WO2019017967A1 - Systèmes de bactériophages modifiés - Google Patents

Systèmes de bactériophages modifiés Download PDF

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WO2019017967A1
WO2019017967A1 PCT/US2017/043298 US2017043298W WO2019017967A1 WO 2019017967 A1 WO2019017967 A1 WO 2019017967A1 US 2017043298 W US2017043298 W US 2017043298W WO 2019017967 A1 WO2019017967 A1 WO 2019017967A1
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phage
population
phages
homogeneous
phage population
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PCT/US2017/043298
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Angela Belcher
Uyanga TSEDEV
Fred LAM
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Angela Belcher
Tsedev Uyanga
Lam Fred
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Priority to PCT/US2017/043298 priority Critical patent/WO2019017967A1/fr
Publication of WO2019017967A1 publication Critical patent/WO2019017967A1/fr

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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
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    • 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
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    • 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
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • C40—COMBINATORIAL TECHNOLOGY
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    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14111—Inoviridae
    • C12N2795/14132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14111—Inoviridae
    • C12N2795/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14111—Inoviridae
    • C12N2795/14151—Methods of production or purification of viral material

Definitions

  • the present application relates generally to the field of biotechnology and more specifically to engineered bacteriophage systems and methods of making and using them.
  • One embodiment is a method of making a homogeneous phage population and/or a homogeneous ssDNA population, comprising: obtaining a first artificial plasmid comprising an fl origin replication sequence; obtaining a second artificial plasmid that does not comprise an fl origin replication sequence; and co-transforming the first artificial plasmid and the second artificial plasmid into a bacterial strain to produce a homogeneous phage population and/or a homogeneous ssDNA population, wherein the first and second artificial plasmid together contain sequences encoding a complete library of phage coat and assembly proteins.
  • Another embodiment is a homogeneous engineered phage population, wherein at least 30% of phages in the phage population have a length within 15% of a length value, which is selected from 10 nm to 10 microns.
  • Yet another embodiment is a homogeneous engineered phage population, wherein at least 30%) of phages in the phage population have a length within 8 nm of a length value, which is selected from 10 nm to 10 microns. Yet another embodiment is a phage comprising chlorotoxin expressed by a coat protein of the phage.
  • kits for making a homogeneous phage population and/or a homogeneous ssDNA population comprising: a first artificial plasmid comprising an fl origin replication sequence; and a second artificial plasmid, which does not comprise an fl origin replication sequence, wherein the first and second artificial plasmid together contain sequences encoding a complete library of phage coat and assembly proteins.
  • FIG. 1 is an Inho construct plasmid map, with site of circular ssDNA production region shown in dark green.
  • FIG. 2 schematically illustrates phage-inho production: inho plasmids when co-transformed with another plasmid (RM13-fl) coding for phage proteins produce short phage that packages the inho genomes, which control the length of the phage.
  • RM13-fl another plasmid
  • FIG. 3A-C are atomic force microscopy (AFM) images of inho 1960, inho 475 and inho 285 phages.
  • FIG. 3D is a plot showing a measured length of phage in nm as a function of packaged ssDNA size.
  • FIG. 4 shows a nucleotide sequence (SEQ ID No. 1) for M13 code modified with chlorotoxin sequence at the p3 gene.
  • FIG. 5 shows an AFM image of chlorotoxin-phage with negatively charged gold particle interaction at p3.
  • FIG. 6A-D show fluorescent images for internalization of CTX Phage in Adult Human U87MG Glioblastoma and Pediatric Human D458 Medulloblastoma Cells.
  • CTX phage red
  • Golgi apparatus green
  • C & D human U87MG glioblastoma
  • C & D human D458 medulloblastoma cells.
  • Nuclei shown in blue, (scale bar 5 ⁇ ).
  • FIG. 7 presents results of Intravital Multiphoton Imaging Showing Tumor Uptake of CTX- Phage in the Brain of a NCR Nude Mice with an Orthotopic Human U87MG Glioma Xenograft.
  • FIG. 8 presents results of Intravital Multiphoton Imaging Showing Tumor Uptake of CTX Phage in the Brain of a C57/BL6 Mouse With an Orthotopic Synergic Mouse GL261 Glioma Xenograft.
  • FIG. 9 relates to the use of CTX phage (long and short) with NIRII imaging of glioma (ex- vivo, post IV injection).
  • FIG. 10 schematically illustrates small phage model.
  • FIG. 11 schematically illustrates passage of small CTX phage across the BBB.
  • FIG. 12 schematically illustrates multi-phage scaffolds & nanostructures.
  • FIG. 13 is a map of CTX insertion.
  • FIG. 14 shows a replication sequence from M13-ori (SEQ ID No. 2).
  • FIG. 15 is a map of RM13-fl, RM13-fl with p8 modification (DDAH), with p3 modification (HIS6), with p8 and p3 modification (DSPH & CTX).
  • FIG. 16 shows sequences of fl replication modified for origin only (SEQ ID No. 3), termination only (SEQ ID No. 4 and SEQ ID No. 5), and the packaging signal as found in M13-ori (SEQ ID No. 6).
  • FIG. 17 schematically depicts ssDNA sequence site of inho construct.
  • FIG. 18 schematically illustrates examples of inho sequence insertions for modified ssDNA length.
  • FIG. 19 schematically illustrates examples of inho ssDNA production sequence combined with RM13-fl protein sequences: with differing plasmid replication origins (pl50ri and pUC Ori) for varying yield effects during e. coli growth.
  • FIG. 20 presents results of analysis of packaged genomes: TBE-PAGE gel of the inho 285, 311, 344, 475, 1310, 1960 constructs, stained with SYBR-Gold. Inho ssDNA run higher than the reference ruler (designed for dsDNA).
  • FIG. 21 presents results of analysis of p3 coat protein: anti-p3 Western Blot from NU-PAGE gel run of inho, CTX, and Wildtype phage.
  • FIG. 22 shows a histogram distribution of inho phage sizes as measured from AFM imaging: for inho475 and inho 1960 phages, about 90% of all measured lengths fall within the base size bin.
  • FIG. 23 provides a list of M13 bacteriophage proteins.
  • a phage may be a filamentous phage.
  • filamentous phages include Ff phages, such as Ml 3 bacteriophage, Ike phage, fl phage and fd phage.
  • a phage may be a non-filamentous phage, such as lambda phage or adeno-associated virus.
  • the method includes obtaining two artificial, i.e. man-made, phage plasmids and co- transforming the two artificial plasmids into a competent bacterial strain to produce a homogeneous phage population.
  • the product of the co-transformation may be amplified to produce the homogeneous phage population.
  • the bacterial strain may be one that carries the F- episome.
  • the bacterial strain may be a strain of E. coli.
  • the bacterial strain may be DH5a, NovaBlue, XL10, ER2738 or XLl .
