Non-replicative transduction particles with one or more non-native tail fibers and transduction particle-based reporter systems

Abstract

The present invention relates to compositions comprising and methods for producing genetically engineered bacteriophages, bacteriophage-like particles, and non-replicative transducing particles (NRTPs) that contain non-native tail fibers that exhibit altered host specificity and / or reactivity. The invention also relates to methods of using these bacteriophages and NRTPs for the development of novel diagnostic, therapeutic, and / or research reagents for bacterial-associated diseases.

Description

[Technical field]

[0001] The present invention relates to methods and compositions for packaging and delivery of non-replicative transducing particles derived from bacteriophage with non-native tail fibers that contain reporter molecules for detecting target organisms. [Background technology]

[0002] Transduction particles are viruses that can deliver non-viral nucleic acid into cells. Viral-based reporter systems have been used to detect the presence of cells and rely on the lysogenic phase of the virus to express reporter molecules from the cells. These virus-based reporter systems use replication-competent transduction particles that express reporter molecules and cause target cells to emit detectable signals.

[0003] However, the lytic cycle of the virus has been shown to be detrimental to virus-based reporter assays. Carriere, C. et al., Conditionally replicating luciferase reporter phages: Improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis. Journal of Clinical Microbiology, 1997. 35(12):3232-3239. Carriere et al. developed an M. tuberculosis / bacillus Calmette-Guerin (BCG) luciferase reporter phage whose lytic cycle is inhibited at 30°C and active at 37°C. Using this system, Carriere et al. demonstrated detection of BCG using a phage reporter with an inhibited lytic cycle.

[0004] However, there are disadvantages associated with suppressing but not eliminating the replication function of the bacteriophage in a bacteriophage-based reporter assay. First, controlling the replication function of the bacteriophage imposes constraints on the assay conditions. For example, the lytic cycle of the reporter phage phAE40 used by Carriere et al. was suppressed when the phage was used to infect cells at the non-permissive temperature of 30°C. This temperature requirement imposed constraints on the reporter assay in that the optimum temperature for the target bacteria is 37°C. These constraints prevent optimal assay performance.

[0005] Furthermore, viral replication functions are difficult to control. Viral replication should be suppressed during the use of transducing particles as reporter systems. For example, the lytic activity of the reporter phage phAE40 reported by Carriere et al. was reduced but not eliminated, leading to a decrease in luciferase signal in the assay. Carrier et al. highlighted possible causes for the resulting decrease in reporter signal, such as intact phage-expressed genes and temperature limitations of the assay, all resulting from the fact that the lytic cycle of the phage reporter was not eliminated.

[0006] Reporter assays that rely on the natural lysogenic cycle of phages can be expected to show lytic activity sporadically, and are prone to superinfection immunity from target cells that have already been lysogenized with similar phages, as well as naturally occurring host restriction systems that target incoming viral nucleic acid, thus limiting the host range of these reporter phages.

[0007] In other examples, transduction particle production systems are designed to package exogenous nucleic acid molecules, but transduction particles often contain a combination of exogenous nucleic acid molecules and natural progeny viral nucleic acid molecules. Natural viruses can exhibit lytic activity that is disruptive to assay performance, and the viral lytic activity must be removed to purify the transduction particles. However, this purification is not generally possible. In U.S. Patent Application Publication No. 2009 / 0155768, entitled "Reporter Plasmid Packaging System for Detection of Bacteria," Scholl et al. describe the development of such a transduction particle system. The product of this system is a combination of a reporter transduction particle and a natural bacteriophage (Figure 8 in the Supporting Information). The authors show that the transduction particle and the natural bacteriophage can be separated by ultracentrifugation, but this separation is only possible in systems in which the transduction particle and the natural virus exhibit different densities that would allow for separation by ultracentrifugation. This feature is exhibited by the bacteriophage T7-based packaging system described in references, but it is not a generally applicable feature to other viral systems. It is common for viral packaging mechanisms to exhibit headful packaging, which is believed to result in native viruses and transducing particles that exhibit indistinguishable densities that cannot be separated by ultracentrifugation. Viral packaging systems also rely on a minimal amount of packaging as a requirement for proper viral structure assembly, which results in native viruses and transducing particles that have indistinguishable densities.

