).
[0054] Fig. 7 is a series of schematics showing genetic maps of lBHI and lBH2 phage.
[0055] Fig. 8 is a series of pictures showing spot assays of 3 pL of ~107 pfu/mL of l, imm933W, imm434, and 7/mmP22dis phages against non-lysogenic E. coli, its l, lBHI, or lBH2 lysogen.
[0056] Fig. 9 is a series of line graphs showing Shigatoxin producing E. coli mixed with various phages (e.g., WT phage, engineered phage) in liquid culture with shigatoxin measured overtime. Of the non-engineered wild type phages (left panel), lambda (temperate) and lambda-immP22dis (temperate) have no marked impact on shigatoxin compared to the control (buffer only). The lytic T4 phage shows an initial suppression of shigatoxin production, but the expansion of T4-resistant E. coli leads to the eventual increase in shigatoxin production. Two types of lambda phage (right panel) were engineered, one that is unable to productively integrate itself into the bacterial genome (vl) and one that successfully integrates itself (v2). The v2 phage shows complete suppression of shigatoxin production.
[0057] Fig. 10 is a series of dot plots showing in vivo testing by colonizing mice with shigatoxin- producing E. coli, then administering no phage, vl or v2. Comparison of the shigatoxin production in stool between phage treatments (left panel) shows that the v2 engineered phage successfully represses shigatoxin in the gut. It was confirmed that a large fraction of the shigatoxin-producing bacteria harbor the v2 engineered phage using a selection marker (right panel).
[0058] Fig. 11 is a series of images showing lambda phage that were engineered to include a deactivated Cas9 (dCas9) that binds to targeted regions using a CRISPR RNA guide sequence. In an E. coli strain that constitutively expresses RFP and GFP (panel ii), the engineered phage with crRNA targeting RFP neutralize RFP fluorescence (panel iii) whereas the same construct without the crRNA has no effect (panel iv).
[0059] Fig. 12 is a plot showing flow cytometry data for mCherry fluorescence in E. coli. E. coli was mixed with buffer or engineered dCas9 phage with and without crRNA for 1 day and analyzed by flow cytometry to quantitate intracellular mCherry fluorescence. E. coli treated with ::dCas9-crRNA showed similar fluorescence levels to those receiving buffer whereas E. coli treated with ::dCas9+crRNA. i.e. engineered phage comprising crRNA targeting the mCherry gene for repression, showed a marked shift in fluorescence towards that of the E. coli control that lacks mCherry.
[0060] Fig. 13A-13E is a series of schematics and graphs showing in vitro gene repression by /.::dCas9rlp Fig. 13A is a scheme of in vitro experiment examining repression by engineered l phage. Fig. 13B-13C is a series of graphs showing E. coli cultures mixed with phage buffer, l::dCas9 phage or l::dCas9Ifp phage tracked for RFP fluorescence (Fig. 13B) and bacterial density (Fig. 13C). Fig. 13D-13E are a series of graphs showing non-lysogenic, l: :dCas9 lysogenic or /.::dCas9rlp lysogenic E. coli cultures tracked for RFP fluorescence (Fig. 13D) and bacterial density (Fig. 13E). Lines represent means and shaded regions represent standard errors.
[0061] Fig. 14A-14F is a series of schematics and graphs showing phage delivered genetic repression in vivo. Fig. 14A is a schematic showing that engineered phage or vehicle was orally administered in bicarbonate solution to mice pre-colonized with E. coli expressing RFP and GFP. Fig. 14B-14D is a series of graphs showing that after oral administration the following measured were quantified: fecal phage (Fig. 14B); total E. coli (Fig. 14C); and percentage of E. coli lysogenized by phage (Fig. 14D). Fig. 14E is a series of representative fluorescence images of colonies of fecal lysogens shown compared to in vitro cultured controls. Fig. 14F is a graph showing relative RFP intensity normalized by GFP intensity of fecal lysogens from mice. Positive and negative controls represent in vitro cultured /.::dCas9rlp and /.::dCas9 lysogens, respectively. Symbols represent individual mice (n = 5) with bars or lines indicating the geometric mean. Significance was calculated for preselected time-matched samples using one-way ANOVA with a Sidak post hoc test (****. P < 0.0001).
[0062] Fig. 15A-15E is a series of schematics and graphs showing that encapsulation protects oral phage but does not impair function. Fig. 15A is a schematic showing that free l::dCas9Gfp phage in bicarbonate buffer (to ensure survival) or encapsulated l::dCas9Ifp phage in water were orally administered to mice pre-colonized with E. coli expressing RFP and GFP. Fig. 15B-15D is a series of graphs showing that after oral administration the following measured were quantified: fecal phage (Fig. 15B); total E. coli (Fig. 15C); and percentage of E. coli lysogenized by phage (Fig. 15D). Symbols represent individual mice (n = 5) with bars or lines indicating the geometric mean. Fig. 15E shows an ensemble view of relative fluorescence from lysogens with in vitro cultured controls. Symbols represent individual colonies (~50 colonies per mouse sample or culture) with lines indicating the median.
[0063] Fig. 16A-16C is a series of schematics and graphs showing plasmid based in vitro gene repression. Fig. 15A is a schematic showing E. coli transformed with ate inducible expression of dCas9 with and without crRNA targeting rfp. Fig. 16B is a graph showing relative fluorescence of E. coli over time after induction with 25 ng ate. Fig. 16C is a graph showing relative fluorescence as a function of ate concentration after 6 h incubation. Symbols represent independent experiments (- crRNA, 0 ng = 2 experiments; -crRNA, 50 ng = 4 experiments; +crRNA, 0 ng = 2 experiments; +crRNA, 100 ng = 5 experiments; all other samples = 6 experiments) and lines represent the mean. [0064] Fig. 17 is a graph showing the ratio of phage to bacteria. The concentration is shown of relative phage to bacteria present in each fecal sample for each mouse. Symbols represent individual mice (n=5 per group) and lines represent the geometric mean.
[0065] Fig. 18 is a graph showing an ensemble view of relative colony fluorescence of fecal E. coli, specifically 50 colonies from mice receiving either l:^Oh89 (n=5; see e.g., dashed boxes) or /.::dCas9rlp phage (n=5; see e.g., dotted boxes). Controls represent lysogens after in vitro culture.
Lines represent the median.
[0066] Fig. 19 is a graph showing the ratio of phage to bacteria. The concentration is shown of phage relative to bacteria present in each fecal sample for each mouse. Symbols represent individual mice (n=5 per group) and lines represent the geometric mean.
DETAILED DESCRIPTION
[0067] The technology described herein is directed to compositions and methods comprising engineered bacteriophage as alternative to an antimicrobial strategy for killing pathogens.
[0068] Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria without unexpected cascading effects. As described herein, bacteriophage were used to deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long- lasting modification. As a real-world therapeutic test, l phage were engineered to transcriptionally- repress shigatoxin, and genetic hybrids between l and other lambdoid phages were used to overcome resistance encoded by the virulent prophage derived from enterohemorrhagic E. coli. A single dose of engineered phage propagated throughout the bacterial community and reduced shigatoxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. Relying on anti -virulence and not anti -bacterial action thus minimizes the selection for resistance. This work reveals a framework for transferring functions to bacteria within their native environment.
[0069] In one aspect, described herein is an engineered bacteriophage. As used herein, the term “bacteriophage” (which can be used interchangeably with the terms “phage” or “bacteria phage”) refers to a virus that infects and replicates within bacteria (e.g., prokaryotes) and/or archaea. In some embodiments of any of the aspects, the bacteriophage does not infect and replicate within eukaryotes. In some embodiments of any of the aspects, the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium. As used herein, the term “lysogenic” refers to a specific viral life cycle characterized by integration of the bacteriophage nucleic acid into the host bacterium's genome or formation of a circular replicon in the bacterial cytoplasm; lysogenic phage can also be referred to herein as temperate phage. In some embodiments of any of the aspects, the engineered bacteriophage is not lytic, which is to say that it does not kill or lyse the bacterium. In some embodiments of any of the aspects, the target bacterium is the natural host of the engineered bacteriophage. In some embodiments of any of the aspects, the target bacterium is not the natural host of the engineered bacteriophage, and the bacteriophage has been further engineered to infect a bacterium (e.g., by insertion or modulation of specific bacterial binding proteins in the phage, as well known in the art).
[0070] In some embodiments of any of the aspects, the bacteriophage is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor. In some embodiments of any of the aspects, the bacteriophage is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, or a heterologous bacteriophage immunity region. In some embodiments of any of the aspects, the bacteriophage is engineered to comprise a heterologous bacteriophage immunity region. In some embodiments of any of the aspects, the bacteriophage is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, and a heterologous bacteriophage immunity region.
[0071] In some embodiments of any of the aspects, the bacteriophage is an engineered lambda (l) phage. In some embodiments of any of the aspects, the bacteriophage is an engineered phage that infects and/or replicates in E. coli. In some embodiments of any of the aspects, the bacteriophage is an engineered phage that infects and/or replicates in at least one of the bacteria described herein.
[0072] In some embodiments of any of the aspects, the un-engineered wild-type lambda (l) phage comprises genome SEQ ID NO: 32 (Escherichia phage Lambda, complete genome GenBank: J02459.1 or NCBI Accession ID NC.001416.1) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 32 which retains the same activity as SEQ ID NO: 32. In some embodiments of any of the aspects, the un-engineered lambda (l) phage genome comprises SEQ ID NO: 32 or a sequence that is at least 95% identical to SEQ ID NO: 32 which retains the same activity as SEQ ID NO: 32. Any of the foregoing un-engineered lambda (l) phage genomes can be engineered as described herein.
[0073] In some embodiments of any of the aspects, the lambda (l) phage genome is engineered through homologous recombination with at least one heterologous nucleic acid sequence comprising two homology arms (also referred to herein as homology regions). Through homologous recombination, the nucleic acid sequence in between the homology arms in the lambda (l) phage genome (e.g., SEQ ID NO: 32) is replaced with the nucleic acid sequence in between the homology arms in the heterologous nucleic acid sequence (e.g., at least one of SEQ ID NOs: 1-6 or 20-21). In some embodiments of any of the aspects, the at least one heterologous nucleic acid sequence is inserted (e.g., through homologous recombination) in place of or within at least one non-essential viral gene in the phage genome. In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises at least one heterologous nucleic acid sequence selected from at least one of SEQ ID NOs: 1-6 or 20-21 or the reverse complement of at least one of SEQ ID NOs: 1-6 or 20-21. In some embodiments of any of the aspects, the heterologous nucleic acid sequence comprises a nucleic acid encoding an inhibitor (e.g., SEQ ID NO: 1 or one of SEQ ID NOs: 20-21). In some embodiments of any of the aspects, the heterologous nucleic acid sequence comprises an immunity region of a heterologous bacteriophage (e.g., one SEQ ID NO: 2-6).
[0074] In some embodiments of any of the aspects, the heterologous nucleic acid comprises one of SEQ ID NOs: 1-6 or 20-21 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 1-6 or 20-21 which retains the same activity as one of SEQ ID NOs: 1-6 or 20-21. In some embodiments of any of the aspects, the heterologous nucleic acid comprises one of SEQ ID NOs: 1-6 or 20-21 or a sequence that is at least 95% identical to one of SEQ ID NOs: 1-6 or 20-21 which retains the same activity as one of SEQ ID NOs: 1-6 or 20-21. See e.g., Fig. 6A-6C, Fig. 7, or Fig.
13A for exemplary schematics of lambda (l) phage engineered with a heterologous nucleic acid through recombination.
[0075] In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises an immunity region of a heterologous bacteriophage (e.g., one of SEQ ID NO: 2-6). In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises a nucleic acid encoding an inhibitor (e.g., SEQ ID NO: 1 or one of SEQ ID NOs: 20-21). In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises an immunity region of a heterologous bacteriophage (e.g., one SEQ ID NO: 2-6) and a nucleic acid encoding an inhibitor (e.g., SEQ ID NO: 1 or one of SEQ ID NOs: 20-21). [0076] In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises an engineered SEQ ID NO: 32, wherein at least one internal region of SEQ ID NO: 32 is replaced through homologous recombination with at least one of SEQ ID NOs: 1-6 or 20-21, or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to the engineered SEQ ID NO: 32 which retains the same activity as the engineered SEQ ID NO: 32. In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises an engineered SEQ ID NO: 32, wherein at least one internal region of SEQ ID NO: 32 is replaced through homologous recombination with at least one of SEQ ID NOs: 1-6 or 20-21, or a sequence that is at least 95%, or at least 99.9% identical to the engineered SEQ ID NO: 32 which retains the same activity as the engineered SEQ ID NO: 32.
[0077] In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises SEQ ID NO: 34 (e.g., lBH2), wherein SEQ ID NO: 1 and SEQ ID NO: 6 have been recombined into the wild-type l phage (e.g., SEQ ID NO: 32). In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises SEQ ID NO: 35 (e.g., lBHI), wherein SEQ ID NO: 1 has been recombined into the wild-type l phage (e.g., SEQ ID NO: 32). In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises SEQ ID NO: 36 (e.g., /xlCas9). wherein SEQ ID NO: 20 has been recombined into the wild-type l phage (e.g.,
SEQ ID NO: 32). In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises SEQ ID NO: 37 (e.g., dCas9rfp), wherein SEQ ID NO: 21 has been recombined into the wild-type l phage (e.g., SEQ ID NO: 32).
[0078] In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises one of SEQ ID NOs: 34-37 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 34-37 which retains the same activity as one of SEQ ID NOs: 34-37. In some embodiments of any of the aspects, the engineered lambda (l) phage genome comprises one of SEQ ID NOs: 34-37 or a sequence that is at least 95% identical to one of SEQ ID NOs: 34-37 which retains the same activity as one of SEQ ID NOs: 34-37.
[0079] In some embodiments of any of the aspects, the inhibitor comprises an inhibitor protein encoded by a phage endogenous to the bacterium. As used herein, “endogenous phage” (also referred to herein as a “prophage”) refers to a phage genome that has been integrated into the bacterial host genome. As a non-limiting example, phage 933 W can be an endogenous phage of specific E. coli strains. In some embodiments of any of the aspects, the inhibitor comprises an inhibitor protein encoded by phage 933 W. [0080] In some embodiments of any of the aspects, the inhibitor comprises cl protein from enterobacteria phage 933 W. In some embodiments of any of the aspects, the 933 W cl protein comprises SEQ ID NO: 9. In some embodiments of any of the aspects, the inhibitor comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 9, which retains the same activity or function as SEQ ID NO: 9 or 933W cl protein. In some embodiments of any of the aspects, the inhibitor comprises a sequence at least 95% identical to SEQ ID NO: 9, which retains the same activity or function as SEQ ID NO: 9 or 933W cl protein. In some embodiments of any of the aspects, the inhibitor comprises SEQ ID NO: 9 with a K178N mutation.
[0081] SEQ ID NO: 9 repressor protein Cl [Enterobacteria phage 933W]; NCBI Reference Sequence: NP_049485.1; 235 amino acids; residue 178 is shown bold and double-underlined.
1 mvqnekvrke faqrlaqack eagldehgrg maiaralsls skgvskwfha eslprqekmn 61 alakflnvdv vwlqhgtsln gandedtlsf vgklkkglvr vvgeailgvd gaiemteerd 121 gwlkiysddp dafglrvkgd smwpriksge yvliepntkv fpgdevfVrt veghnmikvl 181 gydrdgeyqf tsinqdhrpi tlpyhqvakv eyvagilkqs rhlddieare wlkss [0082] In some embodiments of any of the aspects, the engineered bacteriophage comprises SEQ ID NO: 1. In some embodiments of any of the aspects, the engineered bacteriophage comprises the unformatted black text of SEQ ID NO: 1 (see e.g., Table 3 and SEQ ID NO: 11). In some embodiments of any of the aspects, the engineered bacteriophage comprises SEQ ID NO: 10. In some embodiments of any of the aspects, the inhibitor comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 10, which retains the same activity or function as SEQ ID NO: 10 or 933W cl protein. In some embodiments of any of the aspects, the inhibitor comprises a sequence that is at least 95% identical to SEQ ID NO: 10, which retains the same activity or function as SEQ ID NO: 10 or 933W cl protein. In some embodiments of any of the aspects, the inhibitor comprises SEQ ID NO: 10 with a N178K mutation.
[0083] SEQ ID NO: 10: 933W cl protein, 235 amino acids; residue 178 is shown bold and double-underlined
MV QNEKVRKEFAQRLAQACKEAGLDEHGRGMAIARALSLS SKGV SKWFNAESLPRQEKMN
ALAKFLNVDVVWLQHGTSLNGANDEDTLSFVGKLKKGLVRVVGEAILGVDGAIEMTEERD
GWLKIYSDDPDAFGLRVKGDSMWPRIKSGEYVLIEPNTKVFPGDEVFVRTVEGHNMINVLG
YDRDGEYQFTSINQDHRPITLPYHQVAKVEYVAGILKQSRHLDDIEAREWLKSS
[0084] In some embodiments of any of the aspects, the inhibitor is engineered to be non- degradable or less degradable. Such engineering can include mutation of enzymatic action sites, e.g., protease recognition sequences, protease cleavage sites, and the like or the use of non-natural amino acids or peptide backbone structures. In some embodiments of any of the aspects, the 933W cl protein comprises a K178N mutation that causes the protein to be non-degradable or degraded less than the wild-type 933W cl protein. In some embodiments of any of the aspects, the 933W cl protein comprises an asparagine at residue 178 that causes the protein to be non-degradable or degraded less than the wild-type 933W cl protein.
[0085] In some embodiments of any of the aspects, the at least one nucleic acid of the engineered bacteriophage encoding an inhibitor comprises the unformatted black text of SEQ ID NO: 1 (see e.g., Table 3). In some embodiments of any of the aspects, the at least one nucleic acid of the engineered bacteriophage encoding an inhibitor comprises SEQ ID NO: 11 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 11, which retains the same activity as SEQ ID NO: 11, or a codon- optimized version thereof. In some embodiments of any of the aspects, the at least one nucleic acid of the engineered bacteriophage encoding an inhibitor comprises SEQ ID NO: 11 or a sequence that is at least 95% identical to SEQ ID NO: 11, which retains the same activity or function as SEQ ID NO: 11, or a codon-optimized version thereof.
[0086] SEQ ID NO: 11 atggttcagaatgaaaaagtgcgcaaagaattcgcccagcggctagcgcaagcctgtaaagaagctggtcttgatgaacatggtaggggaatggc tatagcccgtgccctttctctttcgtccaaaggcgttagcaaatggtttaatgctgagtctttaccgcgtcaggaaaaaatgaatgcgcttgcgaaatttc taaacgttgatgttgtttggcttcagcacggcacttcgttaaatggagcgaatgatgaagatactctttcatttgttggcaaattaaaaaaagggttagtg cgcgtggttggtgaggcaattcttggtgttgatggtgccatcgagatgaccgaagagcgcgatgggtggctcaaaatttatagcgatgatccagatg cctttggtcttcgtgtgaaaggagacagcatgtggcccagaataaaatcaggagaatatgtactcattgagcctaacaccaaagtattcccgggtgat gaggtgtttgtcagaaccgttgaaggacacaacatgattaacgttcttggctatgacagagatggagaataccaatttacaagcattaaccaggatca caggcctataacgttgccttatcatcaagtagcaaaggtggagtatgtagctggtattctgaagcaatctcgccatctggatgacatcgaggcaagg gagtggctgaaaagttcgtga
[0087] In some embodiments of any of the aspects, the engineered bacteriophage genome further comprises a promoter for the nucleic acid encoding the inhibitor. In some embodiments of any of the aspects, the promoter for the inhibitor is engineered to be non-inducible. In some embodiments of any of the aspects, the promoter for the inhibitor is the LacUV5 promoter. In some embodiments of any of the aspects, the promoter for the inhibitor is mutated for constitutive expression. In some embodiments of any of the aspects, the promoter of the inhibitor comprises SEQ ID NO: 12 (see e.g., Table 3) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 12 which retains the same activity as SEQ ID NO: 12. In some embodiments of any of the aspects, the promoter of the inhibitor comprises SEQ ID NO: 12 (see e.g., Table 3) or a sequence that is at least 95% identical to SEQ ID NO: 12 which retains the same activity as SEQ ID NO: 12.