  • the bacterial strain may be an antibiotic resistant strain, such as a tetracycline resistant bacterial strain.
  • the two artificial plasmids are preferably such that together they contain sequences for a complete library for coat and assembly proteins for a particular type of phage.
  • the two artificial plasmids contain together at least one copy of each protein encoding gene present in a corresponding wildtype phage genome.
  • the two artificial plasmids will contain together sequences for each of pI-pXI proteins, see Fig 23.
  • the first of the artificial plasmids may be a modified (compared to the wildtype) circular double strand (ds) DNA phage plasmid.
  • the first artificial plasmid may contain an f 1 origin replication sequence.
  • the first artificial plasmid may prepared by modifying a wildtype phage plasmid in certain areas.
  • the modification(s) of the dsDNA acting as the first plasmid may be used to control or define a length of phages in the phage population and/or a length of produced ssDNA.
  • the fl origin replication sequence may also function as an fl termination replication sequence. Such sequence is illustrated, for example, in Figure 14.
  • the first artificial plasmid may also contain at least one of an fl termination replication sequence and a packaging signal sequence, such as SEQ ID No. 6.
  • the first artificial plasmid may contain an fl termination replication sequence in addition to the fl origin replication. In such case, the ssDNA's producing section between the fl origin replication sequence and the f 1 termination of replication sequence may be modified. For example, one or more base pairs may be added, replaced or removed.
  • the first artificial plasmid may contain an fl origin replication sequence, a packaging signal sequence and an fl termination of replication sequence.
  • the packaging signal sequence may comprise SEQ ID NO. 6 (FIG. 16) A) ssDNA producing section between the fl origin replication sequence and the packaging signal sequence and/or B) ssDNA producing section between the packaging signal sequence and the fl termination of replication sequence may be modified. For example, one or more bases in one or both of these sections may be added, replaced or removed.
  • the first artificial plasmid may contain an fl origin replication sequence and a packaging signal sequence.
  • the packaging signal sequence may comprise SEQ ID NO. 6 (FIG. 16) A) ssDNA producing section between the end of the fl origin replication sequence and the packaging signal sequence and/or B) ssDNA producing section between the packaging signal sequence and the start of fl origin of replication sequence may be modified. For example, one or more base pairs in one or both of these sections may be added, replaced or removed.
  • the first artificial plasmid leads to the replication of a packageable circular ssDNA, i.e. a plasmid, which may be packaged, after the co-transformation of the first and second artificial plasmids into a bacterial strain.
  • the first artificial plasmid may be modified in such a way that it may be missing one or more phage protein genes. In such case, at least one copy of the phage protein missing gene(s) may be present in the second plasmid.
  • the first artificial plasmid may be modified in such a way that it may be missing all of phage protein genes. In such case, at least one copy of each phage protein gene may be present in the second plasmid.
  • the fl origin replication sequence may be a modified (compared to the wildtype one) fl origin replication sequence.
  • a non-limiting example of a modified fl origin replication sequence is presented in FIG. 16 as SEQ ID No. 3 (Dotto et al., PNAS, 1982, 79(23), 7122-7126).
  • the fl termination of replication sequence may be a modified (compared to the wildtype one) f 1 termination of replication sequence.
  • modified fl termination of replication sequences are presented in FIG. 16 and SEQ ID Nos. 4 and 5 (Horiuchi, Genes to Cells, 1997, 2(7), 425-432).
  • the first artificial plasmid may be missing a packaging signal sequence. Such sequence may be used to produce minimal size phage populations and minimal size ssDNA. When the first artificial plasmid is missing a packaging signal sequence, it may be preferred to use the second artificial plasmid, which also misses a packaging signal sequence.
  • the second of the two artificial plasmids may be a helper phage plasmid, which is modified to disrupt the origin for packageable ssDNA replication.
  • the second artificial plasmid may be a helper phage plasmid which is modified so that it does not contain an fl replication origin sequence (either wildtype or modified).
  • the second artificial plasmid may be a helper phage plasmid which is modified so that it does not also contain a packaging signal sequence (either wildtype or modified).
  • the modified helper phage plasmid used as the second artificial plasmid may include all essential phage assembly components, i.e. contain sequences encoding all phage proteins (coat and assembly proteins). In such case, the first plasmid may or may not be missing all these sequences.
  • the second artificial plasmid may be prepared, for example, from a commercial helper phage plasmid by reassembling the intergenic region to disrupt the origin for packageable ssDNA replication.
  • a sequence comprising SEQ ID No. 2 may be removed from a helper phage plasmid.
  • Examples of commercially available helper phage plasmids for M13 bacteriophage include M13KE , M13K07 and R 408.
  • the modified helper phage plasmid used as the second artificial plasmid may include further modifications.
  • COLE1 (pl5a-ori) plasmid replication sequence may be included. Such inclusion may allow achieving an optimal copy numbers during bacterial growth, such as E. coli growth.
  • the modified helper phage may also include one or more of additional functionalization, such as adding chlorotoxin (CTX) sequence, such as SEQ ID No.
  • the product of the co-transformation which may be a bacterial colony comprising a phage population
  • a bacterial growth media such as a lysogeny broth media
  • the bacterial colony which may be the product of the co-transformation may be grown in the bacterial growth media to produce a resultant bacterial culture.
  • bacteria may be removed from the resultant bacterial culture to produce a supernatant containing a phage product. Such removal may be performed by centrifuging the resultant bacterial culture.
  • the supernatant containing the phage product may be used for precipitating phage particles of the phage population.
  • the phage precipitated phage media may be centrifuged to pellet out the phage phage population.
  • the pelleted phage population may be suspended in a sterile solution, which may be, for example, a sterile buffer, or purified water, such as MilliQ water.
  • the product of the co-transformation may be stored for up to three months prior to amplification.
  • the product of the co-transformation may be stored for at least 1 day up to three months or for at least 2 days up to three months or for at least 3 days up to three months or for at least 5 days up to three months or for at least a week up to three months or for at least 10 days up to three months or for at least two weeks up to three months or for at least 3 weeks up to three months or for at least one month up to three months or for at least 45 days up to three months and any values or subranges within these ranges.
  • the present method may allow production of homogeneous phage populations.
  • a phage population may be homogeneous when a substantial portion of phages of the population, e.g. at least 10% of the population, has a phage length, which is substantially close to a selected phage length value.
  • the selected length value may be determined by selecting a particularly modified first artificial plasmid. In certain embodiments, the selected length value may be determined by selecting a length (and/or a number of base pairs) of the first artificial plasmid.
  • first artificial plasmid when all other conditions are the same, a longer (having more base pairs) first artificial plasmid will result in a longer phage population, while a shorter (having less base pairs) first artificial plasmid will result in a shorter phage population.