[0008] Recently, methods and systems for packaging reporter nucleic acid molecules, also referred to herein as Smarticles, into non-replicating transducing particles (NRTPs) have been described in U.S. Pat. No. 9,388,453 and U.S. Patent Application Publication No. 2017 / 0166907, in which production of replication-competent natural progeny viral nucleic acid molecules was greatly reduced due to disruption of the packaging initiation site in the bacteriophage genome.

[0009] Currently, NRTPs are produced from naturally occurring temperate bacteriophages found in nature, such as φ80α or P1 bacteriophage. It is a time-consuming process to find new bacteriophages, characterize them, and engineer them into NRTPs capable of transducing cells with the desired host range specificity.

[0010] Several recent studies (Ando et al., Cell Systems 2015, 1:187-196; Yosef et al., Molecular Cell 2017, 66:721-728) have revealed that T3 and T7 bacteriophages as well as bacteriophage / transducing particle mixtures can be reprogrammed to recognize a desired host by swapping out tails / tail fibers from T3 / T7-like viral sources that would allow the phages to recognize different hosts. To this end, different tails / tail fibers were constructed on the T3 / T7 chassis and tested for their ability to inject DNA into different hosts. Although these experiments paved the way for the production of hybrid particles with novel host ranges, these effects were only shown with phage / tails / tail fibers found in T3- and T7-like bacteriophages. Notably, these studies do not enable phage genome engineering platforms based on chassis from other bacteriophage families, such as Myoviridae, which, unlike T3 / T7 bacteriophages, have long contractile tails and contain completely different tail fiber genes.

[0011] One of the best-characterized Myoviridae phages is the P1 bacteriophage, which infects Escherichia coli (E. coli) and other bacteria of the Enterobacteriaceae family. The host range specificity of P1 is controlled within the tail fiber operon, which is approximately 6 kb long (Figure 2) and consists of two alternating 3' ends of the tail fiber gene (s v or s v ') to the constant 5' end (s c ) that can be fused to the tail fiber operon. Two alternate genes, u and u', are located outside the S gene and encode alternative chaperone proteins involved in tail fiber assembly. The other two genes in the tail fiber operon are the r gene, whose product R is thought to be involved in tail fiber assembly, and the gene cin, which encodes a sequence-specific recombinase enzyme that can "flip" nucleic acid between Cin recombination sites, known as cix sites, which alter host specificity; these two genes are described in Guidolin et al., 1989 Gene, 76:239-243, and Sandmeier et al., 1992 Journal of Bacteriology, 174(12):3936-3944.

[0012] Thus, there is a need in the art for a rapid, reliable and scalable method to generate new NRTPs derived from the tail fibers of many types of phages. Therefore, the present invention relates to a tail fiber exchange platform (or chassis) derived from P1 bacteriophage that is modifiable solely at the tail fiber to alter or broaden its host specificity, which can utilize tail fibers from many virus families. Additionally, the present invention relates to methods for improving the yield of NRTPs from engineered production strains and for producing NRTPs with multiple native or engineered tail fibers in a single fermentation. This approach can be used for a variety of other bacteriophages to develop new NRTPs with desired inclusion and exclusivity profiles with respect to host range specificity. Summary of the Invention

[0013] The present invention contemplates a bacteriophage lysogen in which the expression of one or more factors essential for native tail fiber production (e.g., genes s, u, u', r and s') has been genetically disrupted. This could be accomplished through any mutagenesis technique known to one of skill in the art, or through the isolation of a tail fiber-deficient phage remnant from a natural source. This genetically disrupted phage lysogen, which lacks the ability to express its own functional tail fiber without complementation, is referred to as a chassis or "Bald" chassis. In one embodiment, the disrupted lysogen has a mutation or deletion in one or more of the genes selected from a gene encoding a tail fiber structural protein involved in binding to a bacterial cell receptor, a gene encoding a chaperone protein required for folding one or more regions of the tail fiber structural protein, a gene encoding a protein required for attachment of the tail fiber structural protein to the tail, or any combination of these genes. In another embodiment, the disrupted lysogen contains a gene encoding a selectable marker that disrupts the expression of one or more of the genes. In yet another embodiment, the disrupted lysogen comprises the nucleotide sequence of SEQ ID NO:97.