[0088] SEQ ID NO: 12, LacUV5 promoter with mutated nucleotides (uppercase; e.g., nucleotides 76, 77, 90, 101 of SEQ ID NO: 12) for constitutive expression gcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatAAtgtgtggaattaTgagcggat aaTaatttcacacaggaaacagct
[0089] In some embodiments of any of the aspects, the inhibitor comprises a Cas9 protein and at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, the inhibitor comprises a Cas9 protein or at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor.
In some embodiments of any of the aspects, the inhibitor comprises a Cas9 protein. In some embodiments of any of the aspects, the inhibitor comprises at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, Cas9 protein is administered or supplied with the engineered bacteriophage comprising at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, the CRISPR guide RNA (also referred to herein as crRNA) can be designed according to methods as known in the art. In some embodiments of any of the aspects, the Cas9 is a deactivated Cas9 (dCas9), in other words the Cas9 is catalytically inactive or lacks nuclease activity.
[0090] In some embodiments of any of the aspects, the inhibitor comprises a CRISPR-Cas protein selected from the group consisting of C2cl, C2c3, Casl, CaslOO, Casl2a, Casl2b, Casl2c, Casl2d, Casl2e, Casl3a, Casl3b, Casl3c, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Casl, CaslB, CaslO, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csa5, Csa5, CsaX, Csbl, Csb2, Csb3, Cscl, Csc2, Csel, Cse2, Csfl, Csf2, Csf3, Csf4, Csm2, Csm3, Csm4, Csm5, Csm6, Csn2, Csxl, CsxlO, Csxl4, Csxl5, Csxl6, Csxl7, Csx3, Csyl, Csy2, Csy3, and homologues thereof, or modified versions thereof. It is noted that the inhibitor can be from an analog or variant of a known CRISPR-Cas protein.
[0091] It is noted that the CRISPR-Cas protein can be from an organism from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Leptospira, Desulfovibrio,
Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium or Acidaminococcus . In some embodiments of any of the aspects, the CRISPR-Cas protein is Cas9 from Staphylococcus aureus. In some embodiments of any of the aspects, the Cas9 protein is modified with nuclease -inactivating mutations in the HNH and/or RuvC catalytic regions. In some embodiments of any of the aspects, the Cas9 protein comprises a D10A mutation and/or aN580A mutation (see e.g., Friedland et al. Genome Biol. 16, 257 (2015); Nishimasu et al. Cell 162, 1113-1126 (2015)).
[0092] In some embodiments of any of the aspects, the engineered bacteriophage genome further comprises a promoter for the nucleic acid encoding the inhibitor (e.g., Cas9). In some embodiments of any of the aspects, the promoter for the inhibitor is inducible. In some embodiments of any of the aspects, the promoter for the inhibitor is inducible by anhydrotetracy cline (ATc). In some embodiments of any of the aspects, the promoter for the inhibitor is the ProC promoter. In some embodiments of any of the aspects, the promoter of the inhibitor comprises SEQ ID NO: 22 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 22 which retains the same activity as SEQ ID NO: 22. In some embodiments of any of the aspects, the promoter of the inhibitor comprises SEQ ID NO: 22 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 22 which retains the same activity as SEQ ID NO: 22.
[0093] SEQ ID NO: 22, ProC Promoter
TTCTAGAGCACAGCTAACACCACGTCGTCCCTATCTGCTGCCCTAGGTCTATGAGTGGTT
GCTGGATAACTTTACGGGCATGCATAAGGCTCGTATGATATATTCAGGGAGTCCACAACG
GTTTCCCTCTACAAATAATTTTGTTTAACTTTTACTAGAG
[0094] In some embodiments of any of the aspects, the inhibitor is encoded by a nucleotide sequence comprising SEQ ID NO: 23, a functional fragment of SEQ ID NO: 23, or a nucleic acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 22, which retains the same activity as SEQ ID NO: 22, or a codon-optimized version thereof. In some embodiments of any of the aspects, the inhibitor is encoded by a nucleotide sequence comprising SEQ ID NO: 23, a functional fragment of SEQ ID NO: 23, or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 22, which retains the same activity as SEQ ID NO: 22, or a codon-optimized version thereof.
[0095] SEQ ID NO: 23, dSaCas9; bold double -underlined text indicates D10A mutation (see e.g., nt 28-30 of SEQ ID NO: 23); italicized double-underlined text indicates N580A mutation (see e.g., nt 1738-1740 of SEQ ID NO: 23).
A TG A A GCGT A A TT A T A TTTTGGG A CTGGCC A TCGGT A TT A CCTCTGTTGGTT A CGGT ATC
ATTGATTACGAGACTCGTGATGTGATTGACGCCGGCGTGCGTTTGTTTAAGGAGGCAAAC
GTGGAAAACAATGAGGGTCGCCGCTCCAAACGTGGAGCGCGCCGCCTGAAACGCCGCCG
CCGTCATCGTATTCAACGTGTTAAGAAATTGCTTTTCGACTATAATTTATTGACAGATCAT
AGTGAACTGTCCGGCATCAATCCCTATGAAGCGCGCGTTAAAGGATTGTCACAGAAGCTT AGCGAGGAGGAATTTTCCGCAGCGTTACTTCACTTAGCCAAGCGTCGTGGTGTCCATAAC
GTGAACGAAGTGGAAGAGGATACTGGTAACGAGTTATCCACCAAAGAACAGATCAGTCG
CAACTCTAAGGCGCTTGAGGAAAAGTATGTAGCCGAGCTGCAACTTGAGCGCTTGAAGA
AGGACGGGGAGGTGCGCGGCAGTATTAACCGTTTTAAAACCAGCGACTATGTCAAAGAA
GCTAAACAATTATTGAAGGTGCAGAAAGCCTACCACCAACTTGACCAGTCATTTATCGAT
ACTTATATTGATCTTTTAGAGACTCGCCGTACTTATTACGAAGGCCCTGGCGAGGGGTCG
CCCTTCGGCTGGAAGGACATCAAGGAATGGTATGAAATGCTGATGGGGCATTGCACGTA
TTTCCCTGAAGAGCTTCGTTCCGTGAAGTATGCCTACAACGCCGATCTTTACAACGCACT
TAATGATTTAAACAACTTAGTAATCACTCGCGATGAGAACGAGAAATTGGAATACTACG
AAAAGTTCCAAATTATTGAGAATGTATTTAAGCAAAAGAAGAAGCCAACACTGAAGCAG
ATTGCAAAAGAAATCTTAGTAAATGAAGAGGACATTAAAGGGTACCGCGTAACGTCGAC
CGGAAAGCCGGAGTTCACGAACCTTAAGGTGTACCATGATATTAAAGATATCACGGCCC
GTAAAGAAATTATCGAAAACGCTGAACTGTTAGACCAGATCGCTAAGATCTTGACGATTT
ATCAGTCTAGCGAAGATATTCAGGAGGAGCTGACGAATCTTAACTCTGAGTTAACGCAG
GAGGAAATTGAGCAGATTAGCAACTTGAAGGGATACACGGGGACCCACAATCTTTCCTT
AAAAGCGATCAACCTTATCCTGGATGAGCTGTGGCATACGAACGACAATCAAATCGCTA
Figure imgf000020_0001
CTACCACTCTTGTCGACGATTTCATTCTGTCGCCCGTAGTGAAGCGTTCATTCATCCAGTC
AATCAAAGTCATCAACGCAATTATTAAGAAATATGGCCTTCCTAACGACATTATCATCGA
ACTTGCGCGTGAGAAGAACTCAAAGGATGCTCAAAAGATGATCAATGAGATGCAGAAAC
GTAATCGCCAGACAAACGAGCGCATCGAAGAAATTATTCGCACGACGGGAAAGGAAAA
TGCTAAATATTTAATTGAGAAAATCAAACTTCACGACATGCAGGAGGGCAAGTGTCTTTA
TTCACTGGAGGCGATCCCTTTGGAGGACCTGCTTAATAATCCGTTCAATTACGAGGTAGA
TCACATCATCCCCCGTAGCGTTTCTTTTGATAATTCTTTCAATAATAAGGTCCTGGTTAAG
CAGGAGGAAfiCrrCCAAGAAGGGGAATCGTACGCCGTTCCAATACTTGTCGTCCTCAGA
CAGCAAGATTTCATACGAAACCTTTAAGAAGCATATCTTAAACCTGGCTAAGGGCAAGG
Figure imgf000020_0002
GGGGTACAAACACCACGCGGAAGATGCTTTAATCATTGCCAACGCTGACTTTATCTTTAA
AGAATGGAAAAAGCTTGACAAGGCCAAGAAAGTTATGGAAAATCAGATGTTTGAGGAA
AAGCAGGCAGAGAGTATGCCAGAAATCGAGACTGAGCAGGAATATAAGGAGATCTTCAT
CACTCCGCATCAAATTAAGCACATTAAAGACTTCAAGGATTATAAATATTCACACCGTGT
GGATAAAAAGCCAAATCGTGAATTAATCAACGACACGTTGTATAGTACTCGTAAGGATG
ACAAGGGTAACACCCTGATCGTAAACAACTTAAACGGCTTATATGACAAAGATAATGAC AAGCTTAAGAAACTTATTAACAAATCCCCAGAGAAACTTCTGATGTACCACCATGACCCT
CAGACTTACCAGAAATTAAAGCTGATTATGGAACAGTATGGGGACGAGAAAAACCCGCT
TTACAAGTATTATGAAGAAACGGGGAACTACCTTACCAAGTATTCTAAAAAGGATAATG
GTCCAGTGATTAAAAAAATCAAGTACTACGGCAATAAGCTGAATGCACACTTGGACATT
ACGGATGACTACCCTAATAGCCGTAACAAAGTCGTAAAGCTGTCTTTAAAGCCTTACCGT
TTCGATGTTTATTTAGATAACGGCGTCTATAAATTTGTGACCGTCAAAAATTTAGACGTA
ATTAAGAAAGAGAATTATTACGAGGTTAATTCTAAATGCTACGAAGAAGCTAAAAAACT
Figure imgf000021_0001
AATTAATGGAGAACTTTACCGCGTTATCGGAGTTAACAATGACTTGCTGAATCGTATTGA
GGTCAACATGATCGATATTACATATCGCGAGTATTTGGAAAACATGAATGATAAGCGCC
CGCCACGCATTATTAAAACAATCGCTTCGAAAACACAGTCGATTAAGAAATACAGCACA
GACATTCTGGGGAATTTATACGAAGTCAAGTCTAAGAAACATCCGCAGATTATTAAGAA
GGGCTAA
[0096] In some embodiments of any of the aspects, the inhibitor comprises SEQ ID NO: 19, a functional fragment of SEQ ID NO: 19, or an amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 19, which retains the same activity as SEQ ID NO: 19. In some embodiments of any of the aspects, the inhibitor comprises SEQ ID NO: 19, a functional fragment of SEQ ID NO: 19, or an amino acid sequence that is at least 95 identical to SEQ ID NO: 19, which retains the same activity as SEQ ID NO: 19.
[0097] SEQ ID NO: 19, dSaCas9; bold double-underlined text indicates D10A mutation; italicized double-underlined text indicates N580A mutation.
MKRNYILGLAIGITSVGYGIIDYETRDVIDAGVRLFKEANVENNEGRRSKRGARRLKRRRRHR
IQRVKKLLFDYNLLTDHSELSGINPYEARVKGLSQKLSEEEFSAALLHLAKRRGVHNVNEVE
EDTGNELSTKEQISRNSKALEEKYVAELQLERLKKDGEVRGSINRFKTSDYVKEAKQLLKVQ
KAYHQLDQSFIDTYIDLLETRRTYYEGPGEGSPFGWKDIKEWYEMLMGHCTYFPEELRSVKY
AYNADLYNALNDLNNLVITRDENEKLEYYEKFQIIENVFKQKKKPTLKQIAKEILVNEEDIKG
YRVTSTGKPEFTNLKVYHDIKDITARKEIIENAELLDQIAKILTIYQSSEDIQEELTNLNSELTQE
EIEQISNLKGYTGTHNLSLKAINLILDELWHTNDNQIAIFNRLKLVPKKVDLSQQKEIPTTLVD
DFILSPVVKRSFIQ SIKVINAIIKKY GLPNDIIIELAREKN SKDAQKMINEMQKRNRQTNERIEEII
RTTGKENAKYLIEKIKLHDMQEGKCLY SLEAIPLEDLLNNPFNYEVDHIIPRS V SFDN SFNNKV
LVKQE SKKGNRTPFQYLSSSDSKISYETFKKHILNLAKGKGRISKTKKEYLLEERDINRFSV
QKDFINRNLVDTRYATRGLMNLLRSYFRVNNLDVKVKSINGGFTSFLRRKWKFKKERNKGY
KHHAEDALIIANADFIFKEWKKLDKAKKVMENQMFEEKQAESMPEIETEQEYKEIFITPHQIK
HIKDFKDYKY SHRVDKKPNRELINDTLY STRKDDKGNTLIVNNLNGLYDKDNDKLKKLINKS PEKLLMYHHDPQTY QKLKLIMEQY GDEKNPLYKYYEETGNYLTKY SKKDNGPVIKKIKYY G NKLNAHLDITDDYPNSRNKVVKLSLKPYRFDVYLDNGVYKFVTVKNLDVIKKENYYEVNSK CYEEAKKLKKISNQAEFIASFYNNDLIKINGELYRVIGVNNDLLNRIEVNMIDITYREYLENMN DKRPPRIIKTIASKTQSIKKYSTDILGNLYEVKSKKHPQIIKKG
[0098] In some embodiments of any of the aspects, the inhibitor comprises a Cas protein in combination with a CRISPR guide RNA (gRNA). In some embodiments of any of the aspects, the CRISPR gRNA comprises a tracrRNA and/or crRNA. In some embodiments of any of the aspects, the engineered bacteriophage genome further comprises at least one tracrRNA and/or at least one crRNA. In some embodiments of any of the aspects, the tracrRNA and crRNA are separate RNA molecules.
In some embodiments of any of the aspects, the tracrRNA and crRNA are comprised by the same RNA molecule.
[0099] The full-length guide nucleic acid strand (e.g., the tracrRNA and/or crRNA) can be any length. For example, the guide nucleic acid strand can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments of the various aspects described herein, a nucleic acid strand is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. For example, the guide nucleic acid sequence is 10-30 nucleotides long. In some embodiments of the various aspects described herein, the guide nucleic acid is designed using a guide design tool (e.g., Benchling™; Broad Institute GPP™; CasOFFinder™; CHOPCHOP™; CRISPOR™; Deskgen™; E-CRISP™; Geneious™; GenHub™; GUIDES™ (e.g., for library design); Horizon Discovery™; IDT™; Off- Spotter™; and Synthego™; which are available on the world wide web).
[00100] In some embodiments of any of the aspects, the engineered bacteriophage genome further comprises a promoter for the at least one tracrRNA and/or at least one crRNA. In some embodiments of any of the aspects, the promoter for the tracrRNA is constitutive. In some embodiments of any of the aspects, the promoter for the crRNA is constitutive. In some embodiments of any of the aspects, the promoter (e.g., for the tracrRNA) comprises SEQ ID NO: 24 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 24 which retains the same activity as SEQ ID NO: 24. In some embodiments of any of the aspects, the promoter (e.g., for the tracrRNA) comprises SEQ ID NO: 24 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 24 which retains the same activity as SEQ ID NO: 24.
[00101] In some embodiments of any of the aspects, the promoter (e.g., for the crRNA) comprises the J23100 promoter. In some embodiments of any of the aspects, the promoter (e.g., for the crRNA) comprises SEQ ID NO: 26 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 26 which retains the same activity as SEQ ID NO: 26. In some embodiments of any of the aspects, the promoter (e.g., for the crRNA) comprises SEQ ID NO: 24 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 26 which retains the same activity as SEQ ID NO: 26.
[00102] In some embodiments of any of the aspects, the trans-activating CRISPR RNA (tracrRNA) specifically binds with the CRISPR-Cas protein (e.g., Cas9). In some embodiments of any of the aspects, the tracrRNA hybridizes with the crRNA. In some embodiments of any of the aspects, the tracrRNA specifically binds with the CRISPR-Cas protein (e.g., Cas9) and hybridizes with the crRNA. In some embodiments of any of the aspects, the tracrRNA comprises SEQ ID NO: 25 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 25 which retains the same activity as SEQ ID NO: 25. In some embodiments of any of the aspects, the tracrRNA comprises SEQ ID NO: 25 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 25 which retains the same activity as SEQ ID NO: 25.
[00103] In some embodiments of any of the aspects, the CRISPR RNA (crRNA) comprises a variable targeting sequence and a region that is substantially complementary to a region of the tracrRNA. In some embodiments of any of the aspects, the CRISPR RNA (crRNA) comprises a variable targeting sequence. In some embodiments of any of the aspects, the variable targeting sequence of the crRNA is substantially complementary to a target nucleic acid. In some embodiments of any of the aspects, the variable targeting sequence of the crRNA is substantially complementary to a nucleic acid encoding a bacterial virulence factor. In some embodiments of any of the aspects, the variable targeting sequence of the crRNA is substantially complementary to RFP (see e.g., SEQ ID NO: 28). In some embodiments of any of the aspects, the variable targeting sequence of a negative control crRNA is substantially complementary to no known nucleic acid sequence (see e.g., SEQ ID NO: 27). In some embodiments of any of the aspects, the variable targeting sequence comprises one of SEQ ID NOs: 27-28 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 27-28 which retains the same activity as one of SEQ ID NOs: 27-28. In some embodiments of any of the aspects, the variable targeting sequence comprises one of SEQ ID NOs: 27-28 (see e.g., Table 4) or a sequence that is at least 95% identical to one of SEQ ID NOs: 27-28 which retains the same activity as one of SEQ ID NOs: 27-28.
[00104] In some embodiments of any of the aspects, the CRISPR RNA (crRNA) comprises a region that is substantially complementary to a region of the tracrRNA. In some embodiments of any of the aspects, the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 (see e.g., Table 4) or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to SEQ ID NO: 29 which retains the same activity as SEQ ID NO: 29. In some embodiments of any of the aspects, the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 (see e.g., Table 4) or a sequence that is at least 95% identical to SEQ ID NO: 29 which retains the same activity as SEQ ID NO: 29.
[00105] SEQ ID NO: 24, tracrRNA promoter
CTGATAAATTTCTTTGAATTTCTCCTTGATTATTTGTTATAAAAGTTATAAAAT [00106] SEQ ID NO: 25, saCas9_tracrRNA
ATTGTACTTATACCTAAAATTACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATC
Figure imgf000024_0001
[00107] SEQ ID NO: 26, J23100 promoter (e.g., promoter for crRNA)
GACAATGAAAACGTTAGTCATGGCGCGCCTTGACGGCTAGCTCAGTCCTAGGTACAGTG
CTAGCT
[00108] SEQ ID NO: 27, Bsal spacer (e.g, control gRNA) GAGACCGACTGAGGTCTCA [00109] SEQ ID NO: 28, rip l gRNA TGGTAACTTTCAGTTTAGCGGT [00110] SEQ ID NO: 29, SaCas9 repeat (e.g., for hybridization with tracrRNA) GTTTTAGTACTCTGTAATTTTAGGTATGAGGTAGAC
[00111] In some embodiments of any of the aspects, a vector comprises at least one of the following: l phage homology arms, a promoter for the CRISPR-Cas protein (e.g., Cas9), the CRISPR- Cas protein (e.g., S. aureus dCas9), the tracrRNA under a constitutive promoter, the crRNA under a constitutive promoter, and a resistance cassette (e.g., pACYCDuet chloramphenicol resistance cassette). In some embodiments of any of the aspects, the vector comprises one of SEQ ID NOs: 30- 31 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 30-31 which retains the same activity as one of SEQ ID NOs: 30-31. In some embodiments of any of the aspects, the vector comprises one of SEQ ID NOs: 30-31 or a sequence that is at least 95% identical to one of SEQ ID NOs: 30-31 which retains the same activity as one of SEQ ID NOs: 30-31.