  • artificial plasmids having 1960, 475 and 285 base pairs when co-transformed with the RM13-fl second plasmid will give phages having lengths of 280 nm, 100 nm and 50 nm.
  • At least 10% of phages or at least 20% of phages or at least 30 % of phages or at least 40% of phages or at least 50 % of phages or at least 60% of phages or at least 70%) of phages or at least 80% of phages or at least 90% of phages may be within 20% or within 15 % or within 12% or within 10% or within 9% or within 8% or within 7% or within 6% or within 5% of the selected phage length value.
  • At least 10% of phages or at least 20% of phages or at least 30 % of phages or at least 40% of phages or at least 50 % of phages or at least 60% of phages or at least 70%) of phages or at least 80% of phages or at least 90% of phages may be within 20 nm or within 15 nm or within 12 nm or within 10 nm or within nm or within 8 nm or within 7 nm or within 6 nm or within 5 nm of the selected phage length value.
  • the selected phage length value may be, for example, a value between 10 nm and 10000 nm (10 microns).
  • the selected phage length value may be from 10 nm to 8000 nm or from 10 nm to 6000 nm or from 10 nm for 4000 nm or from 10 nm to 3000 nm or from 10 nm to 2000 nm or from 10 nm to 1800 nm or from 10 nm to 1600 nm or from 10 nm to 1400 nm or from 15 nm to 8000 nm or from 15 nm to 6000 nm or from 15 nm for 4000 nm or from 15 nm to 3000 nm or from 15 nm to 2000 nm or from 15 nm to 1800 nm or from 15 nm to 1600 nm or from 15 nm to 1400 nm or from 20 nm to 8000 nm or from 20 nm to 6000 nm or from
  • the selected phage length value may be below 50 nm.
  • the selected phage length value may be at least 10 nm but less than 50 nm or at least 10 nm but no more than 45 nm or at least 10 nm but no more than 40 nm or at least 10 nm but no more than 35 nm or at least 15 nm but less than 50 nm or at least 15 nm but no more than 45 nm or at least 15 nm but no more than 40 nm or at least 15 nm but no more than 35 nm.
  • the selected phage length value may be greater than 50 nm.
  • the selected phage length value may be at least 55 nm or at least 60 nm or at least 65 nm or at least 70 nm or at least 75 nm or at least 80 nm or at least 80 nm or at least 85 nm or at least 90 nm or at least 100 nm or least 110 nm or at least 120 nm.
  • the selected phage length value may be greater than 50 nm but less than a wildtype type phage length.
  • the selected phage length value may be greater than 50 nm but less than 880 nm; or at least 55 nm but less than 880 nm or at least 60 nm but less than 880 nm or at least 70 nm but less than 880 nm or at least 80 nm but less than 880 nm or at least 100 nm but less 880 nm or greater than 50 nm but less than 850 nm; or at least 55 nm but less than 850 nm or at least 60 nm but less than 850 nm or at least 70 nm but less than 850 nm or at least 80 nm but less than 850 nm or at least 100 nm but less 850 nm or greater than 50 nm but less than 820 nm; or at least 55 nm but less than 820 nm;
  • the selected phage length value may be greater than a wildtype type phage length.
  • the selected phage length value may be greater than 900 nm or at least 920 nm or at least 940 nm or at least 960 nm or at least 980 nm or at 1000 nm.
  • the selected phage length may be from 10 nm to 150 nm or from 10 nm to 120 nm or from 10 nm to 100 nm or from 10 nm to 40 nm or from 60 nm to 120 nm. Phage populations having such lengths may be preferred for imaging applications, when phages are used a biological probe.
  • the selected phage length value may be from 100 nm to 10 microns or from 100 nm to 5 microns or from 100 nm to 2 microns or from 100 nm to 1 micron or from 100 nm to 800 nm or from 950 nm to 10 microns or from 1 micron to 10 microns. Phage populations having such lengths may be preferred when phages are used in implant applications, such as hydrogel and/or scaffold applications.
  • the produced homogeneous phage population may have a phage count of at least lel2 pfu or at least 3el2 pfu or at least 5el2 pfu or at least lel3 pfu or at least 3el3 pfu or at least 5el3 pfu or at least lel4 pfu or at least 3el4 pfu or at least 5el4 pfu or at least lel5 pfu or at least 3el5 pfu.
  • the homogeneous phage populations may be used for producing homogeneous, high yield populations of ssDNA packaged inside of phages.
  • the ssDNA may be extracted by lysing the phages.
  • a length of ssDNA (or a number of bases in it) may be determined by selecting a length (and/or a number of base pairs) of the first artificial plasmid. In general, when all other conditions are the same, a longer (having more base pairs) first artificial plasmid will result in a longer (having more bases) ssDNA, while a shorter (having less base pairs) first artificial plasmid will result in a shorter (having less bases) ssDNA. Due to the homogeneity of the phage populations, the produced ssDNA may also have a high degree of homogeneity, such as homogeneity in size.
  • the produced ssDNA may contain from 135 to 100,000 bases or from 135 to 80,000 bases or from 135 to 60,000 bases or from 135 to 50,000 bases or from 135 to 40,000 bases or from 30,000 bases or from 135 to 20,000 bases or from 135 to 15,000 bases or from 135 to 12,000 bases or from 135 to 10,000 bases or any integer number or subrange within these ranges.
  • the first artificial plasmid may be shorter than a wildtype plasmid.
  • the produced ssDNA may contain from 179 to 6400 bases or from 135 to 6300 bases or from 135 to 6200 bases or from 135 to 6100 bases or from 135 to 6000 bases or from 200 to 5000 bases or from 200 to 4000 bases or from 200 to 3500 bases or from 200 to 3000 bases or any integer number or subrange within these ranges.
  • the first artificial plasmid may be longer than a wildtype plasmid.
  • the first artificial plasmid may contain from 6450 to 100,000 bases or from 6500 to 100000 bases or from 6500 to 80,000 bases or from 6600 to 70,000 bases or from 6800 to 60,000 bases or from 6800 to 50,000 bases or from 7000 to 40000 bases or from 7000 to 35000 bases or from 7000 to 30000 bases or from 7000 to 20000 bases or from 7000 to 15000 bases or any integer number or subrange within these ranges.
  • the present method may allow producing homogeneous phage populations with a high phage count without a need for purifying a desired portion of a phage population, i.e. a portion, which has a desired phage length value, from undesired phage portions, i.e. phage portions, which are not substantially close to the desired phage length value.
  • the present method does not include enriching desired portion of a phage population by PEG
  • the method may include purification of the produced phage population.
  • the present method may involve purification to remove unwanted and/or contaminating proteins from the phage population. Such purification may be performed, for example, using cesium chloride gradient ultracentrifugation and column separation.
  • Homogeneous phage populations may be used for a number of applications.