[0014] A further aspect of the invention relates to methods involving expression of native new tail fiber genes or hybrid tail fibers in lysogens that have been genetically disrupted for native tail fiber expression. This could be accomplished using packaging plasmids, secondary expression plasmids, genomic expression (phage lysogens or bacterial genomes), or any other method known to those of skill in the art.

[0015] Thus, the present invention provides a Myoviridae lysogen containing a gene disruption that prevents expression of one or more genes essential for the production of a tail fiber native to the Myoviridae bacteriophage lysogen (Myoviridae lysogen), wherein the one or more genes are a gene encoding a tail fiber structural protein involved in binding to a bacterial cell receptor, a gene encoding a chaperone protein required for folding one or more regions of the tail fiber structural protein, a gene encoding a protein required for attachment of the tail fiber structural protein to the tail, or any of these genes. and a complementary nucleic acid molecule comprising one or more genes selected from a gene encoding a tail fiber structural protein involved in binding to a bacterial cell receptor, a gene encoding a chaperone protein required for folding of one or more regions of the tail fiber structural protein, a gene encoding a protein required for attachment of the tail fiber structural protein to the tail, or any combination of these genes, wherein the complementary nucleic acid complements a genetic disruption of the Myoviridae lysogen, thereby producing a functional tail fiber.

[0016] In one embodiment, the Myoviridae lysogen is from a genus selected from Bcep781-like virus, Bcepmu-like virus, FelixO1-like virus, Hapuna-like virus, I3-like virus, Mu-like virus, Puna-like virus, Pbuna-like virus, PhiCD119-like virus, Phih-like virus, Phikz-like virus, Viuna-like virus, Eucampyvirinae, Cp220-like virus, Cp8una-like virus, Peduovirinae, Hpuna-like virus, P2-like virus, Spoonavirinae, Spoonavirinae, Twort-like virus, Tevenvirinae, Schizot4-like virus, or T4-like virus. In one embodiment, the Myoviridae lysogen is from the genus Puna-like virus. In one embodiment, the Myoviridae lysogen is Enterobacteriaceae bacteriophage phage P1 (P1 bacteriophage).

[0017] In one embodiment, the gene or genes or combination of genes on the complementary nucleic acid molecule contain a region not derived from one or more genes essential for the production of the native tail fiber of the Myoviridae lysogen, such that the functional tail fiber produced is not a native tail fiber. In such an embodiment, the tail fiber produced is a functional tail fiber not native to the original bacteriophage lysogen. In another embodiment, the gene or genes or combination of genes contain a region not derived from the P1 bacteriophage, such that the functional tail fiber produced is not a native P1 bacteriophage tail fiber.

[0018] In yet another embodiment, the gene disruption on the Myoviridae lysogen comprises one or more mutations in any of the one or more genes. In some embodiments, the Myoviridae lysogen may comprise one or more mutations in one of the one or more genes. In other embodiments, the Myoviridae lysogen may comprise one or more mutations in at least two of the one or more genes. In one embodiment, the one or more mutations are present in any one of the P1 bacteriophage genes s, s', u, u' and r, in any combination of genes s, s', u, u' and r, or in a homolog, ortholog or analog of genes s, s', u, u' and r.