[00112] In some embodiments of any of the aspects, the bacterium (e.g., targeted by the engineered bacteriophage) is Escherichia coli ( E . coli). In some embodiments of any of the aspects, the bacterium is enterohemorrhagic E. coli (EHEC). In some embodiments of any of the aspects, the bacterium is found within the human intestinal microbiome. In some embodiments of any of the aspects, the following microbes that causes diseases and/or associated microbial matter can be amendable to the compositions and methods of various aspects described herein (e.g., can be targeted by the engineered phage described herein): Bartonella henselae, Borrelia burgdorferi, Campylobacter jejuni, Campylobacterfetus, Chlamydia trachomatis, Chlamydia pneumoniae, Chylamydia psittaci, Simkania negevensis, Escherichia coli (e.g., 0157:H7 and K88), Ehrlichia chafeensis, Clostridium botulinum, Clostridium perfringens, Clostridium tetani, Enterococcus faecalis, Haemophilius influenzae, Haemophilius ducreyi, Coccidioides immitis, Bordetella pertussis, Coxiella burnetii, Ureaplasma urealyticum, Mycoplasma genitalium, Trichomatis vaginalis, Helicobacter pylori, Helicobacter hepaticus, Legionella pneumophila, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium leprae, Mycobacterium asiaticum, Mycobacterium avium, Mycobacterium celatum, Mycobacterium celonae, Mycobacterium fortuitum, Mycobacterium genavense, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium ulcerans, Mycobacterium xenopi, Corynebacterium diptheriae, Rhodococcus equi, Rickettsia aeschlimannii, Rickettsia africae, Rickettsia conorii, Arcanobacterium haemolyticum, Bacillus anthracia, Bacillus cereus, Lysteria monocytogenes, Yersinia pestis, Yersinia enterocolitica, Shigella dysenteriae, Neisseria meningitides, Neisseria gonorrhoeae, Streptococcus bovis, Streptococcus hemolyticus, Streptococcus mutans, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus pneumoniae, Staphylococcus saprophyticus, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, or Treponema pallidum. In yet other embodiments, bioterror agents (e.g., B. Anthracis) can be amendable to the compositions and methods of various aspects described herein (e.g., can be targeted by the engineered phage described herein).
[00113] In some embodiments of any of the aspects, the virulence factor is Shiga Toxin (Stx). Non-limiting examples of virulence factors include bacterial hyaluronidases, proteases, coagulases, lipases, deoxyribonucleases, and enterotoxins. In some embodiments of any of the aspects, the virulence factor is encoded by an endogenous phage, including but not limited to phage 933 W.
[00114] In some embodiments of any of the aspects, the engineered phage further comprises a heterologous bacteriophage immunity region. As used herein, "heterologous" refers to that which is not endogenous to, or naturally occurring in, a referenced sequence, molecule (including e.g., a protein), virus, cell, tissue, or organism. For example, a heterologous sequence of the present disclosure can be derived from a different species, or from the same species but substantially modified from an original form. Also for example, a nucleic acid sequence that is not normally expressed in a virus or a cell is a heterologous nucleic acid sequence. The term "heterologous" can refer to DNA, RNA, or protein that does not occur naturally as part of the organism (which is used herein to comprise viruses) in which it is present or which is found in a location or locations in the genome that differ from that in which it occurs in nature. It is DNA, RNA, or protein that is not endogenous to the virus or cell and has been artificially introduced into the virus or cell.
[00115] As used herein, “immunity region” refers to a nucleic acid locus in a temperate phage that is responsible for maintaining the lysogenic state and, as a consequence, effecting immunity against superinfecting phages. In some embodiments of any of the aspects, the immunity region inhibits infection of the bacteria by other phage, i.e., immunity against superinfection. As used herein, “superinfection” refers to bacteria infected with at least bacteriophage being infected by at least one other different bacteriophage.
[00116] In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is a lambdoid phage immunity region. In some embodiments of any of the aspects, the lambdoid phage is selected from the group consisting of lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22. In some embodiments of any of the aspects, the lambdoid phage is selected from the group consisting of 933W phage, lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22. In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is an immunity region from 933W phage (e.g., SEQ ID NO: 13). In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is an immunity region from lambdoid phage 21 (e.g., SEQ ID NO: 15). In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is an immunity region from lambdoid phage 434 (e.g., SEQ ID NO: 16). In some embodiments of any of the aspects, the heterologous bacteriophage immunity region is an immunity region from lambdoid phage P22 (e.g., SEQ ID NO: 14 or 17).
[00117] In some embodiments of any of the aspects, the heterologous bacteriophage immunity region comprises any one of SEQ ID NOs: 2-6, a portion of any one of SEQ ID NOs: 2-6 (e.g., as indicated in Table 3), or a sequence that is at least 95% identical to any one of SEQ ID NOs: 2-6 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 2-6 or a portion thereof. In some embodiments of any of the aspects, the heterologous bacteriophage immunity region comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to any one of SEQ ID NOs: 2-6 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 2-6 or a portion thereof.
[00118] In some embodiments of any of the aspects, the heterologous bacteriophage immunity region comprises any one of SEQ ID NOs: 13-17, a portion of any one of SEQ ID NOs: 13-17, or a sequence that is at least 95% identical to any one of SEQ ID NOs: 13-17 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 13-17 or a portion thereof. In some embodiments of any of the aspects, the heterologous bacteriophage immunity region comprises a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to any one of SEQ ID NOs: 13-17 or a portion thereof, which retains the same activity or function as any one of SEQ ID NOs: 13-17 or a portion thereof. [00119] SEQ ID NO: 13, immunity region of 933W phage (e.g., in limm933W phage; see e.g., SEQ ID NO: 2 in Table 3)
[00120] SEQ ID NO: 14, immunity region of P22 phage (e.g., in lήhihR22άί8 phage; see e.g.,
SEQ ID NO: 3 in Table 3)
[00121] SEQ ID NO: 15, immunity region of 21 phage (e.g., in lίihih21 phage; see e.g., SEQ ID NO: 4 in Table 3)
[00122] SEQ ID NO: 16, immunity region of 434 phage (e.g., in lύhih434 phage; see e.g., SEQ ID NO: 5 in Table 3)
[00123] SEQ ID NO: 17, immunity region of P22 phage (e.g., in lBH2 phage; see e.g., SEQ ID NO: 6 in Table 3; SEQ ID NO: 17 comprises nucleotides 11,060-16,195 of SEQ ID NO: 14)
[00124] In some embodiments of any of the aspects, the engineered bacteriophage genome further comprises a nucleic acid encoding a selectable marker. Non-limiting examples of selectable markers include a positive selection marker; a negative selection marker; a positive and negative selection marker; resistance to at least one of chloramphenicol, ampicillin, kanamycin, and/or triclosan; or an auxotrophy marker. In some embodiments of any of the aspects, the selectable marker is selected from the group consisting of CmR (a chloramphenicol resistance gene; e.g., the pACYCDuet chloramphenicol resistance cassette) beta-lactamase, Neo gene (e.g., Kanamycin resistance cassette) from Tn5, mutant Fabl gene, and an auxotrophic mutation.
[00125] In some embodiments of any of the aspects, the selectable marker is encoded by a nucleotide sequence comprising one of SEQ ID NOs: 18 or 33 or a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identical to one of SEQ ID NOs: 18 or 33, which retains the same activity or function as one of SEQ ID NOs: 18 or 33, or a codon-optimized version thereof.
[00126] SEQ ID NO: 18, kanamycin resistance cassette (Tn5) gccctgcaaagtaaactggatggctttcttgccgccaaggatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcg catgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctc tgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggc agcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcg aagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatcc ggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacg aagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatg cctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttg gctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgcctt ctatcgccttcttgacgagttcttctaa
[00127] SEQ ID NO: 33, chloramphenicol resistance gene CmR
ATGGAGAAAAAAATCACTGGATATACCACCGTTGATATATCCCAATGGCATCGTAAAGA
ACATTTTGAGGCATTTCAGTCAGTTGCTCAATGTACCTATAACCAGACCGTTCAGCTGGA
Figure imgf000028_0001
GCCAATATGGACAACTTCTTCGCCCCCGTTTTCACTATGGGCAAATATTATACGCAAGGC
GACAAGGTGCTGATGCCGCTGGCGATTCAGGTTCATCATGCCGTCTGTGATGGCTTCCAT
GTCGGCAGAATGCTTAATGAATTACAACAGTACTGCGATGAGTGGCAGGGCGGGGCGTA
A
[00128] In one aspect, described herein is a method of treating a bacterial infection, comprising administering an effective amount of an engineered bacteriophage as described or a pharmaceutical composition as described herein to a patient in need thereof. In some embodiments of any of the aspects, the patient is infected with E. coli or EHEC.
[00129] As described herein, levels of at least one virulence factor can be increased in a bacterial infection and/or in subjects with a bacterial infection. Accordingly, in one aspect of any of the embodiments, described herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising administering at least one engineered bacteriophage as described herein to a subject determined to have a level of at least one virulence factor that is increased relative to a reference. In one aspect of any of the embodiments, described herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising: a) determining the level of at least one virulence factor in a sample obtained from a subject; and b) administering at least one engineered bacteriophage as described herein to the subject if the level of at least one virulence factor is increased relative to a reference.
[00130] In some embodiments of any of the aspects, the method comprises administering at least one engineered bacteriophage as described herein to a subject previously determined to have a level of at least one virulence factor that is increased relative to a reference. In some embodiments of any of the aspects, described herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising: a) first determining the level of at least one virulence factor in a sample obtained from a subject; and b) then administering at least one engineered bacteriophage as described herein to the subject if the level of at least one virulence factor is increased relative to a reference. [00131] In one aspect of any of the embodiments, described herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising: a) determining if the subject has an increased level of at least one virulence factor; and b) administering at least one engineered bacteriophage as described herein to the subject if the level of at least one virulence factor is increased relative to a reference. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise performing or having performed an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise receiving the results of an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise receiving a report, results, or other means of identifying the subject as a subject with an increased level of at least one virulence factor.
[00132] In one aspect of any of the embodiments, described herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising: a) determining if the subject has an increased level of at least one virulence factor; and b) instructing or directing that the subject be administered at least one engineered bacteriophage as described herein if the level of at least one virulence factor is increased relative to a reference. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise performing or having performed an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of determining if the subject has an increased level of at least one virulence factor can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level of at least one virulence factor in the subject. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results.
[00133] In one aspect, described herein is a method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of at least one engineered bacteriophage as described herein or an effective amount of at least one pharmaceutical composition as described herein.
[00134] As an example, described herein are methods of inhibiting or delaying the formation of biofdms, comprising administering to a subject in need thereof or contacting a surface with an effective amount of at least one engineered bacteriophage as described herein or an effective amount of a pharmaceutical composition as described herein.
[00135] As used herein, a “biofilm” refers to mass of microorganisms attached to a surface, such as a surface of a medical device, and the associated extracellular substances produced by one or more of the attached microorganisms. The extracellular substances are typically polymeric substances that commonly include a matrix of complex polysaccharides, proteinaceous substances and glycopeptides. The microorganisms can include, but are not limited to, bacteria, fungi and protozoa. In a "bacterial biofilm," the microorganisms include one or more species of bacteria. The nature of a biofilm, such as its structure and composition, can depend on the particular species of bacteria present in the biofilm. Bacteria present in a biofilm are commonly genetically or phenotypically different than corresponding bacteria not in a biofilm, such as isolated bacteria or bacteria in a colony. "Polymicrobic biofilms" are biofilms that include a plurality of bacterial species.
[00136] As used herein, the terms and phrases "delaying", "delay of formation", and "delaying formation of' have their ordinary and customary meanings, and are generally directed to increasing the period of time prior to the formation of biofilm, or a slow growing bacterial infection in a subject or on a surface. The delay may be, for example, about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, about 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, or more. Inhibiting formation of a biofilm, as used herein, refers to avoiding the partial or full development or progression of a biofilm, for example, on a surface, such as a surface of an indwelling medical device.
[00137] The skilled artisan will understand that the methods of inhibiting and delaying the formation of biofilms can be practiced wherever bacteria, such as persistent, slow-growing, stationary-phase, or biofilm forming bacteria, can be encountered. For example, the methods described herein can be practiced on the surface of or inside of an animal, such as a human; on an inert surface, such as a counter or bench top; on a surface of a piece of medical or laboratory equipment; on a surface of a medical or laboratory tool; or on a surface of an in-dwelling medical device. [00138] In some aspects, provided herein are methods of inhibiting the formation of a biofilm on a surface or on a porous material, comprising applying to or contacting a surface or a porous material upon which a biofdm can form at least one engineered bacteriophage or a pharmaceutical composition as described herein in an amount sufficient to inhibit the formation of a biofilm. In some embodiments of these methods and all such methods described herein, the surface is an inert surface, such as the surface of an in-dwelling medical device.
[00139] In some aspects, provided herein are methods of delaying the formation of a biofilm on a surface or on a porous material, comprising applying to or contacting a surface or a porous material upon which a biofilm can form at least one engineered bacteriophage or a pharmaceutical composition as described herein in an amount sufficient to delay the formation of a biofilm. In some embodiments of these methods and all such methods described herein, the surface is an inert surface, such as the surface of an in-dwelling medical device.
[00140] In some aspects, provided herein are methods of preventing the colonization of a surface by persistent bacteria, comprising applying to or contacting a surface with at least one engineered bacteriophage or a pharmaceutical composition as described herein in an amount sufficient to prevent colonization of the surface by persistent bacteria.
[00141] In the embodiments of the methods described herein directed to inhibiting or delaying the formation of a biofilm, or preventing the colonization of a surface by persistent bacteria, the material comprising the surface or the porous material can be any material that can be used to form a surface or a porous material. In some such embodiments, the material is selected from: polyethylene, polytetrafluoroethylene, polypropylene, polystyrene, polyacrylamide, polyacrylonitrile, poly(methyl methacrylate), polyamide, polyester, polyurethane, polycarbonate, silicone, polyvinyl chloride, polyvinyl alcohol, polyethylene terephthalate, cobalt, a cobalt-base alloy, titanium, a titanium base alloy, steel, silver, gold, lead, aluminum, silica, alumina, yttria stabilized zirconia polycrystal, calcium phosphate, calcium carbonate, calcium fluoride, carbon, cotton, wool and paper.
[00142] In embodiments of the methods described herein of inhibiting or delaying the formation of a biofdm, or preventing the colonization of a surface by persistent bacteria, an antibiotic can be applied concurrently with at least one engineered bacteriophage or a pharmaceutical composition as described herein. Suitable antibiotics include, for example, aminoglycosides (e.g., gentamicin, streptomycin, kanamycin), b-lactams (e.g, penicillins and cephalosporins), vancomycins, bacitracins, macrolides (e.g., erythromycins), lincosamides (e.g., clindamycin), chloramphenicols, tetracyclines, amphotericins, cefazolins, clindamycins, mupirocins, sulfonamides and trimethoprim, rifampicins, metronidazoles, quinolones, novobiocins, polymyxins, gramicidins, or any salts or variants thereof. [00143] In some embodiments of these methods and all such methods described herein, the persistent, slow growing, stationary-phase or biofdm bacteria is any bacterial species or population that comprises persistent cells, can exist in a slow growing or stationary-phase, and/or that can form a biofilm. In some such embodiments, the bacteria is E. coli, Staphylococcus aureus, Staphylococcus epidermidis, a vancomycin-susceptible enterococci, a vancomycin-resistant enterococci, a Staphylococcus species or a Streptococcus species. In some such embodiments, the bacteria is selected from vancomycin (VAN)-susceptible Enterococcus faecalis (VSE), VAN-resistant E. faecalis (VRE), and Staph epidermidis .
[00144] As used herein, the term "contacting" is meant to broadly refer to bringing a bacterial cell and at least one engineered bacteriophage or a pharmaceutical composition as described herein into sufficient proximity that the at least one engineered bacteriophage or a pharmaceutical composition as described herein can exert their effects on any bacterial cell present. The skilled artisan will understand that the term "contacting" includes physical interaction between at least one engineered bacteriophage or a pharmaceutical composition as described herein and a bacterial cell, as well as interactions that do not require physical interaction.
[00145] The methods described herein further encompass surfaces coated by at least one engineered bacteriophage or a pharmaceutical composition as described herein, and/or impregnated with at least one engineered bacteriophage or a pharmaceutical composition as described herein. Such surfaces include any that can come into contact with a persistent, slow growing, stationary-phase, biofilm bacteria. In some such embodiments, such surfaces include any surface made of an inert material (although surfaces of a living animal are encompassed within the scope of the methods described herein), including the surface of a counter or bench top, the surface of a piece of medical or laboratory equipment or a tool, the surface of a medical device such as a respirator, and the surface of an in-dwelling medical device. In some such embodiments, such surfaces include those of an in dwelling medical device, such as surgical implants, orthopedic devices, prosthetic devices and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, but are not limited to, artificial joints, artificial hearts and implants; valves, such as heart valves; pacemakers; vascular grafts; catheters, such as vascular, urinary and continuous ambulatory peritoneal dialysis (CAPD) catheters; shunts, such as cerebrospinal fluid shunts; hoses and tubing; plates; bolts; valves; patches; wound closures, including sutures and staples; dressings; and bone cement.
[00146] As used herein, the term “indwelling medical device,” refers to any device for use in the body of a subject, such as intravascular catheters (for example, intravenous and intra-arterial), right heart flow-directed catheters, Hickman catheters, arteriovenous fistulae, catheters used in hemodialysis and peritoneal dialysis (for example, silastic, central venous, Tenckhoff, and Teflon catheters), vascular access ports, indwelling urinary catheters, urinary catheters, silicone catheters, ventricular catheters, synthetic vascular prostheses (for example, aortofemoral and femoropopliteal), prosthetic heart valves, prosthetic joints, orthopedic implants, penile implants, shunts (for example, Scribner, Torkildsen, central nervous system, portasystemic, ventricular, ventriculoperitoneal), intrauterine devices, tampons, dental implants, stents (for example, ureteral stents), artificial voice prostheses, tympanostomy tubes, gastric feeding tubes, endotracheal tubes, pacemakers, implantable defibrillators, tubing, cannulas, probes, blood monitoring devices, needles, and the like. A subcategory of indwelling medical devices refer to implantable devices that are typically more deeply and/or permanently introduced into the body. Indwelling medical devices can be introduced by any suitable means, for example, by percutaneous, intravascular, intraurethral, intraorbital, intratracheal, intraesophageal, stomal, or other route, or by surgical implantation, for example intraarticular placement of a prosthetic joint.
[00147] According to some embodiments of the methods described herein, the in-dwelling medical device is coated by a solution, such as through bathing or spraying, containing a concentration of about 10 pg/ml to about 500 mg/ml of at least one engineered bacteriophage or a pharmaceutical composition as described herein. In some embodiments, more specific ranges of concentrations of the at least one engineered bacteriophage or a pharmaceutical composition as described herein can be used, including: about 10 pg/ml to about 1 mg/ml, about 1 mg/ml to about 100 mg/ml, about 10 mg/ml to about 500 mg/ml, about 50 mg/ml to about 200 mg/ml, about 10 mg/ml to about 100 mg/ml, about 100 mg/ml to about 500 mg/ml. In some embodiments, specific concentrations of at least one engineered bacteriophage or a pharmaceutical composition as described herein can be used, including: about 10 pg/ml, about 50 pg/ml, about 100 pg/ml, about 250 pg/ml, about 500 pg/ml, about 750 pg/ml, about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 250 mg/ml, about 500 mg/ml, about 600 mg/ml, about 750 mg/ml, and about 900 mg/ml. The in-dwelling medical device can be coated by the solution comprising at least one engineered bacteriophage or a pharmaceutical composition as described herein before its insertion in the body.