  • homogeneous phage populations may be used as a delivery vehicle for delivery an active agent, which may be attached to and/or conjugated with phages of the phage population, to a particular area of a body of a subject, such as a mammal, e.g. a human.
  • the active agent may be, for example, a therapeutic agent and/or an imaging agent
  • the homogeneous phage population may be used in a therapeutic and/or imaging method, which may involve administering the homogeneous phage population to a subject, such as a mammal, e.g. a human being.
  • the homogeneous phage population may be, for example, administered topically, enterally or parenterally.
  • the homogeneous phage population may be, for example, administered topically, enterally or parenterally.
  • homogeneous phage population may be swallowed, injected or inhaled.
  • the homogeneous phage population may be administered
  • the homogeneous phage population may be administered intracranially. In some embodiments, the homogeneous phage population may be administered intradermally, subcutaneously or via intramuscular injections.
  • the homogeneous phage population may be administered, for example, intravascularly or intracranially, for treating and/or imaging a tumor, such as a central nervous system (CNS) tumor.
  • a tumor such as a central nervous system (CNS) tumor.
  • the homogeneous phage population may be useful for treating both adult and pediatric CNS tumors.
  • CNS tumors include tumors of neuroepithelial tissue, such as astrocytic tumors, oligodendroglial tumors, oligoastrocytic tumors, ependymal tumors, choroid plexus tumors, astroblastoma, neuronal and mixed neuronal-glial tumors, tumors of the pineal region, embryonal tumors; tumors of cranial and paraspinal nerves; tumors of the meninges, such as tumors of meningothelial cells, sesenchymal tumors, primary melanocytic lesions, haemangioblastoma; tumors of the haematopoietic system; germ cell tumors; tumors of the sellar region.
  • neuroepithelial tissue such as astrocytic tumors, oligodendroglial tumors, oligoastrocytic tumors, ependymal tumors, choroid plexus tumors, astroblastoma,
  • a homogeneous phage population may be a part of a composition, which may be a pharmaceutically acceptable composition.
  • the composition may further include an acceptable carrier, which may be, for example, a sterile buffer, such as PBS buffer or saline buffer, or purified water, such as Mille-Q water.
  • the composition may also include one or more cell media components, such as blood derived serum, and/or one or more components, such as enzymes, gelatin and amino acids.
  • a phage concentration in the composition may vary.
  • a phage concentration may be from lelO to lel5 pfu/ml or from lei 1 to lei 5 pfu/ml or lel2 to lel5 pfu/ml.
  • the homogeneous phage population may be implanted in a body of a subject, such as a mammal, e.g. a human.
  • the homogeneous phage population may be a part of an implant, which may be, for example, one or more of a scaffold, a hydrogel and a nanostructure.
  • the implant may act as a delivery and/or detector device.
  • Courchesen et al, Advanced Materials, 2014, 26(11), 3398-3404), and Chen et al, Advanced Materials, 2014, 26(30), 5101-5107 demonstrate method of crosslinking phage solutions to create viral scaffolding for various materials.
  • Implant type viral hydrogels may be produced via a variety of building methods, such as glutaraldehyde crosslinking, layer-by-layer deposition, nanoparticle linkers, covalent bonding, DNA/sortase binding, lyophilization, photodynamic or UV crosslinking.
  • Figure 12 illustrates, for example, multiphage scaffolds and/or nanostructures.
  • an active agent may be an imaging agent.
  • An imaging agent may be an agent that is capable to produce a signal, the detection of which may provide an image of an area of the subject's body, such as a tumor area.
  • an imaging agent may be a fluorescent imaging agent, such as a fluorescent label; a magnetic imaging agent, such as a magnetic nanoparticle; a radioactive imaging agent, such as a radioactive label; an infrared imaging agent; a photoluminescent imaging agent.
  • One example of an imaging agent may be carbon nanostructure, such as a carbon nanotube, which may be for example, a single wall carbon nanotube. Nanostructure imaging agents conjugated to phages are disclosed, for example, in US 20130230464, which is incorporated herein in its entirety.
  • a signal from an imaging agent may be used to track and diagnose cells or masses in a body of a subject, such as a human.
  • a signal from an imaging agent may be used to track and diagnose cells or masses in a brain and/or spinal cord of a subject, such as a human.
  • a signal from an imaging agent may be also used to visualize a treatment over time.
  • an active agent may be an infrared imaging agent, such as a carbon nanostructure, such as a carbon nanotube, which may be a carbon nanotube, such as a single wall carbon nanotube.
  • the imaging agent may be an organic or inorganic dye, which may emit in the near infrared range, which may be a range from 700 nm to 2000 nm or from 800 nm to 1900 nm or from 900 nm to 1800 nm or from 800 nm to 1750 nm or from 1100 nm to 1700 nm or value or subrange within these ranges.
  • a dye may be sodium yttrium fluoride doped with ytterbium and erbium.
  • an infrared imaging system disclosed in Ghosh et al, PNAS 2014 111 (38) 13948-13953 may be used. Such system may have imaging depths such as 10 cm.
  • an active agent may be a fluorescent imaging agent, such as, for example, Cy5.5 or Alexa-647.
  • a fluorescent imaging agent may be conjugated directly to a phage coat protein, such as pVI, pVII, pVIII or pIX.
  • a fluorescent imaging agent may be conjugated to another moiety, which is conjugated directly to or expressed by a coat protein of the phage.
  • a fluorescent imaging agent may be conjugated to chlorotoxin, which is directly conjugated to or expressed by pill protein of the phage.
  • Imaging using a fluorescent imaging signal may involve detecting a fluorescent signal from the label after it has been administered to a subject, such as a human, together with the homogeneous phage population.
  • a subject such as a human
  • a multiphoton spectrometer or microscope may be used.
  • an active agent may be a therapeutic agent.
  • a therapeutic agent may be an anticancer agent, such as a chemotherapy agent, e.g. doxorubicin.
  • a chemotherapy agent e.g. doxorubicin.
  • a therapeutic agent may be a small molecule.
  • Non-limiting examples of therapeutic agents include siRNA or small DNA for cell-disruption, adjuvants for immunotherapy, nanoparticles such as gold nanoparticles, or theranostics including photodynamic therapy agents such as IR700.
  • a host of agents may be conjugated to the phage via methods such as EDC chemistry, streptavidin- biotin strategies, zinc-finger sortase strategies, metal chelating linkers such as DOTA- NHSester, thiol alkylation, maleimide modifications, and N-terminal transamination.
  • Other amino acid handles may be used including cysteine residues for di-sulfide bonds and tyrosine residue modifications.
  • Peptide sequences conferring affinity to molecules of interest may also be engineered to the coat proteins of the phage. (Bernard et al, Front Microbiol, 2014, 5(734), PMC4274979) (Hess et al, Bioconjug Chem., 2012, 23(7), 1478-1487)
  • the homogeneous phage population may be a population of phages conjugated with a targeting moiety.