[0019] In one embodiment, the gene disruption on the Myoviridae lysogen comprises one or more deletions in any one of the one or more genes. In some embodiments, the Myoviridae lysogen may comprise one or more deletions in one of the one or more genes. In other embodiments, the Myoviridae lysogen may comprise one or more deletions in at least two of the one or more genes. In certain embodiments, at least one deletion is a deletion of the complete coding sequence of one of the one or more genes. In other embodiments, any of the one or more deletions is a deletion of the complete coding sequence of one of the one or more genes. In one embodiment, the one or more deletions are in any one of the P1 bacteriophage genes s, s', u, u' and r, in any combination of genes s, s', u, u' and r, or in a homolog, ortholog or analog of genes s, s', u, u' and r. In another embodiment, the gene disruption on the Myoviridae lysogen further comprises the insertion of a gene encoding a selectable marker that disrupts expression of any one of the P1 bacteriophage genes. In one embodiment, the Myoviridae lysogen comprises the nucleic acid sequence of SEQ ID NO:97.

[0020] In one embodiment, the complementary nucleic acid molecule is a plasmid molecule. In another embodiment, the complementary nucleic acid molecule is integrated at the site of the gene disruption of the Myoviridae lysogen. In yet another embodiment, the complementary nucleic acid molecule is integrated at a site other than the site of the gene disruption of the Myoviridae lysogen. In yet another embodiment, the complementary nucleic acid molecule is integrated into the genome of a host bacterial cell that contains the Myoviridae lysogen.

[0021] In one embodiment, the Myoviridae lysogen contains an additional disruption in one or more packaging genes involved in packaging the genome of the Myoviridae lysogen, thereby rendering the one or more packaging genes involved in packaging the genome of the Myoviridae lysogen non-functional. In one embodiment, the one or more packaging genes that complement the disruption in one or more packaging genes in the Myoviridae lysogen are contained in a plasmid molecule. In another embodiment, the one or more packaging genes that complement the disruption in one or more packaging genes in the Myoviridae lysogen are contained in a reporter or therapeutic expression plasmid that is separate from the plasmid molecule.

[0022] The present invention also relates to a bacterial cell line comprising a P1 bacteriophage lysogen comprising the nucleic acid sequence of SEQ ID NO:97.

[0023] The present invention relates to a method for producing bacteriophage particles or non-replicating transducing particles (NRTPs) with non-native tail fibers, comprising providing a bacterial cell containing a bacteriophage tail fiber exchange platform of the invention, wherein a complementary nucleic acid molecule comprises a gene encoding a tail fiber structural protein having one or more regions not derived from one or more genes essential for production of native tail fibers of Myoviridae lysogens, and providing the bacterial cell with conditions that induce the lytic phase of Myoviridae lysogens to produce bacteriophage particles with non-native tail fibers.

[0024] In one embodiment, the one or more regions not derived from one or more genes native to the Myoviridae lysogen comprise a variable region of a tail fiber structural protein (tail fiber variable region) obtained from a bacteriophage within the Caudovirales, hi one embodiment, the tail fiber variable region is obtained from a bacteriophage within the Myoviridae. In another embodiment, the tail fiber variable region is obtained from a bacteriophage within a genus selected from Bcep781-like viruses, Bcepmu-like viruses, FelixO1-like viruses, Hapuna-like viruses, I3-like viruses, Mu-like viruses, Puna-like viruses, Pbuna-like viruses, PhiCD119-like viruses, Phih-like viruses, Phikz-like viruses, Viuna-like viruses, Eucampyvirinae, Cp220-like viruses, Cp8una-like viruses, Peduovirinae, Hpuna-like viruses, P2-like viruses, Spoonavirinae, Spoonavirinae, Twort-like viruses, Tevenvirinae, Schizot4-like viruses, or T4-like viruses. In one embodiment, the genus is Puna-like viruses.