[00148] In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having a bacterial infection. Subjects having a bacterial infection can be identified by a physician using current methods of diagnosing bacterial infections. Symptoms and/or complications of a bacterial infection which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to fever; fatigue; swollen lymph nodes in the neck, armpits, or groin; headache; and/or nausea or vomiting. Tests that may aid in a diagnosis of, e.g. a bacterial infection include, but are not limited to, a blood test, a urine test, a culture test, rt-qPCR, MALDI- TOF MS, or other methods as known in the art. A family history of bacterial infections, or exposure to risk factors for bacterial infections (e.g. immune deficiency, hospitalization for another condition or infection, co-infection with another microorganism, etc.) can also aid in determining if a subject is likely to have a bacterial infection or in making a diagnosis of a bacterial infection. [00149] The compositions and methods described herein can be administered to a subject having or diagnosed as having a bacterial infection. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. at least one engineered bacteriophage as described herein, to a subject in order to alleviate a symptom of a bacterial infection. As used herein, "alleviating a symptom of a bacterial infection " is ameliorating any condition or symptom associated with the bacterial infection. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
[00150] The term “effective amount" as used herein refers to the amount of an engineered bacteriophage as described herein or a pharmaceutical composition comprising at least one engineered bacteriophage as described herein needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmaceutical composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of an engineered bacteriophage that is sufficient to provide a particular anti-bacterial effect (e.g., especially with regard to decreasing the level of at least one virulence factor as described herein) when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount". However, for any given case, an appropriate “effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
[00151] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e.. the concentration of an engineered bacteriophage, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for bacteriophage such as a plaque assay as described herein, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. [00152] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising at least one engineered bacteriophage as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise at least one engineered bacteriophage as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of at least one engineered bacteriophage as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of at least one engineered bacteriophage as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include:
(I) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol;
(I I) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; (23) C2-C12 alcohols, such as ethanol; and (24) other non toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. at least one engineered bacteriophage as described herein.
[00153] In some embodiments, the pharmaceutical composition comprising at least one engineered bacteriophage as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient.
Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-re lease parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.
[00154] Suitable vehicles that can be used to provide parenteral dosage forms of at least one engineered bacteriophage as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non- aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
[00155] Pharmaceutical compositions comprising at least one engineered bacteriophage as described herein can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non- aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally,
Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
[00156] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the pharmaceutical composition comprising at least one engineered bacteriophage as described herein can be administered in a sustained release formulation. [00157] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
[00158] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
[00159] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 Bl; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profde in varying proportions.
[00160] In some embodiments of any of the aspects, the pharmaceutical composition comprising at least one engineered bacteriophage as described herein is administered as a monotherapy, e.g., another treatment for the bacterial infection is not administered to the subject.
[00161] The methods described herein can further comprise administering a second antimicrobial agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. In some embodiments of any of the aspects, the antimicrobial agent can be selected from aminoglycosides, ansamycins, beta- lactams, bis-biguanides, carbacephems, carbapenems, cationic polypeptides, cephalosporins, fluoroquinolones, glycopeptides, iron-sequestering glycoproteins, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidinones, penicillins, polypeptides, quaternary ammonium compounds, quinolones, silver compounds, sulfonamides, tetracyclines, and any combinations thereof. In some embodiments of any of the aspects, the antimicrobial agent can comprise an antibiotic.
[00162] Some exemplary specific antimicrobial agents include broad penicillins, amoxicillin (e.g., Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin), Penicillins and Beta Lactamase Inhibitors (e.g., Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam, Benzylpenicillin, Cloxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin), Cephalosporins (e.g., Cephalosporin I Generation, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine),
Cephalosporin II Generation (e.g., Cefaclor, Cefamandole, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Ceftnetazole, Cefuroxime, Loracarbef), Cephalosporin III Generation (e.g., Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone), Cephalosporin IV Generation (e.g., Cefepime), Macrolides and Lincosamides (e.g., Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin, Troleandomycin), Quinolones and Fluoroquinolones (e.g., Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, Perfloxacin), Carbapenems (e.g., Imipenem-Cilastatin, Meropenem), Monobactams (e.g., Aztreonam), Aminoglycosides (e.g., Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin), Glycopeptides (e.g., Teicoplanin, Vancomycin), Tetracyclines (e.g., Demeclocy cline, Doxycycline, Methacycline, Minocycline, Oxytetracy cline, Tetracycline, Chlortetracycline), Sulfonamides (e.g., Mafenide, Silver Sulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole, Sulfamethizole), Rifampin (e.g., Rifabutin, Rifampin, Rifapentine), Oxazolidinones (e.g., Linezolid, Streptogramins, Quinupristin Dalfopristin), Bacitracin, Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazole, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin, Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin, Ethionamide, Pyrazinamide, Para-aminosalicylic acid, Erythromycin ethylsuccinate, and the like.
[00163] The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. By way of non-limiting example, if a subject is to be treated for pain or inflammation according to the methods described herein, the subject can also be administered a second agent and/or treatment known to be beneficial for subjects suffering from pain or inflammation. Examples of such agents and/or treatments include, but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs - such as aspirin, ibuprofen, or naproxen); corticosteroids, including glucocorticoids (e.g. cortisol, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and beclometasone); methotrexate; sulfasalazine; leflunomide; anti-TNF medications; cyclophosphamide; pro-resolving drugs; mycophenolate; or opiates (e.g. endorphins, enkephalins, and dynorphin), steroids, analgesics, barbiturates, oxycodone, morphine, lidocaine, and the like.
[00164] In certain embodiments, an effective dose of a composition comprising at least one engineered bacteriophage as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising at least one engineered bacteriophage as described herein can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more. [00165] In some embodiments of any of the aspects, subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, at least 10 PFU/dose (plaque-forming units per dose), at least 101 PFU/dose, at least 102 PFU/dose, at least 103 PFU/dose, at least 104 PFU/dose, at least 105 PFU/dose, at least 106 PFU/dose, at least 107 PFU/dose, at least 108 PFU/dose, at least 109 PFU/dose, or at least 1010 PFU/dose. In some embodiments of any of the aspects, subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, about 107 PFU/dose. In some embodiments of any of the aspects, subjects can be administered a therapeutic amount of a composition comprising at least one engineered bacteriophage as described herein, such as, about 5xl03 PFU/dose.
[00166] In some embodiments of any of the aspects, a composition (e.g., for administration to a patient in need thereof) comprises 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, or at least 10 engineered phage as described herein.
[00167] In some embodiments of any of the aspects, at least one of the engineered phage as described herein is administered unencapsulated, e.g., in a saline solution or buffer (e.g., bicarbonate buffer). In some embodiments of any of the aspects, at least one of the engineered phage as described herein is administered encapsulated or as part of an encapsulation composition, e.g., to protect it from stomach acid. In some embodiments of any of the aspects, the phage can be administered on a substrate or in/on a composition, e.g., beads, nanoparticles, hydrogel.
[00168] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. a bacterial infection by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more. [00169] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to at least one engineered bacteriophage as described herein. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising at least one engineered bacteriophage as described herein can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
[00170] The dosage ranges for the administration of a composition comprising at least one engineered bacteriophage as described herein, according to the methods described herein depend upon, for example, the form of the engineered bacteriophage, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for at least one virulence factor. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
[00171] The efficacy of a composition comprising at least one engineered bacteriophage as described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. a bacterial infection) can be determined by the skilled clinician. However, a treatment is considered “effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. at least one virulence factor as described herein. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. fever, levels of bacteria in a sample, levels of at least one virulence factor). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of a bacterial infection. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. at least one virulence factor as described herein.
[00172] In vitro and animal model assays are provided herein which allow the assessment of a given dose of a composition comprising at least one engineered bacteriophage as described herein. By way of non-limiting example, the effects of a dose of composition comprising at least one engineered bacteriophage as described herein can be assessed by measurement of at least one virulence factor in cell culture or in an animal model of infection (see e.g., Examples 1 and 2).
[00173] In some embodiments, one or more of the genes described herein is expressed in a recombinant expression vector or plasmid. As used herein, the term "vector" refers to a polynucleotide sequence suitable for transferring transgenes into a host cell. The term “vector” includes plasmids, mini-chromosomes, phage, naked DNA and the like. See, for example, U.S. Pat. Nos. 4,980,285; 5,631,150; 5,707,828; 5,759,828; 5,888,783 and, 5,919,670, and, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press (1989). One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments are ligated. Another type of vector is a viral vector, wherein additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors" . In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" is used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions. [00174] A cloning vector is one which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence can be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence can occur many times as the plasmid increases in copy number within the host cell such as a host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication can occur actively during a lytic phase or passively during a lysogenic phase.
[00175] An expression vector is one into which a desired DNA sequence can be inserted by restriction and ligation such that it is operably joined to regulatory sequences and can be expressed as an RNA transcript. Vectors can further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., b-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). In certain embodiments, the vectors used herein are capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
[00176] As used herein, a coding sequence and regulatory sequences are said to be “operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.
[00177] When the nucleic acid molecule that encodes any of the polypeptides described herein is expressed in a cell, a variety of transcription control sequences (e.g., promoter/enhancer sequences) can be used to direct its expression. The promoter can be a native promoter, i.e., the promoter of the gene in its endogenous context, which provides normal regulation of expression of the gene. In some embodiments the promoter can be constitutive, i.e., the promoter is unregulated allowing for continual transcription of its associated gene. A variety of conditional promoters also can be used, such as promoters controlled by the presence or absence of a molecule. In some embodiments, the promoter is the LacUV5 promoter (e.g., SEQ ID NO: 12).
[00178] The precise nature of the regulatory sequences needed for gene expression can vary between species or cell types, but in general can include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences can also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
[00179] Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA). That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
[00180] In some embodiments, the vector is pJETl .2.
[00181] Without limitations, the genes (e.g., of the engineered bacteriophage) described herein can be included in one vector or separate vectors. For example, the virulence factor inhibitor gene or the immunity region gene can be included in the same vector; or the virulence factor inhibitor gene and the immunity region gene can be included in the same vector.
[00182] In some embodiments, the virulence factor inhibitor gene can be included in a first vector, and the immunity region gene can be included in a second vector.
[00183] In some embodiments, one or more of the recombinantly expressed gene can be integrated into the genome of the cell.
[00184] A nucleic acid molecule that encodes the enzyme of the claimed invention can be introduced into a cell or cells using methods and techniques that are standard in the art. For example, nucleic acid molecules can be introduced by standard protocols such as transformation including chemical transformation and electroporation, transduction, particle bombardment, etc. Expressing the nucleic acid molecule encoding the enzymes of the claimed invention also may be accomplished by integrating the nucleic acid molecule into the genome.
[00185] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
[00186] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
[00187] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction" or “decrease" or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[00188] The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.
[00189] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.
[00190] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of a bacterial infection. A subject can be male or female.
[00191] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. a bacterial infection) or one or more complications related to such a condition, and optionally, have already undergone treatment for a bacterial infection or the one or more complications related to a bacterial infection. Alternatively, a subject can also be one who has not been previously diagnosed as having a bacterial infection or one or more complications related to a bacterial infection. For example, a subject can be one who exhibits one or more risk factors for a bacterial infection or one or more complications related to a bacterial infection or a subject who does not exhibit risk factors.
[00192] A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
[00193] As used herein, the terms “protein" and “polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
[00194] In the various embodiments described herein, it is further contemplated that variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and/or conservative substitution variants of any of the particular polypeptides described are encompassed. As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid and retains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles consistent with the disclosure.
[00195] A given amino acid can be replaced by a residue having similar physiochemical characteristics, e.g., substituting one aliphatic residue for another (such as lie, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, e.g., substitutions of entire regions having similar hydrophobicity characteristics, are well known. Polypeptides comprising conservative amino acid substitutions can be tested in any one of the assays described herein to confirm that a desired activity, e.g. activity and specificity of a native or reference polypeptide is retained.
[00196] Amino acids can be grouped according to similarities in the properties of their side chains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val (V), Leu (L), lie (I), Pro (P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gin (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Alternatively, naturally occurring residues can be divided into groups based on common side-chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Particular conservative substitutions include, for example; Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Val; Leu into He or into Val; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into He; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val, into He or into Leu.
[00197] In some embodiments, the polypeptide described herein (or a nucleic acid encoding such a polypeptide) can be a functional fragment of one of the amino acid sequences described herein. As used herein, a “functional fragment” is a fragment or segment of a peptide which retains at least 50% of the wild-type reference polypeptide’s activity according to the assays described below herein. A functional fragment can comprise conservative substitutions of the sequences disclosed herein.
[00198] In some embodiments, the polypeptide described herein can be a variant of a sequence described herein. In some embodiments, the variant is a conservatively modified variant. Conservative substitution variants can be obtained by mutations of native nucleotide sequences, for example. A “variant," as referred to herein, is a polypeptide substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions or substitutions. Variant polypeptide encoding DNA sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference DNA sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of PCR-based site-specific mutagenesis approaches are known in the art and can be applied by the ordinarily skilled artisan. [00199] A variant amino acid or DNA sequence can be at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a mutant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g. BLASTp or BLASTn with default settings).
[00200] Alterations of the native amino acid sequence can be accomplished by any of a number of techniques known to one of skill in the art. Mutations can be introduced, for example, at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide -directed site-specific mutagenesis procedures can be employed to provide an altered nucleotide sequence having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations are very well established and include, for example, those disclosed by Walder et al. (Gene 42: 133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Pat. Nos. 4,518,584 and 4,737,462, which are herein incorporated by reference in their entireties. Any cysteine residue not involved in maintaining the proper conformation of the polypeptide also can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or facilitate oligomerization.
[00201] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA, cDNA, bacterial DNA, or viral DNA. Suitable RNA can include, e.g., mRNA, bacterial RNA, viral RNA, crRNA, tracrRNA, or gRNA.
[00202] The term “substantial complementary” as used herein refers both to complete complementarity of the two nucleic acids, in some cases referred to as an identical sequence, as well as complementarity sufficient to achieve the desired binding of the two nucleic acids. The substantially complementary region can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments of any of the aspects, substantially complementary region is less than about 75,
50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. For example, the substantially complementary region is 10-30 nucleotides long. In some embodiments of any of the aspects, the substantially complementary region is 15-25 nucleotides long. In some embodiments of any of the aspects, the substantially complementary region is 18-22 nucleotides long. In some embodiments of any of the aspects, the substantially complementary region is about 20 nucleotides long.
[00203] The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. Expression can refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid fragment or fragments of the invention and/or to the translation of mRNA into a polypeptide.
[00204] In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are tissue-specific. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is/are global. In some embodiments, the expression of a biomarker(s), target(s), or gene/polypeptide described herein is systemic.
[00205] "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).
[00206] "Marker" in the context of the present invention refers to an expression product, e.g., nucleic acid or polypeptide which is differentially present in a sample taken from subjects having a bacterial infection, as compared to a comparable sample taken from control subjects (e.g., a healthy subject). The term "biomarker" is used interchangeably with the term "marker."
[00207] In some embodiments, the methods described herein relate to measuring, detecting, or determining the level of at least one marker. As used herein, the term "detecting" or “measuring” refers to observing a signal from, e.g. a probe, label, or target molecule to indicate the presence of an analyte in a sample. Any method known in the art for detecting a particular label moiety can be used for detection. Exemplary detection methods include, but are not limited to, spectroscopic, fluorescent, photochemical, biochemical, immunochemical, electrical, optical or chemical methods. In some embodiments of any of the aspects, measuring can be a quantitative observation.
[00208] In some embodiments of any of the aspects, a polypeptide, nucleic acid, or cell as described herein can be engineered. As used herein, “engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be “engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as “engineered" even though the actual manipulation was performed on a prior entity.
[00209] In some embodiments of any of the aspects, the engineered bacteriophage described herein is exogenous. In some embodiments of any of the aspects, the engineered bacteriophage described herein is ectopic. In some embodiments of any of the aspects, the engineered bacteriophage described herein is not endogenous.
[00210] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g. a nucleic acid encoding a polypeptide) or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively, “exogenous” can refer to a nucleic acid or a polypeptide that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell. As used herein, “ectopic” refers to a substance that is found in an unusual location and/or amount. An ectopic substance can be one that is normally found in a given cell, but at a much lower amount and/or at a different time. Ectopic also includes substance, such as a polypeptide or nucleic acid that is not naturally found or expressed in a given cell in its natural environment.
[00211] In some embodiments, a nucleic acid encoding a polypeptide as described herein (e.g. a virulence factor inhibitor) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence encoding a given polypeptide as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term “vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
[00212] In some embodiments of any of the aspects, the vector is recombinant, e.g., it comprises sequences originating from at least two different sources. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different species. In some embodiments of any of the aspects, the vector comprises sequences originating from at least two different genes, e.g., it comprises a fusion protein or a nucleic acid encoding an expression product which is operably linked to at least one non-native (e.g., heterologous) genetic control element (e.g., a promoter, suppressor, activator, enhancer, response element, or the like).
[00213] In some embodiments of any of the aspects, the vector or nucleic acid described herein is codon-optimized, e.g., the native or wild-type sequence of the nucleic acid sequence has been altered or engineered to include alternative codons such that altered or engineered nucleic acid encodes the same polypeptide expression product as the native/wild-type sequence, but will be transcribed and/or translated at an improved efficiency in a desired expression system. In some embodiments of any of the aspects, the expression system is an organism other than the source of the native/wild-type sequence (or a cell obtained from such organism). In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a mammal or mammalian cell, e.g., a mouse, a murine cell, or a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a human cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a yeast or yeast cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in a bacterial cell. In some embodiments of any of the aspects, the vector and/or nucleic acid sequence described herein is codon-optimized for expression in an E. coli cell.
[00214] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
[00215] As used herein, the term “viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
[00216] It should be understood that the vectors described herein can, in some embodiments, be combined with other suitable compositions and therapies. In some embodiments, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
[00217] As used herein, the terms "treat,” "treatment," "treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. a bacterial infection. The term “treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a bacterial infection. Treatment is generally “effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective" if the progression of a disease is reduced or halted. That is, “treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
[00218] As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
[00219] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. In some embodiments, administration comprises physical human activity, e.g., an injection, act of ingestion, an act of application, and/or manipulation of a delivery device or machine. Such activity can be performed, e.g., by a medical professional and/or the subject being treated.
[00220] As used herein, “contacting" refers to any suitable means for delivering, or exposing, an agent to at least one cell. Exemplary delivery methods include, but are not limited to, direct delivery to cell culture medium, perfusion, injection, or other delivery method well known to one skilled in the art. In some embodiments, contacting comprises physical human activity, e.g., an injection; an act of dispensing, mixing, and/or decanting; and/or manipulation of a delivery device or machine.
[00221] The term “statistically significant" or “significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
[00222] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.
[00223] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.
[00224] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[00225] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[00226] As used herein, the term “corresponding to” refers to an amino acid or nucleotide at the enumerated position in a first polypeptide or nucleic acid, or an amino acid or nucleotide that is equivalent to an enumerated amino acid or nucleotide in a second polypeptide or nucleic acid. Equivalent enumerated amino acids or nucleotides can be determined by alignment of candidate sequences using degree of homology programs known in the art, e.g., BLAST.
[00227] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third non-target entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
[00228] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00229] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00230] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties. [00231] Other terms are defined herein within the description of the various aspects of the invention.
[00232] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the fding date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[00233] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[00234] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
[00235] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
[00236] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A bacteriophage engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium.
2. The engineered bacteriophage of paragraph 1, wherein the bacteriophage is an engineered lambda (l) phage. 3. The engineered bacteriophage of any of paragraphs 1-2, wherein the inhibitor comprises an inhibitor protein encoded by a phage endogenous to the bacterium.
4. The engineered bacteriophage of any of paragraphs 1-3, wherein the inhibitor comprises cl protein from enterobacteria phage 933 W.
5. The engineered bacteriophage of any of paragraphs 1-4, wherein the inhibitor is engineered to be non-degradable.
6. The engineered bacteriophage of any of paragraphs 1-5, wherein the inhibitor comprises a Cas9 protein and at least one CRISPR guide R A that selectively targets a nucleic acid encoding a bacterial virulence factor.