  • the targeting moiety may be such that it preferentially binds to a particular type of cells compared to other cells.
  • the targeting moiety may be a tumor targeting moiety, i.e. a targeting moiety that binds preferentially to tumor cells compared to other types of cells, e.g. normal cells.
  • the targeting moiety may be chlorotoxin. Chlorotoxin may selectively and/or preferentially bind to certain types of tumors, such as gliomas and tumors of neuroectrodermal origin such as
  • homogeneous phage populations may be used for treating a plaque caused by or associated with a neurogenerative disease, such as an Alzheimer's disease and other types of dementia; Parkinson's disease (PD) and PD-related disorders; prion disease; motor neurone diseases; Huntington's disease, spinocerebellar ataxia; spinal muscular atrophy.
  • a neurogenerative disease such as an Alzheimer's disease and other types of dementia; Parkinson's disease (PD) and PD-related disorders; prion disease; motor neurone diseases; Huntington's disease, spinocerebellar ataxia; spinal muscular atrophy.
  • the present inventors also developed a complex comprising a phage, such as a filamentous phage, and chlorotoxin, which may be conjugated to the phage or expressed by the phage during phage assembly.
  • chlorotoxin may be conjugated to or expressed by the phage's coat protein, such as pill protein, or expressed by the phage's coat protein, such as pill protein, during the phage assembly through engineering phage sequence.
  • the phage may be a wildtype phage.
  • the phage may be an engineered phage, such as a phage, which is a part of a homogeneous phage population discussed above.
  • chlorotoxin may be a labeled chlorotoxin, i.e.
  • chlorotoxin with one or more labels attached to and/or conjugated with it.
  • the label may be, for example, a radioactive label or a fluorescent label.
  • labeled chlorotoxin may be Cy5.5 labeled chlorotoxin, which is a combination of chlorotoxin and a fluorescent material Cy5.5.
  • Chlorotoxin-phage may be prepared by incorporating a chlorotoxin encoding sequence, such as SEQ ID No. 1, into one of phage coat protein genes of a plasmid, so that the chlorotoxin will be expressed by the corresponding coat protein.
  • the chlorotoxin encoding sequence may be incorporated into pill gene of Ml 3 bacteriophage and thus, chlorotoxin may be expressed by pill protein.
  • a phage for expressing chlorotoxin may be any phage whose genome may be edited.
  • a phage for expressing chlorotoxin may be any of the phages disclosed above.
  • the phage may be one of filamentous or filamentous phages disclosed above.
  • a phage for expressing chlorotoxin may be T4 phage, T7 phage, tobacco mosaic virus or potato virus.
  • the chlorotoxin encoding sequence may be incorporated into a phage coat protein gene, such as pill gene, of a wildtype helper phage to produce a helper phage construct containing the chlorotoxin encoding sequence.
  • the chlorotoxin encoding sequence may be incorporated into a phage coat protein gene, such as pill gene, of the first or second artificial plasmid discussed above.
  • the chlorotoxin encoding sequence may be incorporated into the modified helper phage plasmid, which acts as the second artificial plasmid, to produce a modified helper phage construct containing the chlorotoxin encoding sequence.
  • the plasmid containing the chlorotoxin encoding sequence may be transformed through a bacterial strain, such as the ones discussed above.
  • the plasmid containing the chlorotoxin encoding sequence may be transformed by itself without other plasmids.
  • the plasmid containing the chlorotoxin encoding sequence may be co-transformed with the other plasmid disclosed above.
  • the second artificial plasmid contains the chlorotoxin encoding sequence it may be co- transformed with the first artificial plasmid.
  • the first artificial plasmid contains the chlorotoxin encoding sequence it may be co-transformed with the second artificial plasmid.
  • the products of transformation and or co-transformation may be amplified as discussed above and also in examples below.
  • chlorotoxin-phages may be further conjugated with an active agent, which may be an imaging agent and/or a therapeutic agent.
  • an imaging agent such as a fluorescent label or a carbon nanostructure may be conjugated or attached to the pVIII protein of the phage.
  • a fluorescent label such as AlexaFlour may be conjugated with the pVIII protein through a reaction with side chains of lysine groups.
  • a carbon nanostructure such as a single wall carbon nanotube
  • DSPH or other carbon nanotube complexing peptide may be conjugated with or expressed by the pVIII protein of the phage. This may be accomplished by incorporating a sequence for DSPH or a sequence for a carbon nanotube complexing peptide at the pVIII gene of a respective helper phage sequence before its transformation into a bacterial strain.
  • the homogeneous phage populations and/or CTX-phage populations may have an ability to penetrate a blood brain barrier in subject such as a human, see Figure 11 as well as additional discussion in Example below.
  • phage populations having lengths of no more than 900 nm or no more than 800 nm or no more than 700 nm or no more than 600 nm or no more than 500 nm or nor more than 400 nm or no more than 300 nm or no more than 200 nm or no more than 150 nm or no more than 100 nm may have an ability to penetrate a blood brain barrier when administered intravascularly.
  • the homogeneous phage populations may be also used in a method of mass producing ssDNA of varying lengths/sequences.
  • homogenous phage populations produced with desired length or sequence of ssDNA packaged within may be produced at titers of at least lel4 or at least lei 5 or at least 3e 15 or at least lei 6 phage from 10L growths. These phage may be lysed and high yields of ssDNA extracted for use.
  • the present application also provides a kit for making a homogeneous phage population, which may include the first artificial plasmid disclosed above; and
  • the kit may include a bacterial strain, which may be used for to con-transforming the first and the second artificial plasmids into it to produce a homogeneous phage population.
  • Ml 3 phage assembly system for optimal blood trafficking, tumor penetration, and passage across the blood brain barrier
  • M13 bacteriophage as an engineerable biomaterial for medical imaging and therapy applications particularly in the case of tumor diagnosis.
  • M13 phages of engineered sizes (less than ⁇ 50nm to above a micron in range), termed 'inho' phages were successfully assembled.
  • M13 phages displaying chlorotoxin peptide on the tail capsid of Ml 3 were also successfully assembled.
  • the construction of shorter phage may improve on the extravasation and blood trafficking of Ml 3 probe systems while retaining its multi-functional capsid proteins which may allow simultaneous targeting, detecting, and/or delivery of various agents, such as active agent, e.g.
  • phage expressing chlorotoxin as well as its reduced size may allow for the passage of Ml 3 probe systems across the blood-brain barrier (BBB) and the potential targeting to adult and pediatric brain tumors.