[0025] In one embodiment, the complementary nucleic acid molecule further comprises a gene encoding a chaperone protein. In one embodiment, the gene encoding the chaperone protein is obtained from a bacteriophage within the Caudovirales. In another embodiment, the gene encoding the chaperone protein is obtained from a bacteriophage within the Myoviridae. In yet another embodiment, the gene encoding the chaperone protein is obtained from a bacteriophage from a genus selected from Bcep781-like virus, Bcepmu-like virus, FelixO1-like virus, Hapuna-like virus, I3-like virus, Mu-like virus, Puna-like virus, Pbuna-like virus, PhiCD119-like virus, Phih-like virus, Phikz-like virus, Viuna-like virus, Eucampyvirinae, Cp220-like virus, Cp8una-like virus, Peduovirinae, Hpuna-like virus, P2-like virus, Spoonavirinae, Spoonavirinae, Twort-like virus, Tevenvirinae, Schizot4-like virus, or T4-like virus. In one embodiment, the genus is Puna-like virus. In one embodiment, the chaperone protein is obtained from a bacterial genome encoding a non-replicating virus-derived structure or from a nucleic acid delivery particle. In one embodiment, the gene encoding the chaperone protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-96 and 113-117.

[0026] In one embodiment, the one or more regions not derived from one or more genes native to the Myoviridae lysogen comprise a variable region of a tail fiber structural protein (tail fiber variable region) obtained from a bacterial genome encoding a phage tail-like particle or structure or from a nucleic acid delivery particle. In one embodiment, the gene encoding the tail fiber structural protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-66 and 108-112. In another embodiment, the gene encoding the tail fiber structural protein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-34, 98, 100, 102, 104 and 106.

[0027] The present invention further relates to a method for producing a non-replicating transducing particle (NRTP) having a non-native tail fiber comprising the steps of providing a bacterial cell containing a bacteriophage tail fiber exchange platform of the invention, wherein the Myoviridae lysogen comprises an additional disruption in one or more packaging genes involved in packaging of the Myoviridae lysogen genome, and the complementary nucleic acid molecule comprises a gene encoding a tail fiber structural protein that contains one or more regions not derived from the one or more genes essential for production of the native tail fiber of the Myoviridae lysogen, and the bacterial cell further comprises a reporter plasmid comprising the one or more packaging genes that have been disrupted in the Myoviridae lysogen, and providing the bacterial cell with conditions that induce a lytic phase of the Myoviridae lysogen to produce an NRTP having a non-native tail fiber.

[0028] In one embodiment, the one or more regions not derived from one or more genes native to the Myoviridae lysogen comprise a variable region of a tail fiber structural protein (tail fiber variable region) obtained from a bacteriophage within the Caudovirales, hi one embodiment, the tail fiber variable region is obtained from a bacteriophage within the Myoviridae. In another embodiment, the tail fiber variable region is obtained from a bacteriophage within a genus selected from Bcep781-like viruses, Bcepmu-like viruses, FelixO1-like viruses, Hapuna-like viruses, I3-like viruses, Mu-like viruses, Puna-like viruses, Pbuna-like viruses, PhiCD119-like viruses, Phih-like viruses, Phikz-like viruses, Viuna-like viruses, Eucampyvirinae, Cp220-like viruses, Cp8una-like viruses, Peduovirinae, Hpuna-like viruses, P2-like viruses, Spoonavirinae, Spoonavirinae, Twort-like viruses, Tevenvirinae, Schizot4-like viruses, or T4-like viruses. In one embodiment, the genus is Puna-like viruses.

[0029] In one embodiment, the complementary nucleic acid molecule further comprises a gene encoding a chaperone protein. In one embodiment, the gene encoding the chaperone protein is obtained from a bacteriophage within the Caudovirales. In another embodiment, the gene encoding the chaperone protein is obtained from a bacteriophage within the Myoviridae. In yet another embodiment, the gene encoding the chaperone protein is obtained from a bacteriophage from a genus selected from Bcep781-like virus, Bcepmu-like virus, FelixO1-like virus, Hapuna-like virus, I3-like virus, Mu-like virus, Puna-like virus, Pbuna-like virus, PhiCD119-like virus, Phih-like virus, Phikz-like virus, Viuna-like virus, Eucampyvirinae, Cp220-like virus, Cp8una-like virus, Peduovirinae, Hpuna-like virus, P2-like virus, Spoonavirinae, Spoonavirinae, Twort-like virus, Tevenvirinae, Schizot4-like virus, or T4-like virus. In one embodiment, the genus is Puna-like virus. In one embodiment, the chaperone protein is obtained from a bacterial genome encoding a non-replicating virus-derived construct or from a nucleic acid delivery particle. In one embodiment, the gene encoding the chaperone protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-96 and 113-117.