7. The engineered bacteriophage of any of paragraphs 1-6, wherein the bacterium is Escherichia coli ( E . coll).
8. The engineered bacteriophage of any of paragraphs 1-7, wherein the bacterium is enterohemorrhagic . coli (EHEC).
9. The engineered bacteriophage of any of paragraphs 1-8, wherein the virulence factor is Shiga Toxin (Stx).
10. The engineered bacteriophage of any of paragraphs 1-9, wherein the engineered bacteriophage further comprises a heterologous bacteriophage immunity region.
11. The engineered bacteriophage of any of paragraphs 1-10, wherein the heterologous bacteriophage immunity region is a lambdoid phage immunity region.
12. The engineered bacteriophage of any of paragraphs 1-11, wherein the lambdoid phage is selected from the group consisting of lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22.
13. The engineered bacteriophage of any of paragraphs 1-12, wherein the engineered bacteriophage further comprises a selectable marker.
14. A pharmacological composition comprising the engineered bacteriophage of any of paragraphs 1-13 and an acceptable carrier.
15. A method of treating a bacterial infection, comprising administering an effective amount of an engineered bacteriophage of any of paragraphs 1-13 or a pharmaceutical composition of paragraph 14 to a patient in need thereof.
16. The method of paragraph 13, wherein the patient is infected with E. coli or EHEC.
17. A method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of an engineered bacteriophage of any of paragraphs 1-13 or a pharmaceutical composition of paragraph 14.
[00237] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs: A bacteriophage, wherein the bacteriophage genome is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium. The engineered bacteriophage of paragraph 1, wherein the bacteriophage is an engineered lambda (l) phage. The engineered bacteriophage of any of paragraphs 1-2, wherein the inhibitor comprises an inhibitor protein encoded by a phage endogenous to the bacterium. The engineered bacteriophage of any of paragraphs 1-3, wherein the inhibitor comprises cl protein from enterobacteria phage 933 W. The engineered bacteriophage of any of paragraphs 1-4, wherein the inhibitor comprises one of SEQ ID NOs: 9-10 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 9-10 that maintains the same function. The engineered bacteriophage of any of paragraphs 1-5, wherein the inhibitor is engineered to be non-degradable. The engineered bacteriophage of any of paragraphs 1-6, wherein the cl protein from enterobacteria phage 933 W comprises a K178N mutation that causes the protein to be non- degradable. The engineered bacteriophage of any of paragraphs 1-7, wherein the inhibitor comprises a Cas protein and at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor. The engineered bacteriophage of any of paragraphs 1-8, wherein the Cas protein comprises S. aureus Cas9. The engineered bacteriophage of any of paragraphs 1-9, wherein the Cas protein comprises SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 that maintains the same function. The engineered bacteriophage of any of paragraphs 1-10, wherein the CRISPR guide RNA comprises a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA). The engineered bacteriophage of any of paragraphs 1-11, wherein the tracrRNA comprises SEQ ID NO: 25 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 25 that maintains the same function. The engineered bacteriophage of any of paragraphs 1-12, wherein the crRNA comprises a variable targeting sequence and a region that is substantially complementary to a region of the tracrRNA. The engineered bacteriophage of any of paragraphs 1-13, wherein variable targeting sequence of the crRNA is substantially complementary to a nucleic acid encoding a bacterial virulence factor. The engineered bacteriophage of any of paragraphs 1-14, wherein the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 29 that maintains the same function. The engineered bacteriophage of any of paragraphs 1-15, wherein the bacterium is Escherichia coli ( E . coli). The engineered bacteriophage of any of paragraphs 1-16, wherein the bacterium is enterohemorrhagic E. coli (EHEC). The engineered bacteriophage of any of paragraphs 1-17, wherein the virulence factor is Shiga Toxin (Stx). The engineered bacteriophage of any of paragraphs 1-18, wherein the engineered bacteriophage genome further comprises a heterologous bacteriophage immunity region. The engineered bacteriophage of any of paragraphs 1-19, wherein the heterologous bacteriophage immunity region is a lambdoid phage immunity region. The engineered bacteriophage of any of paragraphs 1-20, wherein the lambdoid phage is selected from the group consisting of lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22. The engineered bacteriophage of any of paragraphs 1-21, wherein the heterologous bacteriophage immunity region comprises one of SEQ ID NOs: 13-17 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 13-17 that maintains the same function. The engineered bacteriophage of any of paragraphs 1-22, wherein the heterologous bacteriophage immunity region comprises SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 that maintains the same function. The engineered bacteriophage of any of paragraphs 1-23, wherein the engineered bacteriophage genome further comprises a nucleic acid encoding a selectable marker. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and b. a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium. cteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium. cteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, b. a heterologous bacteriophage immunity region wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium. cteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and b. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium. cteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, b. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, and c. a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium. 31. A pharmaceutical composition comprising the engineered bacteriophage of any of paragraphs 1-30 and an acceptable carrier.
32. A method of treating a bacterial infection, comprising administering an effective amount of an engineered bacteriophage of any of paragraphs 1-30 or a pharmaceutical composition of paragraph 31 to a patient in need thereof.
33. The method of paragraph 32, wherein the patient is infected with E. coli or EHEC.
34. A method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of an engineered bacteriophage of any of paragraphs 1-30 or a pharmaceutical composition of paragraph 31.
35. A composition of any of paragraphs 1-31, for use in a method of treating a bacterial infection, the method comprising administering an effective amount of an engineered bacteriophage of any of paragraphs 1-30 or a pharmaceutical composition of paragraph 31 to a patient in need thereof.
36. The composition of paragraph 35, wherein the patient is infected with E. coli or EHEC.
37. A composition of any of paragraphs 1-31, for use in a method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of an engineered bacteriophage of any of paragraphs 1-30 or a pharmaceutical composition of paragraph 31.
EXAMPLES
Example 1
[00238] Stable neutralization of virulent bacteria using temperate phage in the mammalian gut
[00239] Temperate phage lysogenized gut bacteria
[00240] Phages typically replicate by a lytic mechanism in which a bacterium infected by a phage turns its cellular machinery towards producing phage components that assemble into viral particles and are released upon cell lysis. This produces hundreds of progeny phage to infect new bacterial hosts (see e.g., Fig. 1A sections i-iii). Lytic phages replicate solely by this life cycle and the decimation of phage -susceptible bacteria selects for phage-resistant mutants that can repopulate over time (see e.g., Fig. 1A upper panel). In contrast, temperate phages can also integrate their genetic material into the host chromosome as a prophage to co-replicate with the bacterial genome during cell proliferation (see e.g., Fig. 1A sections iv-v). In a bacterial population, this leads to the lysogenic conversion of phage -susceptible species that coexist with phage -resistant species (see e.g., Fig. 1A lower panel). Because anti-bacterial approaches can enrich for resistance, including phage therapy which typically utilizes lytic phages, temperate phages were engineered to deliver an anti-virulence payload that neutralizes virulence in a manner that minimizes the selection for resistance. [00241] To illustrate the feasibility of using a temperate phage, bacteriophage l were shown to transduce a substantial fraction of targeted bacteria in the mammalian gut. As shown in Fig. IB, a streptomycin-treated mouse model was used to quantitate temperate phage lysogeny on E. coli colonizing the mammalian gastrointestinal tract. One day after colonization with E. coli MG1655, lBHI phage was introduced by oral gavage and daily stool samples were collected for analysis of bacterial and phage titers. lBHI were constructed from l phage by inserting an antibiotic resistance cassette for quantification of lysogens (see e.g., Fig. 2D). After oral administration of lBHI phage, fecal phage levels reached equilibrium approximately two days later and persisted at substantial concentrations (> 106 plaque forming units per gram of stool (pfu/g stool)) for the duration of the experiment (see e.g., Fig. 1C). As phage in the absence of its cognate bacterial host is undetectable in the stool of mice ~2 days after administration, these results indicate that lBHI phage is capable of continuous replication in the gut, permitting its expansion throughout the bacterial population from a single dose. Furthermore, introduction of lBHI phage did not significantly alter fecal E. coli concentrations (see e.g., Fig. ID), which is in sharp contrast to lytic phages that can cause substantial reduction. Using antibiotic selection, the number of fecal E. coli harboring the lBHI prophage was quantified and a substantial fraction (~17 to 30%) remained lysogenized by days 7 to 10 (see e.g., Fig. IE). Overall, these results indicate that the temperate phage l is capable of widespread modification of its cognate bacteria in the gut.
[00242] Phage hybridization overcame superinfection exclusion [00243] As gut bacteria harbor numerous prophages including those encoding virulence, overcoming superinfection exclusion mechanisms is crucial for achieving efficacious in situ transduction. For the foodbome pathogen Enterohemorrhagic E. coli (EHEC), the lambdoid prophage, 933 W, both produces Stx2 and inhibits phage superinfection by other lambdoid phages. To demonstrate the efficacy of the in situ phage-based anti-virulence strategy, experiments were undertaken to neutralize Stx2 production from E. coli MG 1655 lysogenized by 933 W ( E . coli 933W). [00244] E. coli 933W excludes l phage infection but genetic hybrids of l with other lambdoid phages restore infectivity. As shown schematically in Fig. 2A, the 933W prophage inhibits infection from l phage by recognition of its immunity region, e. indispensable genes responsible for the lysis- lysogeny decision in the phage life cycle. Because lambdoid phages have similarities in genetic function and organization despite dissimilar sequences, it is feasible to replace the l immunity region with orthologous immunity regions from other lambdoid phages to overcome the superinfection exclusion. The efficiency of plating (EOP) of l phage against E. coli 933W was ~106-fold lower than that of the non-lysogen (see e.g., Fig. 2B), confirming its superinfection exclusion. This effect was not due to a cl-based immunity (see e.g., Fig. 5). The EOP was then determined for genetic hybrids of l phage in which the l immunity region was swapped with that of other lambdoid phages (e.g. 21, 434, and P22; see e.g., Fig. 6A-6C and Table 3). As shown in Fig. 2B, these hybrid phages had substantially improved EOPs against E. coli 933W with 6.0% for Ximmll and 6.7% for imm434 phages. Moreover, hybridization with the Salmonella phage P22 resulted in near complete recovery of EOP to 78% for /2mmP22dis phage, indicating that lambdoid phages from non-cognate bacterial hosts can be a reservoir for genetic orthologs that maintain phage function while circumventing superinfection exclusion mechanisms.
[00245] Genetic-based anti- virulence encoded by hybrid temperate phage
[00246] Expression of Stx2 is dependent upon induction of the 933 W prophage. As schematically depicted in Fig. 2C, panel i), the 933W prophage in E. coli 933W maintains a dormant state by expression of its repressor protein, cl, which blocks expression of cro and consequently lytic genes including Stx2. Induction, which occurs spontaneously and by stimuli such as antibiotics, causes activation of the bacterial SOS response and RecA-mediated degradation of Cl (see e.g., Fig. 2C panel ii). This ultimately leads to expression of the lytic genes that produce phage progeny and Stx2. As the phage encoded repressor for the 933W prophage, cl, is key to blocking lytic induction and maintaining the dormant lysogenic state, constitutive expression of a non-degradable mutant of this repressor ( 933W-cTnd~ ) that contains a Lysl78Asn mutation blocks induction of the 933W prophage and consequently neutralizes production of progeny phage and Stx2 (see e.g., Fig. 2C panel Hi), ultimately demonstrating anti-virulence at a genetic level.
[00247] Efficient gene transduction permits the delivery of anti-virulence genes. Genes for 933W-cFnd~ and a kanamycin resistant cassette (to quantitate lysogeny) were inserted into the non- essential b2 region of l,30 producing lBHI (see e.g., Fig. 2D and Fig. 7). We confirmed that 933W-cFnd~ expressed from lBHI was functional (see e.g., Fig. 8). To overcome superinfection exclusion from the 933 W prophage, a P22 immunity region was utilized instead of a l immunity region. A phage cross between lBHI and l/m/i? P22dis resulted in the replacement of ~6 kb of the immunity region of lBHI with a ~5 kb portion of that from / «?«?P22dis while retaining 933W-cEnd~ and KarP genes (see e.g., Fig. 2D, Fig. 7, and Table 3). This new phage, lBH2, showed improved EOP to 90% (see e.g., Fig. 2D) and demonstrated a functional loss of l immunity and gain of P22 immunity, as well as expression of functional 933W-cFnd~ (see e.g., Fig. 8).
[00248] Anti-virulence phage inhibited Stx2 production in vitro
[00249] Transcriptional repression delivered by lBH2 phage neutralizes Stx2 production. As outlined in Fig. 3A, the efficacy of lBH2 phage to inhibit Stx2 production from E. coli 933W was tested by mixing them at equal concentrations (multiplicity of infection (MOI) ~1) and culturing for 8 h. Significantly less Stx2 was produced in E. coli 933W cultures treated with lBH2 phage compared to those untreated (“buffer”) or treated with ' /.immPlldis phage, the parental phage of lBH2 that is capable of infecting E. coli 933W but lacks the 933W-cTnd~ gene (see e.g., Fig. 3B). Quantification of bacterial concentrations over time show that E. coli 933W steadily grew over 8 h in the absence of phage (“buffer”) whereas introduction of /J«?«?P22dis resulted in an initial drop in titer during the first 4 h followed by a recovery (see e.g., Fig. 3D, non-induced) . For lBH2 phage, a similar drop in bacterial concentration was associated with increased lysogenic conversion of E. coli 933W that reached 70% by 4 h, indicating that both decreased bacterial titers and repressed Six 2 expression can contribute to the overall reduction of Stx2 concentration. To confirm that the latter provides sustained anti -virulence effect, we isolated lBH2 lysogens of . coli 933 w. i.e. E. coli containing prophages of both 933W and lBH2 (see e.g., Fig. 3F) and measured the Stx2 produced in culture. While an E. coli 933W culture accumulated 13.1 ng/mL of Stx2 over 8 h, there was no Stx2 detected for lBH2 lysogens (see e.g., Fig. 3G). Similarly, lBHI lysogens did not produce detectable concentrations of Stx2 despite their poor ability to initially infect E. coli 933W, confirming that once lysogenic conversion occurs the resultant lysogens do not produce Stx2.
[00250] Stx2 repression is maintained under inducing conditions. DNA damaging agents such as antibiotics can induce lambdoid prophages towards lysis (see e.g., Fig. 1A, lower panel) by activating the bacterial SOS response leading to RecA-mediated degradation of Cl (see e.g., Fig. 2C). To test whether lBH2 phage remains effective under these more aggressive lytic conditions, Stx2 produced in cultures of lBH2 phage mixed with E. coli 933W (see e.g., Fig. 3 A) was measured in the presence of an inducing agent, mitomycin c. As shown in Fig. 3 C, E. coli 933 w receiving buffer alone produced substantially more Stx2 when incubated with mitomycin c due to induction of the 933 W prophage. This induction also directed other phages towards primarily lytic replication and so the introduction of /. mm P22dis phage significantly reduced E. coli 933W concentrations (see e.g., Fig. 3D induced) and consequently Stx2 concentrations (see e.g., Fig. 3C). Ultimately, lBH2 phage treatment achieved significantly lower Stx2 concentrations than those measured for buffer and /J«?«?P22dis phage conditions (see e.g., Fig. 3C) because it was capable of repressing six 2 expression from a large fraction of E. coli 933W as shown by the substantial lysogenic conversion (see e.g., Fig. 3E induced).
To confirm that once lysogeny was established by lBHI or lBH2 phage, six 2 repression was maintained even in the presence of mitomycin c, lBH2 lysogens of E. coli 933W were cultured for up to 8 h in the presence of mitomycin c (see e.g., Fig. 3F). In the case of lBH2 lysogens of A. co// 933W, the toxin was undetectable, indicating repression was maintained under inducing conditions (see e.g., Fig. 3H).
[00251] Anti-virulence phage reduced Stx2 production in vivo
[00252] lBH2 reduced fecal Stx2 concentrations in mice. To determine if the phage-based anti virulence strategy was effective in vivo, a mouse model of enteric Stx2 intoxication from Stx- producing E. coli was used. While it is challenging to model the effect of enteric pathogens including Stx-producing E. coli in mice, mitomycin c injections can induce substantial quantities of Stx that is otherwise too low to be detected in stool. Mice pre-colonized by E. coli 933W were orally treated with lBH2 phage and then received three doses of mitomycin c by intraperitoneal injection to induce stx2 expression (see e.g., Fig. 4A). Daily fecal samples were collected for analysis of bacterial and Stx2 concentrations. After mitomycin c injections, fecal Stx2 was quantified, and it was found that lBH2 treatment reduced fecal Stx2 titers compared to buffer on days 3 and 4 with the latter showing statistical significance (see e.g., Fig. 4B). Furthermore, mice receiving lBH2 phage showed a significant reduction in fecal Stx2 from day 3 to 4, whereas buffer treated mice did not (p = 0.547; Wilcoxen test). Although lBH2 phage did not completely repress Stx2 production, these were highly inducing non-physiological conditions with fecal Stx2 concentrations (~102 to 103 ng Stx2/g mouse stool) in excess of those encountered in human Stx-producing E. coli infections (-2-50 ng Stx2/mL human stool).
[00253] lBH2 phage lysogenized E. coli 933W and did not affect its titer in the murine gut. Quantification of total fecal E. coli 933W did not reveal markedly different concentrations between buffer and lBH2 treated mice (see e.g., Fig. 4C). Quantification of fecal E. coli 933W lysogenized by lBH2 (see e.g., Fig. 4D) showed that a substantial fraction of the population was transduced, with geometric means between -0.9% to 2.6% and individual samples reaching as high as 71%. Notably, there was a large spread in lysogeny between individual mice with daily means lower than what was found in a previous experiment with E. coli lacking the 933W prophage (see e.g., Fig. IE). However, a high level of induction can disfavor lysogeny, and the use of a mitomycin c mouse model may underestimate the achievable degree of lysogeny in more typical, less-inducing conditions such as those shown in Fig. 1B-1E.
[00254] Lysogeny by lBH2 phage reduced fecal concentrations of Stx2. Though treatment with lBH2 phage showed a reduction in the average fecal Stx2 between treated and untreated groups, it was confirmed that this reduction was indeed associated with lysogenic conversion by lBH2 phage. By plotting the fecal concentration of Stx2 as a function of lysogeny by lBH2, for both days 3 and 4 there was a significantly non-zero inverse correlation (see e.g., Fig. 4E and 4F) confirming that reduced fecal Stx2 was caused by anti-virulence effect from lBH2 phage.
[00255] DISCUSSION
[00256] Herein is demonstrated a genetic strategy for in situ anti -virulence treatment of bacteria colonizing the gut. Temperate phage l were genetically engineered to express a repressor that neutralizes Stx production in E. coli and take advantage of the genetic mosaicism of lambdoid phages to create a hybrid phage that is capable of overcoming phage resistance mechanisms. The anti virulence phage not only efficiently infected, lysogenized, and inhibited Stx2 production from E. coli in vitro, but it also was effective at propagating in the murine gut from a single dose to significantly reduce Stx2 production in vivo.
[00257] With the complexity and interconnectedness of microbes in the gut, perturbations can have unexpected consequences. Modulating the impact of a bacterial species by manipulating its concentration can lead to unintended cascading effects mediated by inter-bacterial or bacterial-host interactions. While the typical strategy is to eliminate a particular bacteria, it is usually a specific function performed by this bacteria that is deleterious. By precisely and robustly modifying this individual function, a therapeutic effect can persist while minimizing disruption to the surrounding microbiota and avoiding the selection for resistance. Described herein is a framework for making precise genetic modifications that can be practically applied to bacteria within a complex biological system such as the gut microbiome.
[00258] For treating pathogenic bacterial infections, disarming their pathogenicity by targeting virulence factors provides a direct therapeutic strategy. With the impending crisis of antimicrobial resistant infections, new strategies for combating pathogens are desperately needed. By aiming to repress virulence factors instead of relying on anti-bacterial effect, the selection pressures for resistance are minimized. Furthermore, using a genetic-based approach makes it feasible to rationally design anti-virulence strategies that target one or multiple virulence factors to improve therapeutic efficacy.