  • Ml 3 filamentous bacteriophage is composed of a circular single stranded DNA (ssDNA) encapsulated by the major coat protein p8 and minor cap proteins p3, p6, p7, p9 [1, see section "References” below]. These proteins can be engineered to display or attach various targeting sequences and nanoparticles or drug molecules— effectively creating a phage shuttle that carry imaging or therapy agents to specifically targeted cancer cells [2]. In our system, we can control the length (or the number of p8 coat) of the Ml 3 phage by manipulating the length of the circular ssDNA plasmid packaged during the Ml 3 assembly process.
  • ssDNA circular single stranded DNA
  • a set of plasmids that generate package-able viral genomes with desired sizes (i.e. 100s to 1000s base-pairs), which may be much smaller than the 6407 nucleotides observed in wildtype Ml 3 ( ⁇ 880nm in length), was created.
  • These package-able genomes may be of varying lengths and contain the phage packaging signal and the f 1 origin and termination of replication but none of the phage protein genes.
  • construct with the inserts produces ssDNA of a given length that signal to be packaged ( Figure 1).
  • the packaging signal region may also be removed and the packaging genome retaining the fl origin may be enough to begin assembly.
  • a second plasmid which expresses all essential phage assembly components but itself lacks the packaging signal and fl replication origin, was constructed. Only in the presence of both of these two plasmids is phage production of the given size observed ( Figure 2), where inho plasmids are packaged by the proteins translated from the unpackaged large RM13-fl plasmid.
  • manipulations such as the addition of chlorotoxin display that may further improve on the specificity of phage delivery of an active agent, such as an imaging agent and/or a therapeutic agent.
  • CTX Chlorotoxin
  • GBM malignant gliomas
  • tumors of neuroectodermal origin such as medulloblastomas, neuroblastomas, melanomas, P ETS, and small cell lung carcinoma [3].
  • native CTX as well as fluorophore conjugated CTX tumor paint display the ability to cross the blood-brain barrier (BBB) in both animals and humans with brain tumors, which may make it an ideal trafficking peptide for nanoprobes designed for brain tumor diagnosis and theragnosis.
  • BBB blood-brain barrier
  • a phage clone that encodes for CTX on the p3 capping protein of the Ml 3 phage both wildtype and inho or short phage was developed.
  • the 36 amino acid sequence of the CTX peptide is incorporated into the gene for p3 as illustrated in Figure 4.
  • CTX peptide is highly positive in charge, an attribute which may be part of the reason for its ability to cross the BBB and target to tumor areas with extracellular matrix which is particularly negative in charge [3].
  • CTX phage electrostatically interacts with the nanoparticles at its CTX-p3 end ( Figure 5).
  • Chlorotoxin also has a distinct in vitro cellular localization and uptake pattern in human glioma versus normal cells, where it localizes near the golgi apparatus in glioma cells .
  • CTX phage particles in vitro cellular uptake and colocalization of phage particles to the Golgi apparatus in human adult U87MG glioma cells ( Figure 6A & B) as well as human pediatric D458 medulloblastoma cells ( Figure 6C & D) as early as 6 hrs following incubation of cells CTX phage with continued accumulation at 24 hrs, were shown, demonstrating the versatility of the CTX phage to target multiple types of brain tumors.
  • Ml 3 bacteriophage can be used as a molecular shuttle for the delivery of various therapy molecules and imaging agents (i.e. single walled nanotubes, SWNT) to the site of tumors.
  • imaging agents i.e. single walled nanotubes, SWNT
  • NIRII near infrared
  • Figure 9 Illustrated here ( Figure 9) is the NIRII brain tumor signals from the dual phage SWNT probe and NIR imaging system which may give surgeons the information needed to remove tumors at the sub-millimeter size level— and those tumors that were not detectable by the naked eye. These preliminary data may reveal successful localization to glioma masses from long SWNT complexed CTX phage (880nm) as well as short SWNT complexed CTX phage (300nm).
  • filamentous phages of shorter lengths by constructing a set of small viral ssDNA that are packaged by M13 capsid proteins (these smaller phage retains the M13 major and minor coat proteins) were enginerred. Now with the ability to control the aspect ratio of these rigid, rod-like phages one can further improve on Ml 3 based cancer/disease detection or panning by optimizing for phage blood circulation and tumor extravasation.
  • collection by cloning for chlorotoxin display on the tail p3 capsid protein of Ml 3 has been added.
  • CTX may induce passage across the BBB and target glioma tumors in vivo.
  • CTX on Ml 3 may allow capitalizing on its strong affinity for tumors of neuroectodermal origin, increasing its usage for the detection and delivery of novel therapies to treat adult and pediatric brain tumors with the potential to expand the platform for use in other diseases of the central nervous system.
  • Preceding work by Specthrie et al. created a plasmid that generates a minimal packaging genome and produced a mixed population of full length phage and smaller phage that are 50nm in length (though only 1-3% by mass of total population) [4, 5, 6].
  • the present work differs from Specthrie et al. in two ways. The first is to construct a set of plasmids that generate package-able genomes with any needed size. The second is to generate populations of a shorter size by removing the helper phage using a plasmid that contains all phage proteins but is unable to package its DNA.
  • Figure 22 provides evidence of a high degree of homogeneity in length for inho phages.
  • Figure 22 presents a histogram distribution of inho phage sizes as measured from AFM imaging: for inho475 and inhol960 phages, about 90% of all measured lengths fall within the base size bin
  • nanoparticles Upon systemic injection, nanoparticles must: 1) Evade uptake by circulating immune macrophages; 2) Achieve marginalization and escape the circulation to reach blood vessel walls; 3) Extravasate into the tumor interstitium; and finally, 4) bind to or be internalized by cancer cells [9].
  • spherical particles have been the norm in nanomedicine research, non-spherical nanoparticles (i.e. rods, chains, ellipsoids) may be more effective in these areas [10]. Not only are chain or rod-like structures more likely to avoid internalization by macrophages, their shape subjects them to certain torque and tumbling motion that increases contact with the vessel walls.
  • oblong shaped particles are more likely to form multivalent occurances essential for targeting - in the case of the filamentous bacteriophage, avidity of binding can be highly enhanced by the display of materials on all 2700 copies of the body p8 protein.
  • size considerations must be made to accommodate for the high interstitial flow pressure typical of tumor masses. Due to the increased leakiness of tumor vessels and reduced lymphatic drainage,
  • extravasation and delivery of nanoparticles to tumor tissues can be achieved via enhanced permeation and retention (EPR) effects, which is typically seen with smaller particles ⁇ 100 nm in diameter.
  • EPR permeation and retention
  • the permeability of the BBB to nanoparticles is affected by a number of factors including size, charge, and surface chemistry of the particles.
  • Ml 3 bacteriophage has been postulated to be able to cross the BBB. While long in length, the structure of M13 is extremely narrow in diameter ( ⁇ 5-6nm) and easily falls under the hydrodynamic size limit of particles that freely pass the BBB [3, 11].