[00259] As greater insight is gained into the importance of the gut microbiota to health and its inter-individual diversity and complexity, modification of specific bacteria within this community requires a thoughtful and nuanced approach that maximizes therapeutic efficacy and minimizes collateral effect. This work illustrates a framework by which bacteria can be specifically modulated in situ with rationally designed function in a manner alternative to present approaches and can be used in strategies for treating recalcitrant bacterial infections.
[00260] MATERIALS AND METHODS
[00261] Animal studies. Female BALB/c mice (Charles River Laboratories™) 6-7 week old were acclimated for one week prior to experiments.
[00262] To study of the effect of temperate phage on non-pathogenic E. coli in the mouse gut (Figure 2), mice received 5 g/L of streptomycin sulfate (Gold Bio™) in their drinking water which was replaced every 2-3 days. On day 0, 100 pL of streptomycin-resistant A. coli MG 1655 was administered to mice by oral gavage. The bacterial gavage solution was prepared from an overnight culture in LB, washed twice with PBS, and then diluted 100-fold into PBS, yielding ~107 cfu/mL (colony forming units per milliliter). One day later (day 1), mice received 100 pL of lBHI phage which consisted of a 5 x 107 pfu/rnL solution diluted 1:10 into 100 mM sodium bicarbonate immediately prior to gavage. Daily stool samples were collected for microbial quantification. To quantify fecal phage, fresh non-frozen samples were gently suspended into 1 mL of phage buffer, incubated at 4°C for ~10 min with a few drops of chloroform, and then pelleted at 4000 rpm at 4°C. Phage concentration was determined using a double-agar overlay plaque assay in which serially diluted phage solutions were incubated for 20 min at r.t. (room temperature) with a hardened overlay of E. coli MG1655 in 0.3% agar in TNT media over a 1.5% agar in TNT base. After aspiration, plates were incubated at 37°C overnight after which plaques were counted. To quantify fecal E. coli, frozen stool was thawed from -80°C and suspended into 1 mL of PBS by vortexing for 10 min at 4°C followed by low-speed centrifugation at 200 rpm for 20 min to settle fecal debris. The fecal suspension was then serially diluted into PBS and 100 pL was plated onto MacConkey agar (Remel™) plates supplemented with 100 pg/mL streptomycin sulfate to quantify total E. coli or supplemented with 100 pg/mL streptomycin and 50 pg/mL kanamycin to quantify lBHI lysogens of E. coli.
[00263] To study the effect of the engineered temperate phage on Stx2 -producing E. coli, mice were treated with similar conditions as described above with the following modifications. On day 0, mice received 100 pL of similarly prepared streptomycin-resistant E. coli 933W in PBS by oral gavage. On day 1, mice received 100 pL of lBH2 phage, which was a 5 x 1010 pfu/mL solution diluted 1:10 into 100 mM sodium bicarbonate immediately prior to gavage. On day 2, to induce Stx2 expression from engrafted E. coli, mice received three intraperitoneal injections of 0.25 mg/kg of mitomycin c at 3 h intervals. Stool samples were collected daily and stored at -80°C until analysis. Fecal E. coli was quantified by plating as described above, and fecal Stx2 was quantified from the same suspension of stool in PBS by mixing 10: 1 with 20 mg/mL of polymyxin B, incubating at 37°C for ~20 min and then storing at -20°C until analysis by ELISA as described below.
[00264] Bacterial strains. A table of bacteria used herein is listed in Table 1. E. coli 933W was generated by a previously described method (see e.g., O’Brien et al. Science 226, 694 (1984))., in which 933W phage was produced from the supernatant of a log phase culture of E. coli 0157:H7 strain edl933 in a modified LB media (10 g/L tryptone, 5 g/L yeast extract, 5 mM sodium chloride, 10 mM calcium chloride, and 0.001% thiamine) and then stored at 4°C. Molten top agar containing 100 pL of E. coli MG 1655 and 3 mL of modified LB media with 0.3% agar at 45 °C was poured onto plates of modified LB agar and allowed to harden. Supernatants of E. coli 0157:H7 cultures were then spotted onto the top agar and incubated at 37°C overnight. Resulting plaques were picked and re streaked onto LB. Successful 933W lysogens of E. coli were identified by screens for resistance to imm933W and the PCR amplification of the cl to cro region of 933 W (fwd (forward primer) - agccactcccttgcctcg (SEQ ID NO: 7); rev (reverse primer)-gcttatttcaagcatttcgcttgc (SEQ ID NO: 8)). E. coli lysogens of l and Hmni933W were generated similarly using TNT media instead of modified LB media and screened for successful lysogeny by resistance to l or Hmni933W, respectively, and ability to produce phage progeny.
[00265] Preparation of high titer phage stocks. Phage was propagated via the double agar overlay method where 100 pL of serially-diluted phage in phage buffer was mixed with 100 pL of E. coli MG1655 for ~20 min at r.t., then mixed with 3 mL of molten top agar (TNT media with 0.3% top agar at 45°C) and poured onto pre-warmed plates of TNT agar. After incubation overnight at 37°C, top agar from plates with the highest density of plaques were suspended into 5 mL of phage buffer and then gently rocked at 4°C for ~2 hrs. Supernatants were sterile filtered to yield ~109 to 1010 pfu/mL of phage. Phage stocks were stored at 4°C.
[00266] Phage strains. A table of phage used herein is listed in Table 2. lBHI phage was generated using the inherent '/.red recombination system of l phage expressed during lytic replication. A crude phage lysate containing recombinant phage was produced by mixing 100 pL of E. coli C600, containing a plasmid vector with Tn5-933W-cImd flanked by 400 bp homology to ea59 and ea47 in a pJET1.2 backbone (see e.g., Table 3), with 100 pL of serially-diluted l phage, incubated for 20 min at r.t. followed by addition with 3 mL of molten top agar (TNT media with 0.3% top agar at 45 °C) and poured onto TNT agar plates. After overnight incubation at 37°C, top agar from the plate with the greatest plaque density was resuspended into 5 mL of phage buffer (50 mM tris, 100 mM sodium chloride, 10 mM magnesium sulfate, and 0.01% gelatin, pH 7.5), sterile filtered, and stored at 4°C. To isolate the recombinant phage, 50 pL of crude phage lysate was mixed with 50 pL of E. coli C600 grown to log-phase in LB and incubated for ~3 h at 37°C. After incubation, 100 pL was plated onto LB containing 50 pg/mL of kanamycin and grown overnight at 37°C with individual colonies re streaked twice. To additionally purify by plaque purification, colonies were grown overnight in LB, and their sterile filtered supernatants were spotted onto TNT top agar of E. coli C600. Individual plaques were streaked onto LB containing 50 pg/mL of kanamycin and sequenced to confirm insertion in the correct locus of l phage.
[00267] lBH2 phage was generated by a phage cross between lBHI phage and /./m«?P22dis phage. 200 pL of a log phase E. coli C600 culture (7 x 107 cfii/mL) in T-broth (l%tryptone and 0.5% sodium chloride) with 0.4% maltose was mixed with a 200 pL solution of lBHI phage (1.5 x 108 pfu/mL) and /./m«?P22dis (1.5 x 108 pfu/mL) in phage buffer. After static incubation at 37°C for 20 min, this mixture was diluted 100-fold into pre-warmed T-broth with 1% glucose and cultured with shaking at 37°C for 90 min. The culture was treated with drops of chloroform, pelleted, and then the supernatant was sterile-filtered to produce a crude phage lysate. Residual chloroform was minimized by crossflowing air (Millipore Steriflip™) at r.t. for 1 h. 10 mL of this phage lysate was mixed with 1 mL of mid-log culture of a l lysogen of E. coli C600 grown in LB with 0.4% maltose, incubated at 37°C for 20 min. After ~30-fold concentration by centrifugation, 200 pL was plated onto LB containing 50 pg/mL of kanamycin. Resulting colonies were re-streaked twice and then tested for phage immunity by spot testing 5 pL of phage against a top agar containing each candidate colony. Correctly engineered phages, as lysogens, were identified from colonies by susceptibility to /./m«?434 (positive control) but resistance to Hmni933W (presence of 933WcTnd~ gene) and resistance to limmFUdis (presence of P22 immunity region). Phage lysates were prepared by culturing colonies overnight in TNT media, pelleting and sterile filtering the supernatant, and then incubating 100 pL of this phage mixture with 100 pL of E. coli C600 (MOI-0.1) at 37°C for 20 min and then plating onto LB with 50 mg/mL of kanamycin. After overnight incubation at 37°C, phage was plaque purified by preparing phage lysates from individual colonies as described above and streaking 10 pL onto hardened top agar containing E. coli C600. After overnight incubation at 37°C, individual plaques were picked and restreaked onto LB with kanamycin. The resultant lBH2 lysogen of E. coli was confirmed susceptible to l and 'kimmA3A as well as resistant to Hmni933W and '/.immPlldis (see e.g., Fig. 8). Sequencing confirmed the presence of the P22 immunity region and 933Wc nd~ gene (see e.g., Fig. 7 and Table 3).
[00268] Quantifying phage and efficiency of plating. The infectivity of phage against E. coli was quantified by the double overlay agar method in which E. coli MG 1655 or E. coli 933W was cultured overnight in TNT media, diluted 1: 100 into fresh TNT media and cultured until mid-log phase of which 50 pL was mixed with 700 pL of molten top agar (TNT media with 0.5% agar at 45°C) and poured into individual wells of a 6-well plate containing pre-poured TNT media with 1.5% agar. After hardening, 100 pL of phage serially-diluted in phage buffer was added and incubated for 20 min at r.t. followed by aspiration. Plates were incubated at 37°C overnight and then examined for titers of plaque forming units. Efficiency of plating was calculated as the titer of phage on the E. coli 933 w divided by its titer on the non-lysogenic E. coli.
[00269] In vitro assay of phage effect. E. coli 933W was cultured overnight in TNT media at 37°C, then cells were washed once with fresh TNT media and diluted to ODMI(lnm = 0.1 (~8 x 107 cfii/mL).
At t = 0, 5 mL of E. coli suspension was mixed with 1 mL of 4 x 108 pfii/mL of lBH2 or / «?«?P22dis phage solution. To quantify E. coli concentrations in solution, aliquots were collected, serially-diluted into PBS and the spotted (10 pL) onto LB or LB with 50 pg/mL kanamycin plates to quantify total E. coli and lBH2 lysogens, respectively. After 8 h, aliquots were mixed 10: 1 with 20 mg/mL of polymyxin B, incubated at 37°C for ~20 min and stored at -20°C for quantification of Stx2 by ELISA. [00270] Stx2 concentrations in cultures of E. coli 933W, its lBHI lysogen, or its lBH2 lysogen were prepared similarly as described to above, where overnight cultures were washed with fresh TNT media and diluted to Oϋboohih = 0.1. During incubation at 37°C, aliquots were collected, mixed with polymyxin B and stored at -20°C for quantification of Stx2 by ELISA.
[00271] ELISA quantification of Stx2. Maxisorp plates (ThermoScientific™) were incubated with 100 pL/we 11 of mouse monoclonal Stx2 antibody (Santa Cruz Biotechnology™, sc-52727) diluted 1:2500 into PBS for 1.5 h at r.t.. Plates were thrice washed with PBST (PBS with 0.05% Tween20™), then incubated with 200 pL of 1% BSA in PBS overnight at 4°C. After washing thrice with PBST, 100 pL/well of samples and a standard curve of diluted Stx2 (List Biological Labs™, #164) were incubated at r.t. for 2 h. Following sample incubation, plates were washed thrice with PBST and incubation with 100 pL/well of anti-Stx2 Ab-HRP conjugate for 1 h at r.t.. The antibody- enzyme conjugate was previously prepared using an HRP conjugation kit (Abeam™ #ab 102890) with a rabbit anti-Stx2 antibody (List Biological Labs™, #765L) according to the manufacturer’s protocol. After washing thrice with PBST, 100 pL/well of colorimetric reagent Ultra TMB™ (ThermoFisher™) was incubated at 37°C for 30 min prior to the addition of 50 pL/well of 2M H2SO4 to stop the reaction. Absorbance was measured at 450 nm.
[00272] Table 1: Bacteria
Figure imgf000068_0001
[00273] Table 2: Phage
Figure imgf000068_0002
[00274] Table 3: Sequences
Figure imgf000068_0003
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Example 2
[00275] Modulation of bacterial gene expression using engineered phages [00276] As shown herein, temperate phages can be engineered to modulate bacterial gene expression. Key is that this approach does not kill the targeted bacterium and instead induces targeted gene knockdown. This approach can be seen as a modular anti-virulence approach that reduces the encouragement of resistance development typically seen with bacteriolytic approaches like lytic phages and antibiotics. In some cases, antibiotics are not prescribed because they can make the bacterial infection worse (e.g., shigatoxin producing bacteria, enterohemorrhagic E. coli).
[00277] Temperate phages can infect a bacterium and predominantly integrate their genomes into the bacterial genome (e.g., as a prophage), replicating along with the bacterium. In one instance, an engineered phage was used to intracellularly produce a repressor of shigatoxin (e.g., cl protein of 933 W prophage) and demonstrate that this neutralizes shigatoxin expression in vitro and in enteric mouse models of infection. In another instance, engineered phage using a deactivated Cas9 were used to allow the customization of the target gene for repression. As shown herein, this phage can repress expression of a fluorescent protein in E. coli.
[00278] Shigatoxin producing E. coli was mixed with various phages in liquid culture with shigatoxin measured over time. Of the non-engineered wild type phages (see e.g., Fig. 9 left panel), lambda (temperate) and lambda-immP22dis (temperate) have no marked impact on shigatoxin compared to the control (buffer only). The lytic T4 phage shows an initial suppression of shigatoxin production, but the expansion of T4-resistant E. coli leads to the eventual increase in shigatoxin production. Two types of lambda phage (see e.g., Fig. 9 right panel) were engineered, one that is unable to productively integrate itself into the bacterial genome (vl) and one that successfully integrates itself (v2). The v2 phage shows complete suppression of shigatoxin production.
[00279] This approach was also tested in vivo by colonizing mice with these shigatoxin-producing E. coli, then administering no phage, vl or v2 (see e.g., Fig. 10). Comparison of the shigatoxin production in stool between phage treatments (see e.g., Fig. 10 left panel) shows that the v2 engineered phage successfully represses shigatoxin in the gut. It was confirmed that a large fraction of the shigatoxin-producing bacteria harbor the v2 engineered phage using a selection marker (see e.g., Fig. 10 right panel).
[00280] In another demonstration, lambda phage were engineered to include a deactivated Cas9 (dCas9) that binds to targeted regions using a crispr RNA guide sequence. In an E. coli strain that constitutively expresses RFP and GFP (see e.g., Fig. 11 panel ii), the engineered phage with crRNA targeting RFP neutralize RFP fluorescence (see e.g., Fig. 11 panel iii) whereas the same construct without the crRNA has no effect (see e.g., Fig. 11 panel iv).
[00281] An example of flow cytometry for mCherry fluorescence in E. coli is shown in Fig. 12. E. coli was mixed with buffer or engineered dCas9 phage with and without crRNA for 1 d and analyzed by flow cytometry to quantitate intracellular mCherry fluorescence. E. coli treated with ::dCas9- crRNA showed similar fluorescence levels to those receiving buffer whereas E. coli treated with ::dCas9+crRNA. i.e. engineered phage comprising crRNA targeting the mCherry gene for repression, showed a marked shift in fluorescence towards that of the E. coli control that lacks mCherry.
[00282] This strategy can be used to selectively repress any number of genes. The selectivity of phage allows the specific targeting of a particular species within a complex mixture, such as the gut microbiome, as demonstrated herein. This method is also adaptable using an activating Cas9 construct to increase gene expression as well. The targeted anti-virulence of pathogens can be used as an alternative to antibiotics that minimizes the development of resistance.
Example 3
[00283] In situ reprogramming of gut bacteria by oral delivery
[00284] Abundant links between the gut microbiota and human health indicate that modification of bacterial function can be a powerful therapeutic strategy. The inaccessibility of the gut and inter connections between gut bacteria and the host make it difficult to target bacterial functions without disrupting the microbiota and/or host physiology. Described herein is an approach to modulate the expression of a specific bacterial gene within the gut by oral administration. An engineered temperate phage l expressing a programmable dCas9 represses a targeted E. coli gene in the mammalian gut. To facilitate phage administration while minimizing disruption to host processes, an encapsulation formulation was developed, and it facilitates oral delivery of phage in vivo. Bacterial gene expression in the mammalian gut can be precisely modified in situ with a single oral dose.
[00285] Introduction
[00286] The gut microbiome has numerous associations with human health. This bacterial community contains hundreds of densely colonizing species with a composition that varies along the gastrointestinal tract, between individuals, and overtime. The complexity of this ecosystem makes it challenging to precisely target specific bacteria without unintended impacts to the microbiota. To permit the interrogation and therapeutic modification of the interactions between microbes and the host, generalizable tools are needed, especially ones capable of modifying specific bacterial functions while minimizing disruption to non-targeted genes, microbes, and host physiology.
[00287] Phages are viewed as possible therapeutics due to their capability of targeting specific bacteria, even when the bacteria are part of complex consortia. Lytic phages, which kill their cognate bacteria during phage propagation, have been of particular interest due to the prevalence of antibiotic resistant infections. Temperate phages have been of less interest therapeutically because they do not primarily pursue lysis and can integrate themselves into the bacterial genomes as prophages. While temperate phages may have some utility as antibacterial agents on their own or as adjuvants, their specificity and lysogenic conversion of bacteria offers an alternative non-lytic strategy: introducing new genes or reprogramming endogenous gene expression in precisely targeted bacteria within their natural ecosystems such as the gut microbiota. As described in Example 1, bacterial virulence can be repressed in vitro and in the mammalian gut using a lysogenic phage engineered to specifically repress shigatoxin expression.
[00288] Described herein is a non-invasive strategy to modify gene expression of specific bacteria in the mammalian gut via oral delivery. First, temperate phage l were engineered to express a nuclease-deactivated Cas9 (dCas9) that specifically represses gene expression in bacteria both in vitro and when colonizing the mouse gut. To improve survival against gastric acid and proteases, allow release into the lower GI tract, and minimize potential physiological disruption to host and microbial processes, an encapsulation formulation was developed to protect phage during oral delivery. Finally, described herein is a non-invasive and minimally-disruptive in situ modification of bacteria in the mammalian gut.
[00289] Engineered phage modifies gene expression in vitro
[00290] Engineered phage containing dCas9 represses target gene function. As shown in Figure 13A, the engineered construct was inserted into the l genome by replacing a portion of the non- essential b2 region. The system was tested in E. coli containing genomically-integrated rfp and gfp genes. The plasmid-based S. aureus dCas9 was effective in repressing fluorescence (see e.g., Fig. 16A-16C). When phage containing crRNA targeting rfp (7::dCas9rlp) was added to E. coli culture, RFP fluorescence was markedly reduced compared to phage lacking this crRNA (7::dCas9 phage) (see e.g., Fig. 13B). E. coli cultures receiving buffer showed a transitory decrease in fluorescence from ~2h to ~6h compared to those receiving phage, which was due to an absence of initial phage propagation that occurs with the l::dCas9 and /.::dCas9rlp phage treated samples as confirmed by the bacterial density (see e.g., Fig. 13C). Furthermore, the presence of crRNA in /.::dCas9rlp phage did not markedly affect bacterial growth compared to l::dCas9 phage. To confirm gene repression was maintained once lysogeny was established, RFP fluorescence from lysogens of E. coli was measured and reduced fluorescence was confirmed in /.::dCas9rlp lysogens compared to both non-lysogens and l:^Oh89 lysogens (see e.g., Fig. 13D). Lysogeny did not affect early exponential growth but may affect bacterial density at stationary phase (see e.g., Fig. 13E).