  • Engineering the M13 platform to enable easy passage across the BBB may be particularly advantageous in concert with deep tissue imaging technologies.
  • the brain is an organ which is especially difficult to image in depth-imaging the CTX-phage probes may be performed using the near infrared imaging system which has depths up to 10cm. Ghosh et al, PNAS 2014 111 (38) 13948-1395; Dang et al, PNAS, 2016, 113(19), 5179-5184.
  • the inho and CTX designs may allow one to further achieve penetration into tumors, such as glioma and medulloblastoma tumors.
  • Small sizes achievable by inho phage may allow penetration of inho phage across intact/heathly BBB of non-tumor bearing mice.
  • the Ml 3 shuttles may be particularly important as a means of increasing distribution of drugs past the BBB and for reducing nonspecific systemic effects.
  • the multifunctional M13 shuttle may play a significant role in the imaging detection, drug treatment, and surgical removal of brain tumors which require extremely precise handling for patient safety.
  • Ml 3 phage has been studied as a means of reversing the formation of plaques derived from amyloid-like structures in the brain related to Alzheimer, Parkinson and other neurodegenerative diseases [12].
  • the P3 capsid protein was identified as a major agent with curative potential and was engineered as a recombinant bivalent G3P molecule called General Amyloid Interactive Motif (GAIM) by NeuroPhage Pharmaceuticals or more recently renamed Proclara Biosciences [13].
  • GIM General Amyloid Interactive Motif
  • This small motif was mainly developed in order to overcome the hurdle of delivering Ml 3 bacteriophage across the BBB while retaining the ability of the bacteriophage to bind to beta amyloid protein in the brain.
  • the effectiveness of Ml 3 phage to clear amyloid plaques is not completely captured with GAIM, which suggests that CTX phage as well as inho-phage may be a more powerful alternative to targeting plaques in the brain.
  • Helper phage template M13KE was used as the basis of the addition of CTX peptide display at the c-terminal end of the p3 capsid protein (see Figure 4 for insertion sequence).
  • CTX insert was achieved through restriction enzyme sites at the p3 region of the New England Biolabs (NEB) M13KE vector.
  • the 108 base-pair CTX insertion and oligos designed with Acc65I and Eagl enzyme cut sites were purchased through IDT.
  • the CTX template was used in a PCR reaction (KAPA HiFi Kit) to amplify CTX inserts with the previously mentioned enzyme cut sites and purified through a 1.2% agarose gel run and extraction (QIAquick Gel Extraction). Both the host vector M13KE and the CTX PCR product were digested overnight at 37°C with Acc65I and Eagl NEB enzymes in Buffer 3.1.
  • Dephosphorylation of the host vector post digestion was conducted at 37°C for 1 hour with 2.5 ⁇ . of rSAP enzyme to reduce recircularization events in the host vector.
  • the final CTX and M13KE digestion products were further purified through agarose gel run and extraction. T4 ligation with the prepped CTX insert and M13KE vector was performed overnight at 16°C.
  • ligation products were transformed according to manufacturer's instructions for competent cells (i.e. XL-1 Blue), prepared in 3ml of agar top and ⁇ . of overnight bacterial culture (XL-1 Blue), and incubated on IPTG-X-Gal agar plates overnight at 37°C . Phage plaques from the plates were picked, grown overnight, and prepped for sequencing (QIAprep). Finally, full sequencing of the purified plasmids were verified for desired final M13KE-CTX construct. A map of CTX insertion is shown in Figure 13.
  • RM-13-fl Constructs Cloning RM-13-fl Constructs.
  • Commercially available helper phage templates M13KE, M13K07, and R408 were used to create RM13-fl constructs where the intergenic region is reassembled to disrupt the origin for packageable ssDNA replication (fl site in particular was removed, Figure 14 presents a part of the removed sequence).
  • Kanamycin resistance site was added to the constructs for identification purposes and ColEl (pl5a-ori) plasmid replication site is included to achieve optimal copy numbers during e.coli growth.
  • Additional functionalization i.e. CTX on p3, DSPH or CNT complexing peptide display on p8, HIS-tag on p3, separation of overlapping capsid sequences
  • Plasmid cloning by PCR with restriction enzyme or gibson master assembly techniques were used to create constructs from vector fragments of interest or to add insertions important to functionalizing the capsid proteins. All oligos and small DNA inserts were purchased through IDT. PCR reactions were performed (KAPA HiFi Kit) to amplify inserts and vectors with/out the enzyme cut sites or gibson overlapping overhangs. PCR products were purified through a 1.2% agarose gel run and extraction (QIAquick Gel Extraction) or DNA spin columns. Standard ligation or Gibson reactions were conducted as appropriate. 1-2 ⁇ . of the ligation or Gibson products were transformed according to manufacturer's instructions for competent cells (i.e.
  • FIG. 15 is a map of RM13-A, RM13-A with p8 modification (DDAH), with p3
  • HIS6 HIS6
  • DSPH & CTX p8 and p3 modification
  • Inho construct were assembled via the incorporation of the f-1 origin of replication (modified for origin of ssDNA replication only), f-1 termination site (a repeat of the f-1 origin with deletions that render ineffective the nicking of the plasmid for ssDNA production), and the packaging signal (PS). Ampicillin resistance site was added to the constructs for identificationon purposes and ColEl plasmid replication site is included to achieve optimal copy numbers during e.coli growth. Addition and deletion of base-pairs in the ssDNA region (as demonstrated in the case of inhol960, inho475 etc) may be easily achieved via the cloning procedures described.
  • Figure 16 shows sequences of fl replication modified for origin only (SEQ ID No.
  • Figure 17 identifies sections of ssDNA, which may be manipulated by for example, adding and/or replacing and/or deleting bases therein.
  • Plasmid cloning by PCR with restriction enzyme or gibson master assembly techniques were used to create constructs from fragments of interest or to add insertions important to ssDNA packaging and to change the length of the inho-phage. Insertions such as EGFP and GLuc sequences were used to test the efficacy of inho constructs with different ssDNA production region (See Figure 18). All oligos and small DNA inserts were purchased through IDT. PCR reactions were performed (KAPA HiFi Kit) to amplify inserts and vectors with/out the enzyme cut sites or gibson overlapping overhangs. PCR products were purified through a 1.2% agarose gel run and extraction (QIAquick Gel Extraction) or DNA spin columns.
  • Phage Plates 50ng each of inho construct of desired base pair length and RM13-fl with desired capsid proteins, such as e.g. CTX and/or DSPH, sequences are co-tranformed through heat shock in tetracycline resistant bacterial strain (i.e. XL-1 Blue) and grown SOC Media. The SOC growth is spread over kanamycin+ampicillin agar plates and incubated overnight at 37°C. Resulting bacterial colonies are stored up to 3 months for amplication of phage.