[00291] Engineered phage represses function of gut bacteria in situ
[00292] Engineered phage lysogenizes bacteria in the mouse gut. The efficacy of the engineered phage was tested in vivo, by administration of /.::dCas9 /.::dCas9rlp or vehicle (phage buffer) to mice pre-colonized with RFP-expressing E. coli. The fecal phage and E. coli concentrations were then tracked overtime (see e.g., Fig. 14A). To minimize degradation during gastric transit, phage solutions and vehicle were diluted 10-fold into a sodium bicarbonate solution immediately prior to oral gavage. As shown in Figure 14B, fecal phage were detectable at high concentrations soon after administration. Despite introduction of phage, total E. coli concentrations remained largely consistent (see e.g., Fig. 14C) indicating that a moderate dose (e.g., 107 pfii) of temperate phage did not markedly affect concentrations of cognate bacteria in the gut. The ratio of fecal phage to fecal E. coli showed initially high proportions of phage that progressively decreased for both l::dCas9 and /.::dCas9rlp treated mice (see e.g., Fig. 17), indicating an initial burst of phage amplification that reduced overtime, likely due to an increasing fraction of E. coli lysogens. A substantial fraction of fecal E. coli were l::dCas9 or l::dCas9Ifp lysogens soon after phage administration and remained so for the duration of the experiment (see e.g., Fig. 14D).
[00293] Lysogenized E. coli have reduced fluorescence. Functional gene repression by the engineered phage was assessed by measuring the relative RFP fluorescence of l:^Oh89 and /.::dCas9rlp lysogens isolated from mouse stool after oral phage administration. As shown in the representative culture plates in Figure 14E, fecal E. coli colonies from mice receiving /.::dCas9rlp phage demonstrated a maintained GFP and reduced RFP fluorescence compared to fecal colonies from mice receiving /.::dCas9 phage. These features were similar to in vitro cultured /.::dCas9rlp or l::dCas9 lysogens, respectively. An ensemble view of the relative fluorescence of ~50 fecal colonies from each mouse at each timepoint shows that RFP repression was maintained in each mouse over time (see e.g., Fig. 18) with significant fluorescence reduction by /.::dCas9rlp phage compared to l::dCas9 phage (see e.g., Fig. 14F).
[00294] Non-invasive and minimally disruptive modification of gut bacteria [00295] Encapsulated phage modifies gut bacteria in situ without compromising the gastric barrier. As shown in Fig. 14A-14F, /.::dCas9rlp phage significantly repressed RFP fluorescence in gut bacteria compared to l: :dCas9 phage, which lacks targeting to the rfp gene of E. coli. Phage administered in water is readily inactivated during oral administration unless the stomach acid is neutralized, phage is given at very high concentration, or phage is encapsulated (data not shown). To determine whether encapsulation impairs /.::dCas9rlp phage performance in situ, a low concentration (5 xlO3 pfu) of this phage was administered either as encapsulated phage suspended in water or as free phage mixed with bicarbonate buffer to neutralize the stomach acid (see e.g., Fig. 15A). Examination of mouse stool after oral phage administration revealed similar results in the following measurements for the bicarbonate-buffered phage compared to the encapsulated phage: fecal phage concentration (see e.g., Fig. 15B); total fecal E. coli concentration (see e.g., Fig. 15C); lysogenic conversion of fecal E. coli (see e.g., Fig. 15D); and ratio of fecal phage to E. coli (see e.g., Fig. 19). When examining the relative RFP fluorescence of individual /.::dCas9rlp lysogen colonies, buffered and encapsulated phage both provided similar levels of RFP repression for the duration of the study (see e.g., Fig. 15E). [00296] DISCUSSION
[00297] Demonstrated herein are microbiological and biomaterial advances for precise targeting of bacteria in the gut. Described herein is a precise and programmable approach for the in-situ modification of bacteria and bacterial transcription using temperate phage and RNA-guided dCas9. In some embodiments the phage are administered using an encapsulation formulation that releases engineered phage in the lower GI tract and maximizes the efficacy of oral delivery while minimizing potential physiological disruption. Described herein is a non-invasive, durable, and targeted approach towards modifying bacterial function in the gut.
[00298] Manipulating bacterial processes in situ is a powerful tool for dissecting the causal relationship between microbial and host physiology. Current approaches, such as the use of small molecules to interrogate the impact of microbial metabolites are limited by off target effects. The approach described herein allows for a fine tuned genetic control of a single gene within a single microbe, which makes it possible to systematically screen diverse genetic targets, characterize cause- effect relationships, and identify cascading effects to the surrounding microbiota and host. The phage described herein can be used as a therapeutic, with durable modification of gut bacteria using a single, self-propagating dose.
[00299] The life cycles of temperate phage — lysogeny and lysis — are important factors in the approach described herein. Lysogeny allows for the integration of genetic material into the bacterial genome while lysis permits phage amplification to reach more bacterial hosts beyond the initial dose. This work leverages these advantages to achieve a high rate of lysogenic conversion in the murine gut from a single relatively low dose: ~103 pfu phage compared to ~108 cfti/g stool of A. coli. Gene repression was demonstrated for at least 6 days. [00300] While there is great interest in the human gut, the microbiomes of other bodily regions such as the mouth, skin, bladder, nose, ears, eyes, and lungs can also be targeted by the engineered phage described herein. In addition, microbiomes in animals, plants, insects, soils, and bodies of water are all be targets for the modification of specific microbes using the engineered phage described herein.
[00301] METHODS
[00302] Molecular cloning and phage engineering
[00303] Golden Gate Assembly was used for cloning plasmids (see e.g., SEQ ID NOs: 30-31). Q5 Hot Start™ polymerase was used to amplify the proC promoter, l phage homology arms, pACYCDuet origin of replication, and pACYCDuet chloramphenicol resistance cassette. Restriction sites were also added during PCR. A DNA sequence including the coding sequence of S. aureus dCas9, the dCas9’s tracrRNA under a constitutive promoter, and the dCas9’s crRNA under a constitutive promoter, was ordered from IDT as two gBlocks. Chloramphenicol acetyltransferase was used as the antibiotic resistance and Cas9 from Staphylococcus aureus because of their minimal size. Cas9 was modified with nuclease -inactivating mutations in the HNH and RuvC catalytic regions (see e.g., Friedland et al. Genome Biol. 16, 257 (2015); Nishimasu et al. Cell 162, 1113-1126 (2015)). Golden Gate reactions were run using 10xT4 Ligase Buffer (Promega™), T4 Ligase (2,000,000 units/mL, NEB), and bovine serum albumin (BSA) (10 mg/mL, NEB) as well as the appropriate restriction enzyme, either Eco31I, Esp3I, or Sapl (Thermo FastDigest™). Golden Gate reactions were desalinated using drop dialysis (for a minimum of 10 minutes) and electroporated in DH 10b Electrocompetent Cells (Thermo Fischer™). Plasmids were verified by sequencing all junctions as well as the entirety of the gBlocks. The gRNA spacers were synthesized as complementary oligos then added to plasmids by Golden Gate after being annealed and phosphorylated. This was done by incubating the oligos with T4 Ligase Buffer (NEB) and T4 Poly Nucleotide Kinase (NEB), heating to boiling, and then slowly cooled to room temperature. Annealed oligos were added to plasmids using Eco31I. The gRNA spacers were verified by sequencing.
[00304] To generate a crude phage lysate containing the desired recombinant phage, cultures of log-phase E. coli C600 containing dCas9 plasmids grown in TNT (tryptone-NaCl-thiamine) media with 25 ug/mL chloramphenicol were pelleted and resuspended into an equal volume of TNT media. Plasmids contained 40-bp homology regions to ea59 and orf-314 genes to facilitate homologous recombination of the dCas9 construct into l phage. In a double agar layer plaque assay method, 100 pL of this culture was mixed with 100 pL of serially diluted l phage in phage buffer (e.g., 50 mM tris, 100 mM sodium chloride, 10 mM magnesium chloride, and 0.01% gelatin at pH 7.5) and then mixed with 3 mL of molten top agar (e.g., TNT with 0.3% agar) and immediately poured onto TNT agar plates to harden. After overnight culture at 37°C, the top agar from plates containing the highest density of individual plaques were resuspended into 5mL of phage buffer with gentle rocking at 4°C for 2h. These suspensions were then pelleted with the supernatant filtered through 0.45 um syringe filters, yielding crude phage lysates.
[00305] E. coli C600 grown to late log in TNT with 0.4% maltose was concentrated by pelleting and resuspension to ~1010 cfti/mL. 200 pL of this bacterial suspension was mixed with 200 pL of crude phage lysate and incubated at 37°C for 2.5 h, statically. Cultures were then plated onto LB with 34 ug/mL chloramphenicol and incubated overnight at 37°C. For plaque purification, phage was isolated by culturing streak-purified colonies in TNT overnight at 37°C, then treated with chloroform and pelleted. Plaques were then generated from serial dilutions of the supernatant by double overlay plaque assay with E. coli ( rfp+ , gfp+). After overnight incubation at 37°C, plaque centers were picked and streaked onto LB with chloramphenicol. Resultant colonies were checked for GFP fluorescence and PCR amplicons to confirm the correct bacterial host and presence of phage, respectively.
[00306] In vitro fluorescence measurements
[00307] In flat bottom 96-well fluorescence plates, 180 uL of log -phase E. coli ( rfp+ , gfp+) cultured in TNT and diluted to OD600 ~ 0.05 (1-cm pathlength) were mixed with 20 pL of /.::dCas9 phage or l::dCas9Ifp phage for a final multiplicity of infection (MOI) ~ 1.0, or phage buffer. Plates were shaken at 37°C for 10 h with measurements of ODMI(l. GFP fluorescence (ex 485nm/em 528nm), and RFP fluorescence (ex 555nm/ em 584 nm) at 5 min intervals (BioTek Synergy H1MF™). Studies of non-lysogens, l:AOh89 lysogens or /.::dCas9rlp lysogens were setup similarly except that 200 pL of log-phase cultures were used.
[00308] Animal studies
[00309] All animal work was approved by an Institutional Animal Care and Use Committee (IACUC) under an animal protocol. Upon arrival, 6-7 week old female BALB/c mice (Charles River™) were acclimated for a week prior to experiments. A solution of 5 g/L streptomycin sulfate USP grade (Goldbio™) was provided in the drinking water one day prior to oral gavage with 100 pL of streptomycin resistant A. coli MG 1655 or A. coli ( rfp+ , gfp+) using 20 gauge polytetrafluoroethylene (PTFE) animal feeding needles (Cadence Science™). The gavage solution of E. coli was prepared by inoculating an overnight culture (-16-20 h) in LB with 100 pg/mL streptomycin, then washing twice by pelleting and then re-suspending in an equal volume of PBS, and then diluting 10-fold into PBS to yield -107 - 108 cfii/mL. One day after administration of E. coli, 100 pL of phage solution was administered by oral gavage.
[00310] Testing the efficacy of engineered phage for repressing RFP fluorescence (see e.g., Fig. 14A-14F). Solutions of vehicle (phage buffer), either 109 pfii/mL of 7::dCas9 phage or 109 pfii/mL of /.::dCas9rlp phage, were diluted 10-fold into 0.1 M sodium bicarbonate followed by immediate administration of 100 pL to mice by oral gavage.
[00311] Testing the efficacy of encapsulated engineered phage in repressing RFP fluorescence (see e.g., Fig. 15A-15E). To test the efficacy of encapsulated k::dCas9rfp phage, 100 pL of a low-dose (5 x 103 pfu) of free phage or phage encapsulated at 710 pfu/encapsulation were administered to mice by oral gavage. Free /.::dCas9rlp phage was diluted 10-fold into 0.1 M sodium bicarbonate immediately before administration to mice. Encapsulated phage was resuspended into water immediately before administration to mice. Solutions and suspensions were administered by oral gavage using 16 gauge polyurethane feeding needles (Instech Labs™) to permit administration. [00312] To quantify the colonization within the mouse gut, daily stool samples were obtained. To quantify phage, within 30 min of excretion stool was suspended into 1 mL of phage buffer using sterile hospital sticks and then kept on ice. After 10 min, a few drops of chloroform was added to kill bacteria without affecting the bacteriophage. After an additional 10 min, the solution was pelleted at 2,100 x g for 10 min and then the supernatant diluted into phage buffer and quantified by plaque assay against the indicator bacteria, E. coli C600 using a double agar layer approach. To quantify bacteria, stool was frozen at -80°C within 30 min of excretion and then immediately prior to analysis, thawed at room temperature, re-suspended into 1 mL of PBS with vortexing for 10 min at 4°C and then gently centrifuged at ~2.5 x g for 20 min to allow debris to settle while leaving bacteria in suspension. E. coli was quantified by plating lOOuL of serial 10-fold dilutions in PBS onto MacConkey agar (Remel™) supplemented with (100 pg/mL) streptomycin to quantify total E. coli or MacConkey agar supplemented with (100 pg/mL) streptomycin sulfate and (50 pg/mL) kanamycin sulfate to quantify bacteriophage lysogens. Fluorescence from E. coli colonies was measured by plating onto LB with 34ug/mL chloramphenicol and incubated at 37°C for 2 d. Fluorescence images were taken with a Bio- Rad™ alpha imager and fluorescence intensity quantified by ImageJ.
[00313] To determine the gastric pH, mice were allowed free access to food and water, then sacrificed under CO2 and cervical dislocation, dissected, and a pH probe was immediately inserted into the gastric contents for measurement.
[00314] SEQUENCES
[00315] Table 4: Regions of SEQ ID NOs: 20-21
Figure imgf000086_0001
Figure imgf000087_0001
[00316] SEQ ID NO: 20, 4865 nt, 991-5855 of IP 567 (SEQ ID NO: 30); l phage homology arms, proC promoter, S. aureus dCas9, the dCas9’s tracrRNA under a constitutive promoter, a control crRNA (Bsal spacer) under a constitutive promoter, and pACYCDuet chloramphenicol resistance cassette for insertion into l. See e.g., Table 4 for region descriptions.
TA (1(IT(ΆAAAA(IAA(IAA(T/AA(1(Ά CTTA TT 'A TTAA 74 G4GCTAGC AGAGTTTGTAGAAACG CAAAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAATTTGATGCCTGGCAGTTTATG GCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCCTTCTAGAGCACAGCTAACAC CACGTCGTCCCT ATCTGCTGCCCT AGGTCT ATGAGTGGTTGCTGGATA ACTTTACGGGCA TGCATA AGGCTCGT ATGAT AT ATTCAGGGAGTCCACA ACGGTTTCCCTCTACA A ATA ATT TTGTTTA AGTTTTAGT ACiAGAA GGA GGAAAAAAAA ATGAAGCC7T AATT AT ATTTTGGGA GTGGGG ATGGGT ATT AGGTGTGTTGGTTAGGGTATG ATTGATTAGGAGAGTGGTGAT GTGATTGAGGGGGGGGTGGGTTTGTTTA AGGAGGGA AAGGTGGAAAAGAATGAGGG TGGGGGGTGGAAAGGTGGAGGGGGGGGGGTGAAAGGGGGGGGGGGTGATGGTATTG AAGGTGTTAAGAAATTGGTTTTGGAGTATAATTTATTGAGAGATGATAGTGAAGTGT GGGGGATGAATGGGTATGAAGGGGGGGTTAAAGGATTGTGAGAGA AGGTTAGGGAG GAGGA ATTTTGGGGAGGGTT AGTTGAGTT AGGGA AGGGTGGTGGTGTGGATA AGGT GA AGGA AGTGGA AGAGGAT AGTGGTA AGGAGTT ATGGAGGA A AGA AG AGATGAGTG GGAAGTGTAAGGGGGTTGAGGAAAAGTATGTAGGGGAGGTGGAAGTTGAGGGGTTG AAGAAGGACGGGGAGGTGCGCGGCAGTATTAACCGTTTTAAAACCAGCGACTATGT
CAAAGAAGCTAAACAATTATTGAAGGTGCAGAAAGCCTACCACCAACTTGACCAGT
CATTTATCGATACTTATATTGATCTTTTAGAGACTCGCCGTACTTATTACGAAGGCC
CTGGCGAGGGGTCGCCCTTCGGCTGGAAGGACATCAAGGAATGGTATGAAATGCTG
ATGGGGCATTGCACGTATTTCCCTGAAGAGCTTCGTTCCGTGAAGTATGCCTACAA
CGCCGATCTTT ACA ACGCACTTA ATGATTTA A ACA ACTT AGTA ATC ACTCGCGATGA
GAACGAGAAATTGGAATACTACGAAAAGTTCCAAATTATTGAGAATGTATTTAAGCA
AAAGAAGAAGCCAACACTGAAGCAGATTGCAAAAGAAATCTTAGTAAATGAAGAGG
ACATTAAAGGGTACCGCGTAACGTCGACCGGAAAGCCGGAGTTCACGAACCTTAAG
GTGTACCATGATATTAAAGATATCACGGCCCGTAAAGAAATTATCGAAAACGCTGA
ACTGTTAGACCAGATCGCTAAGATCTTGACGATTTATCAGTCTAGCGAAGATATTCA
GGAGGAGCTGACGAATCTTAACTCTGAGTTAACGCAGGAGGAAATTGAGCAGATTA
GCA ACTTGA AGGGAT ACACGGGGACCCACA ATCTTTCCTTA A A AGCGATC A ACCTT
ATCCTGGATGAGCTGTGGCATACGAACGACAATCAAATCGCTATTTTTAATCGCCTG
AAATTAGTGCCCAAGAAGGTGGACTTATCCCAGCAGAAGGAGATTCCTACCACTCT
TGTCGACGATTTCATTCTGTCGCCCGTAGTGAAGCGTTCATTCATCCAGTCAATCAA
AGTCATCAACGCAATTATTAAGAAATATGGCCTTCCTAACGACATTATCATCGAACT
TGCGCGTGAGAAGAACTCAAAGGATGCTCAAAAGATGATCAATGAGATGCAGAAAC
GTAATCGCCAGACAAACGAGCGCATCGAAGAAATTATTCGCACGACGGGAAAGGAA
AATGCTAAATATTTAATTGAGAAAATCAAACTTCACGACATGCAGGAGGGCAAGTG
TCTTTATTCACTGGAGGCGATCCCTTTGGAGGACCTGCTTAATAATCCGTTCAATTA
CGAGGTAGATCACATCATCCCCCGTAGCGTTTCTTTTGATAATTCTTTCAATAATAA
GGTCCTGGTTAAGCAGGAGGAAGCTTCCAAGAAGGGGAATCGTACGCCGTTCCAAT
ACTTGTCGTCCTCAGACAGCAAGATTTCATACGAAACCTTTAAGAAGCATATCTTAA
ACCTGGCTA AGGGCA AGGGCCGCATT AGCA A A ACCA AGA AGGA ATACCTTTT AGAG
GAGCGTGACATTA ACCGTTTTTCTGTCCA A A A AGACTTCATC A ATCGTA ACCTTGTG
GATACTCGCTACGCAACTCGTGGTCTTATGAATTTGCTTCGCTCGTACTTCCGTGTC
AACAACCTTGATGTTAAAGTGAAAAGTATTAACGGTGGGTTCACTTCATTTTTGCGT
CGTAAGTGGAAATTTAAGAAGGAACGCAACAAGGGGTACAAACACCACGCGGAAGA
TGCTTTAATCATTGCCAACGCTGACTTTATCTTTA AAGA ATGGAAAAAGCTTGACA A
GGCCAAGAAAGTTATGGAAAATCAGATGTTTGAGGAAAAGCAGGCAGAGAGTATGC
CAGAAATCGAGACTGAGCAGGAATATAAGGAGATCTTCATCACTCCGCATCAAATT
AAGCACATTAAAGACTTCAAGGATTATAAATATTCACACCGTGTGGATAAAAAGCCA
AATCGTGAATTAATCAACGACACGTTGTATAGTACTCGTAAGGATGACA AGGGTAA
CACCCTGATCGTAAACAACTTAAACGGCTTATATGACAAAGATAATGACAAGCTTAA
GAAACTTATTAACAAATCCCCAGAGAAACTTCTGATGTACCACCATGACCCTCAGAC TTACCAGAAATTAAAGCTGATTATGGAACAGTATGGGGACGAGAAAAACCCGCTTT
ACAAGTATTATGAAGAAACGGGGAACTACCTTACCAAGTATTCTAAAAAGGATAAT GGTCCAGTGATTAAAAAAATCAAGTACTACGGCAATAAGCTGAATGCACACTTGGA CATTACGGATGACT AGGGTA AT AGCCGTA ACA A AGTCGTA A AGCTGTCTTTA A AGCC TTACCGTTTCGATGTTTATTTAGATAACGGCGTCTATAAATTTGTGACCGTCAAAAA TTTAGACGTAATTAAGAAAGAGAATTATTACGAGGTTAATTCTAAATGCTACGAAGA AGCTAAAAAACTGAAAAAAATCTCCAATCAGGCAGAGTTCATCGCCAGTTTTTACAA CAATGACTTAATTAAAATTAATGGAGAACTTTACCGCGTTATCGGAGTTAACAATGA CTTGCTGAATCGTATTGAGGTCAACATGATCGATATTACATATCGCGAGTATTTGGA AAACATGAATGATAAGCGCCCGCCACGCATTATTAAAACAATCGCTTCGAAAACAC AGTCGATTAAGAAATACAGCACAGACATTCTGGGGAATTTATACGAAGTCAAGTCT A A G A A A C A TCCGC A G A TT A TT A A G A A GGGCT A A GGGGGTGA TAA A TTTCTTTGA A TTTCT CCTTGA TTA TTTGTTA TAAAAGTTA TAAAA /"A ATCTT A TTGTAGTTA TAGGTAAAA TTAGAGA A TGTA GTA AAA CA A GGGA AAA TGGGGTGTTTA TGTGGTGA A GTTGTTGGGGA GA TTT IJpATACTYCTATYCTACTCTGACTGCAAACCAAAAAAACAAGCGCTTTCAAAACGCTTGTTT 7> /- / /7777>4GGGAAATTAATCTCTTAATCCTTTTATCATTCTACATTTAGGCGCTGCCAT CTTGGGACAATGAAAACGTTAGTCATGGCGCGCCTTGACGGCTAGCTCAGTCCTAG GTACAGTGCTAGCTGAGACCGACTGAGGTCTCA GTTTTA GTA CTCTGTAA TTTTA GGT A TGA GGT A GA C T G C A G A C A A G C C C G G C C G G C C C C A G G C A T C A A A T A A A A G G A A A G G GTGAGTGGA A AGAGTGGGGGTTTGGTTTTATGTGTTGTTTGTGGGTGA AGGGTGTGT AGTAGAGTGAGAGTGGGTGAGGTTGGGGTGGGGGTTTGTGGGTTTATA AAA7,7XGGG CCCGCCCTGCCA CTCA TCGCA GTA CTGTTGTAA TTCA TTA A GCA TTCTGCCGA CA TGGAAGCCA TGA CA GA CGGCA TGA TGAACCTGAA TCGCCA GCGGCA TGA GCA CCTTGTCGCCTTGCGTA TAA TA TTTGGGGA TA GTGAAAAGGGGGGGGAAGAAGTTGTGGA TA TTGGGGA GGTTTAAA TGA A A AG TGGTGAAAGTGA GGGA GGGA TTGGGTGA GA GGAAAAAGA TA TTGTGAA TAAAGGGTTTA GGGAA A TA GGGGA GGTTTTGA GGGTA A GA GGGGA GA TGTTGGGA A TA TA TGTGTA GAAA GTGGGGGA A A TGGTGGTGGTA TTGA GTGGA GA GGGA TGAAAA GGTTTGA GTTTGGTGA TGGA AAA GGGTGTA A G A A GGGTGA AG ACT A TGGGA TA TGA GGA GGTGA GGGTGTTTGA TTGGGA TA GGGA A GTGGGGA TG A GGA TTGA TGA GGGGGGGA AG A A TGTGA A TAA A GGGGGGA TAAAA GTTGTGGTTA TTTTTGTTTA GGGTGTTTAAAAAGGGGGTAA TA TGGA GGTGAAGGGTGTGGTTA TA GGT A GA TTGA GGAAGTGA GTGAAA TGGGTGAAAA TGTTGTTTA GGA TGGGA TTGGGA TA TA TGAAGGGTGGTA TA TGGA GTGA
Figure imgf000089_0001
A CGTCTCA TTTTCGCCTTA TGGA A GGGTGA GA A TA TA GTTAAA TGGA A TGTTTTTG
[00317] SEQ ID NO: 21, 4868 nt, 991-5858 of IP 568 (SEQ ID NO: 31); l phage homology arms, proC promoter, S. aureus dCas9, the dCas9’s tracrRNA under a constitutive promoter, the dCas9’s crRNA under a constitutive promoter, and pACYCDuet chloramphenicol resistance cassette for insertion into l. See e.g., Table 4 for region descriptions.
TA OGTCAAAAAOAAOAAGTAAOCA (ΊΊA ΊΊCΆ ΊΊAA 74 fAGCTAGC AGAGTTTGTAGAAACG CAAAAAGGCCATCCGTCAGGATGGCCTTCTGCTTAATTTGATGCCTGGCAGTTTATG GCGGGCGTCCTGCCCGCCACCCTCCGGGCCGTTGCTTCCTTCTAGAGCACAGCTAACAC CACGTCGTCCCT ATCTGCTGCCCT AGGTCT ATGAGTGGTTGCTGGATA ACTTTACGGGCA TGCATA AGGCTCGT ATGAT AT ATTCAGGGAGTCCACA ACGGTTTCCCTCTACA A ATA ATT TTGTTTA AGTTTTAGT AGAGAA GGA GGAAAAAAAA
Figure imgf000090_0001
GTGGGG ATGGGT ATT AGGTGTGTTGGTTAGGGTATG ATTGATTAGGAGAGTGGTGAT GTGATTGAGGGGGGGGTGGGTTTGTTTA AGGAGGGA AAGGTGGAAAAGAATGAGGG TGGGGGGTGGAAAGGTGGAGGGGGGGGGGTGAAAGGGGGGGGGGGTGATGGTATTG AAGGTGTTAAGAAATTGGTTTTGGAGTATAATTTATTGAGAGATGATAGTGAAGTGT GGGGGATGAATGGGTATGAAGGGGGGGTTAAAGGATTGTGAGAGA AGGTTAGGGAG GAGGA ATTTTGGGGAGGGTT AGTTGAGTT AGGGA AGGGTGGTGGTGTGGATA AGGT GA AGGA AGTGGA AGAGGAT AGTGGTA AGGAGTT ATGGAGGA A AGA AG AGATGAGTG GGAAGTGTAAGGGGGTTGAGGAAAAGTATGTAGGGGAGGTGGAAGTTGAGGGGTTG AAGAAGGAGGGGGAGGTGGGGGGGAGTATTA AGGGTTTTAAAAGGAGGGAGTATGT GAAAGAAGGTAAAGAATTATTGAAGGTGGAGAAAGGGTAGGAGGAAGTTGAGGAGT GATTT ATGGAT AGTT ATATTGATGTTTT AGAGAGTGGGGGTAGTT ATT AGGA AGGGG GTGGGGAGGGGTGGGGGTTGGGGTGGAAGGAGATGA AGGAATGGTATGAAATGGTG ATGGGGGATTGGAGGTATTTGGGTGAAGAGGTTGGTTGGGTGAAGTATGGGTAGAA GGGGGATGTTT AGA AGGGAGTTA ATGATTTA A AGA AGTT AGTA ATG AGTGGGGATGA GAAGGAGAAATTGGAATAGTAGGAAAAGTTGGAAATTATTGAGAATGTATTTAAGGA AAAGAAGAAGGGA AGAGTGAAGGAGATTGGAAAAGAAATGTTAGTAAATGAAGAGG AGATTAA AGGGTAGGGGGTAAGGTGGAGGGGAAAGGGGGAGTTGAGGAAGGTTAAG GTGTAGGATGATATTAAAGATATGAGGGGGGGTAAAGAAATTATGGAAAAGGGTGA AGTGTTAGAGGAGATGGGTA AGATGTTGAGGATTTATGAGTGTAGGGAAGATATTGA GGAGGAGGTGAGGAATGTTAAGTGTGAGTTAAGGGAGGAGGA AATTGAGGAGATTA GGA AGTTGA AGGGAT AGAGGGGGAGGGAGA ATGTTTGGTTA A A AGGGATG A AGGTT ATGGTGGATGAGGTGTGGGATAGGAAGGAGAATGA AATGGGTATTTTTAATGGGGTG AAATTAGTGGGGA AGAAGGTGGAGTTATGGGAGGAGAAGGAGATTGGTAGGAGTGT TGTGGAGGATTTGATTGTGTGGGGGGTAGTGAAGGGTTGATTGATGGAGTGAATGAA AGTGATGAAGGGAATTATTAAGAAATATGGGGTTGGTAAGGAGATTATGATGGAAGT TGGGGGTGAGAAGA AGTGAAAGGATGGTGAAAAGATGATGAATGAGATGGAGAAAG GTAATGGGGAGAGAAAGGAGGGGATGGAAGA AATTATTGGGAGGAGGGGAAAGGAA AATGGTAAATATTTAATTGAGAAAATGAAAGTTGAGGAGATGGAGGAGGGGAAGTG TCTTTATTCACTGGAGGCGATCCCTTTGGAGGACCTGCTTAATAATCCGTTCAATTA
CGAGGTAGATCACATCATCCCCCGTAGCGTTTCTTTTGATAATTCTTTCAATAATAA
GGTCCTGGTTAAGCAGGAGGAAGCTTCCAAGAAGGGGAATCGTACGCCGTTCCAAT
ACTTGTCGTCCTCAGACAGCAAGATTTCATACGAAACCTTTAAGAAGCATATCTTAA
ACCTGGCTAAGGGCAAGGGCCGCATTAGCAAAACCAAGAAGGAATACCTTTTAGAG
GAGCGTGACATTA ACCGTTTTTCTGTCCA A A A AGACTTCATC A ATCGTA ACCTTGTG
GATACTCGCTACGCAACTCGTGGTCTTATGAATTTGCTTCGCTCGTACTTCCGTGTC
AACAACCTTGATGTTAAAGTGAAAAGTATTAACGGTGGGTTCACTTCATTTTTGCGT
CGTAAGTGGAAATTTAAGAAGGAACGCAACAAGGGGTACAAACACCACGCGGAAGA
TGCTTTAATCATTGCCAACGCTGACTTTATCTTTAAAGAATGGAAAAAGCTTGACAA
GGCCAAGAAAGTTATGGAAAATCAGATGTTTGAGGAAAAGCAGGCAGAGAGTATGC
CAGAAATCGAGACTGAGCAGGAATATAAGGAGATCTTCATCACTCCGCATCAAATT
AAGCACATTAAAGACTTCAAGGATTATAAATATTCACACCGTGTGGATAAAAAGCCA
AATCGTGAATTAATCAACGACACGTTGTATAGTACTCGTAAGGATGACAAGGGTAA
CACCCTGATCGTAAACAACTTAAACGGCTTATATGACAAAGATAATGACAAGCTTAA
GAAACTTATTAACAAATCCCCAGAGAAACTTCTGATGTACCACCATGACCCTCAGAC
TTACCAGAAATTAAAGCTGATTATGGAACAGTATGGGGACGAGAAAAACCCGCTTT
ACAAGTATTATGAAGAAACGGGGAACTACCTTACCAAGTATTCTAAAAAGGATAAT
GGTCCAGTGATTAAAAAAATCAAGTACTACGGCAATAAGCTGAATGCACACTTGGA
CATTACGGATGACT AGGGTA AT AGCCGTA ACA A AGTCGTA A AGCTGTCTTTA A AGCC
TTACCGTTTCGATGTTTATTTAGATAACGGCGTCTATAAATTTGTGACCGTCAAAAA
TTTAGACGTAATTAAGAAAGAGAATTATTACGAGGTTAATTCTAAATGCTACGAAGA
AGCTAAAAAACTGAAAAAAATCTCCAATCAGGCAGAGTTCATCGCCAGTTTTTACAA
CAATGACTTAATTAAAATTAATGGAGAACTTTACCGCGTTATCGGAGTTAACAATGA
CTTGCTGAATCGTATTGAGGTCAACATGATCGATATTACATATCGCGAGTATTTGGA
AAACATGAATGATAAGCGCCCGCCACGCATTATTAAAACAATCGCTTCGAAAACAC
AGTCGATTAAGAAATACAGCACAGACATTCTGGGGAATTTATACGAAGTCAAGTCT
A A A A A C A TCCGC A G A TT A TT A A A A GGGCT A A GGGGCTGA TAAA TTTCTTTGA A TTTCT
CCTTGATTATTTGTTATAAAAGTTATAAAATAATCTTA TTGTAGTTA TACCTAAAA TTACAGA
A TCTA GTA AAA GA A GGGA AAA TGGGGTGTTTA TGTGGTGA A GTTGTTGGGGA GA TTT
IJpATACTYCTATYCTACTCTGACTGCAAACCAAAAAAACAAGCGCTTTCAAAACGCTTGTTT
/^ /r^ /r/T/^GGGAAATTAATCTCTTAATCCTTTTATCATTCTACATTTAGGCGCTGCCAT
CTTGGGACAATGAAAACGTTAGTCATGGCGCGCCTTGACGGCTAGCTCAGTCCTAG
GTACAGTGCTAGCTTGGTAACTTTCAGTTTAGCGGT GTTTTA GTA CTCTGTAA TTTTA G
GTA TGA GGTA GA GC T G C A G A C A A G CCC G G C C G G C C C C A G G C A T C A A A T A A A A G G A A A
GGGTGAGTGGA A AGAGTGGGGGTTTGGTTTTATGTGTTGTTTGTGGGTGA AGGGTGT CTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAAAATTIC
GCCCCGCCCTGCCA CTCA TCGCA GTA CTGTTGTAA TTCA TTAAGCA TTCTGCCGA CA TGGAAGC CA TCA CA GA CGGCA TGA TGA A CCTGA A TCGCCA GCGGCA TCA GCA CCTTGTCGCCTTGCGTA T
Figure imgf000092_0001
GA CTGAAA TGCCTCAAAA TGTTCTTTA CGA TGCCA TTGGGA TA TA TCA A CGGTGGTA TA TCCA GT GA TTTTTTTCTCCA T TTTA GGTTGGTTA GGTGGTGA AAA TGTGGA TA A CTCA AAAAA TAG GGGGGGTA GTGA TGTTA TTTGA TTA TGGTGA A A GTTGGA A CCTCTTA GGTGGGGA TG
A A GGTGTGA TTTTGGGC TA TGGA A GCCTCA CA A TA TA GTTAAA TGGA A TGTTTTTG

Claims

CLAIMS What is claimed herein is:
1. A bacteriophage, wherein the bacteriophage genome is engineered to comprise at least one nucleic acid encoding an inhibitor of a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects a target bacterium without killing the bacterium.
2. The engineered bacteriophage of claim 1, wherein the bacteriophage is an engineered lambda (l) phage.
3. The engineered bacteriophage of any of claims 1-2, wherein the inhibitor comprises an inhibitor protein encoded by a phage endogenous to the bacterium.
4. The engineered bacteriophage of any of claims 1-3, wherein the inhibitor comprises cl protein from enterobacteria phage 933 W.
5. The engineered bacteriophage of any of claims 1-4, wherein the inhibitor comprises one of SEQ ID NOs: 9-10 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 9-10 that maintains the same function.
6. The engineered bacteriophage of any of claims 1-5, wherein the inhibitor is engineered to be non-degradable.
7. The engineered bacteriophage of any of claims 1-6, wherein the cl protein from enterobacteria phage 933 W comprises a K178N mutation that causes the protein to be non- degradable.
8. The engineered bacteriophage of any of claims 1-7, wherein the inhibitor comprises a Cas protein and at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor.
9. The engineered bacteriophage of any of claims 1-8, wherein the Cas protein comprises S. aureus Cas9.
10. The engineered bacteriophage of any of claims 1-9, wherein the Cas protein comprises SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 that maintains the same function.
11. The engineered bacteriophage of any of claims 1-10, wherein the CRISPR guide RNA comprises a trans-activating CRISPR RNA (tracrRNA) and a CRISPR RNA (crRNA).
12. The engineered bacteriophage of any of claims 1-11, wherein the tracrRNA comprises SEQ ID NO: 25 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 25 that maintains the same function.
13. The engineered bacteriophage of any of claims 1-12, wherein the crRNA comprises a variable targeting sequence and a region that is substantially complementary to a region of the tracrRNA.
14. The engineered bacteriophage of any of claims 1-13, wherein variable targeting sequence of the crRNA is substantially complementary to a nucleic acid encoding a bacterial virulence factor.
15. The engineered bacteriophage of any of claims 1-14, wherein the region of the crRNA that is substantially complementary to a region of the tracrRNA comprises SEQ ID NO: 29 or a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 29 that maintains the same function.
16. The engineered bacteriophage of any of claims 1-15, wherein the bacterium is Escherichia coli ( E . coll).
17. The engineered bacteriophage of any of claims 1-16, wherein the bacterium is enterohemorrhagic E. coli (EHEC).
18. The engineered bacteriophage of any of claims 1-17, wherein the virulence factor is Shiga Toxin (Stx).
19. The engineered bacteriophage of any of claims 1-18, wherein the engineered bacteriophage genome further comprises a heterologous bacteriophage immunity region.
20. The engineered bacteriophage of any of claims 1-19, wherein the heterologous bacteriophage immunity region is a lambdoid phage immunity region.
21. The engineered bacteriophage of any of claims 1-20, wherein the lambdoid phage is selected from the group consisting of lambdoid phage 21, lambdoid phage 434, and lambdoid phage P22.
22. The engineered bacteriophage of any of claims 1-21, wherein the heterologous bacteriophage immunity region comprises one of SEQ ID NOs: 13-17 or an amino acid sequence that is at least 95% identical to one of SEQ ID NOs: 13-17 that maintains the same function.
23. The engineered bacteriophage of any of claims 1-22, wherein the heterologous bacteriophage immunity region comprises SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 17 that maintains the same function.
24. The engineered bacteriophage of any of claims 1-23, wherein the engineered bacteriophage genome further comprises a nucleic acid encoding a selectable marker.
25. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
26. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and b. a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
27. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
28. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, b. a heterologous bacteriophage immunity region wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
29. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, and b. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises: i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
30. A bacteriophage, wherein the bacteriophage genome is engineered to comprise: a. at least one nucleic acid encoding an inhibitor protein encoded by a phage endogenous to a target bacterium, b. at least one nucleic acid encoding an inhibitor, wherein the inhibitor comprises i. a Cas protein and ii. at least one CRISPR guide RNA that selectively targets a nucleic acid encoding a bacterial virulence factor, and c. a heterologous bacteriophage immunity region, wherein the engineered bacteriophage is lysogenic and infects the target bacterium without killing the bacterium.
31. A pharmaceutical composition comprising the engineered bacteriophage of any of claims 1-
30 and an acceptable carrier.
32. A method of treating a bacterial infection, comprising administering an effective amount of an engineered bacteriophage of any of claims 1-30 or a pharmaceutical composition of claim
31 to a patient in need thereof.
33. The method of claim 32, wherein the patient is infected with E. coli or EHEC.
34. A method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of an engineered bacteriophage of any of claims 1-30 or a pharmaceutical composition of claim 31.
35. A composition of any of claims 1-31, for use in a method of treating a bacterial infection, the method comprising administering an effective amount of an engineered bacteriophage of any of claims 1-30 or a pharmaceutical composition of claim 31 to a patient in need thereof.
36. The composition of claim 35, wherein the patient is infected with E. coli or EHEC.
37. A composition of any of claims 1-31, for use in a method of inhibiting bacterial growth or activity on a surface, the method comprising contacting a surface with an effective amount of an engineered bacteriophage of any of claims 1-30 or a pharmaceutical composition of claim 31.
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WO2024052907A1 (en) * 2022-09-06 2024-03-14 Trobix Bio Ltd. Systems for production of transducing particles, methods, kits, compositions and uses thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023195008A1 (en) * 2022-04-05 2023-10-12 Mybiotics Pharma Ltd. Bacterial compositions and methods for growing bacteria on particles
WO2024052907A1 (en) * 2022-09-06 2024-03-14 Trobix Bio Ltd. Systems for production of transducing particles, methods, kits, compositions and uses thereof

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