  • desired capsid proteins such as e.g. CTX and/or DSPH
  • Sterile LB Media is prepared with tetracycline, kanamycin, and ampicillin at working concentrations of lC ⁇ g/mL, 5C ⁇ g/mL, and 10C ⁇ g/mL respectively.
  • Bacterial colony picked from plate is grown overnight in LB at 37°C and 225rpm.
  • the supernatant containing the phage product is stored overnight at 4°C with 10% PEG/NaCl to precipitate out the inho-phage particles.
  • the phage media is centrifuged again for 40min at 8000rpm to pellet out the phage.
  • CTX construct 50ng of the CTX construct was transformed according to manufacturer's instructions for tetracycline resistant, competent cells (i.e. XL-1 Blue), prepared in 3ml of agar top and ⁇ of overnight bacterial culture (XL-1 Blue), and incubated on IPTG-X-Gal agar plates overnight at 37°C . Resulting phage plaques are stored up to 3 months for amplication of phage. Amplification:
  • Sterile LB Media is prepared with tetracycline at working concentrations of 10 ⁇ g/mL.
  • Phage plaque picked from plate is grown overnight in LB at 37°C and 225 rpm.
  • the supernatant containing the phage product is stored overnight at 4°C with 2.5% PEG/NaCl to precipitate out the inho-phage particles.
  • the phage media is centrifuged again for 40 min at 8000 rpm to pellet out the phage.
  • CNT/phage complexes were fabricated as previously described in published work, Dang et al. Nature Nanotechnology 6, 377-384 (2011) with p8 coat protein insert sequence DSPHTELP selected for SWNT binding and pH control allowing for efficient dispersion of the complex.
  • Fluorescent dyes from the AlexaFlour NHS Ester line where picked for labeling of phage for imaging.
  • the amine reactive chemistry of the dyes allows for easy labeling of side chains of lysine groups exposed by the p8 coat protein. Steps followed per manufacturer's instructions. Labeled phage were processed under sterile conditions and dialyzed against buffer for 72 hrs before usage in vivo or in vitro studies.
  • CNT complexed phage at varying concentrations [lel3, 6el3, 9el3 pfu/mL] and lengths [300, 880 nm] were prepared for 200 ⁇ L tail vein injections in NCR-NU male mice
  • mice were monitored post injection at 16hr, 48hr, 72hr on the Belcher NIR-II Imager (setup at 808nm laser, 2x1100 longpass, 2x1300 longpass, 1 second exposure). NIR-II imaging as previously described by Ghosh et al, PNAS 2014 111 (38) 13948-13953. At 71hrs end of trial, mice were sacrificed and dissected and the brain was imaged for phage localization at the tumor site.
  • Immunofluorescence was performed on cells using Golgin 1 (Cell Signaling Technologies) as a Golgi marker, and DAPI (ThermoFisher) as a DNA stain for colocalization. Images were captured on an EVOS fluorescent microscope (Life Technologies) or a Nikon Eclipse 80i fluorescence microscope.
  • U87MG human glioma cells were purchased through ATCC (ATCC No. HTB-14) and maintained in DMEM media (Gibco) with 10% FBS (Hyclone).
  • DMEM media Gibco
  • FBS Hyclone
  • GL261 mouse glioma cells Szatmari T et al, Cancer Sci, 2006, 97(6):546-553 were maintained in DMEM (Gibco) with 10% FBS (Hyclone), IX Glutamine (Gibco), and IX Pen/Strep (Sigma).
  • mice were transduced with a lentiviral pLMP-GFP-Luc vector to allow for stable expression of GFP and firefly luciferase prior to implantation.
  • Six week old NCR nude (Taconic) or C57/BL6 male mice (Taconic) were used to generate intracranial orthotopic U87MG or GL261 gliomas, respectively.
  • mice were anesthetized using 2% isoflurane and their heads
  • a burr hole was made using a steel drill bit (Plastics One, Roanoke, VA, USA) 1.4 mm right of the sagittal and 1 mm anterior to the lambdoid suture.
  • 105 glioma cells were stereotactically injected 3 mm deep from the dura mater into the brain using a 33-guage Hamilton syringe. Tumors were allowed to grow for 14 days prior to commencement of treatment. Intracranial tumor growth was monitored in vivo using bioluminescence IVIS® imaging (Xenogen, Almeda, CA) equipped with LivinglmageTM software (Xenogen).
  • the skull was thinned away using a sterile stainless steel 2 mm diameter cylindrical drill bit attached to a highspeed hand drill until the underlying dura mater is exposed.
  • Multiphoton imaging was performed on an Olympus FV-1000MPE multiphoton microscope (Olympus Americas, Waltham, MA) using a 25X, N.A 1.05 water objective. Excitation was achieved using a DeepSee Tai-sapphire femtosecond pulse laser (Spectro-Physics, Santa Clara, CA) at 840 nm. The emitted fluorescence was collected by PMTs with emission filters of 425/30 nm for Collagen 1, 525/45 nm for GFP-labeled tumor cells and 668/20 nm for Alexa-647
  • Collagen 1 was excited by second harmonic generation and emits as polarized light at half the excitation wavelength. Images were taken 24 hrs post-IV injection of

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  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne des populations de phages modifiés, qui sont homogènes en longueur, ainsi que des procédés de fabrication et des procédés d'utilisation de ces phages. L'invention concerne également des phages de chlorotoxine modifiés ainsi que leurs procédés de fabrication et d'utilisation. Les populations de phages homogènes et les phages de chlorotoxine selon l'invention peuvent être utilisés, par exemple, pour traiter et/ou imager des tumeurs, telles que des tumeurs du système nerveux central.
PCT/US2017/043298 2017-07-21 2017-07-21 Systèmes de bactériophages modifiés WO2019017967A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0805877B1 (fr) * 1995-01-17 2001-08-16 Bioinvent International Ab Procede perfectionne de selection de bacteriophages specifiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0805877B1 (fr) * 1995-01-17 2001-08-16 Bioinvent International Ab Procede perfectionne de selection de bacteriophages specifiques

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAGENS, S ET AL.: "Genetically Modified Filamentous Phage as Bactericidal Agents: a Pilot Study", LETTERS IN APPLIED MICROBIOLOGY, vol. 37, no. 4, 2003, pages 318 - 323, XP055563371 *
JIN, HE ET AL.: "Collagen Mimetic Peptide Engineered M13 Bacteriophage for Collagen Pargeting and Imaging in Cancer", BIOMATERIALS, vol. 35, no. 33, 10 August 2014 (2014-08-10), pages 9236 - 9245, XP055563374 *
TSEDEV, U: "Engineering M13 Bacteriophage Platforms for Cancer Therapy Applications", June 2015 (2015-06-01), XP055563362, Retrieved from the Internet <URL:https://dspace.mit.edu/handle/1721.1/103838#files-area> [retrieved on 20171204] *

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