[0030] In one embodiment, the one or more regions not derived from one or more genes native to the Myoviridae lysogen comprise a variable region of a tail fiber structural protein (tail fiber variable region) obtained from a bacterial genome encoding a phage tail-like particle or structure or from a nucleic acid delivery particle. In one embodiment, the gene encoding the tail fiber structural protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 35-66 and 108-112. In another embodiment, the gene encoding the tail fiber structural protein comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-34, 98, 100, 102, 104 and 106.

[0031] In another aspect, the invention contemplates expressing chimeric tail fiber genes that create bacteriophages and / or NRTPs that exhibit host range specificity and / or reactivity that differs from that of the native bacteriophage chassis. Thus, the invention relates to a method of creating an engineered bacteriophage or non-replicating transducing particle (NRTP) that exhibits bacterial host cell specificity that differs from that exhibited by a native bacteriophage, comprising fusing a first nucleotide sequence from one tail fiber structural protein-encoding gene with a second nucleotide sequence from a second tail fiber structural protein-encoding gene from a different source to create a chimeric tail fiber structural protein-encoding gene, expressing the chimeric tail fiber structural protein gene in a bacterial cell containing a bacteriophage tail fiber exchange platform of the invention, and providing conditions in the bacterial cell that induce the lytic phase of a Myoviridae lysogen to create the engineered bacteriophage or NRTP.

[0032] In one embodiment, the Myoviridae lysogen is from a genus selected from Bcep781-like viruses, Bcepmu-like viruses, FelixO1-like viruses, Hapuna-like viruses, I3-like viruses, Mu-like viruses, Puna-like viruses, Pbuna-like viruses, PhiCD119-like viruses, Phih-like viruses, Phikz-like viruses, Viuna-like viruses, Eucampyvirinae, Cp220-like viruses, Cp8una-like viruses, Peduovirinae, Hpuna-like viruses, P2-like viruses, Spoonavirinae, Spoonavirinae, Twort-like viruses, Tevenvirinae, Schizot4-like viruses, or T4-like viruses. In one embodiment, the Myoviridae lysogen is from the genus Puna-like viruses. In another embodiment, the Myoviridae lysogen is Enterobacteriaceae bacteriophage phage P1 (P1 bacteriophage).

[0033] In one embodiment, the first nucleotide sequence is from a Myoviridae lysogen containing a gene disruption. In another embodiment, the first nucleotide sequence is from the tail fiber gene s from P1 bacteriophage. c In yet another embodiment, the first nucleotide sequence comprises a sequence having at least 90% sequence identity to a nucleotide sequence encoding amino acid sequence 1-250 of SEQ ID NO:53. In yet another embodiment, the first nucleotide sequence comprises a sequence having at least 90% sequence identity to a nucleotide sequence encoding amino acid sequence 1-500 of SEQ ID NO:53. In one embodiment, the first nucleotide sequence comprises a sequence having at least 90% sequence identity to a nucleotide sequence encoding amino acid sequence 1-500 of SEQ ID NO:53. In one embodiment, the first nucleotide sequence comprises a sequence having at least 90% sequence identity to a nucleotide sequence encoding amino acid sequence 1-500 of SEQ ID NO:53. c Includes the area.

[0034] In another aspect, the invention relates to a method of making an engineered bacteriophage or non-replicating transducing particle (NRTP) with multiple types of tail fibers from a single bacterial cell line, where at least one of the tail fiber types is a non-native tail fiber, comprising the steps of providing a bacterial cell containing a bacteriophage tail fiber exchange platform of the invention, wherein a complementary nucleic acid molecule comprises at least two receptor binding regions from a tail fiber structural protein-encoding gene fused to one or more tail interacting regions from the tail fiber structural protein-encoding gene, where at least one of the receptor binding regions is from a source different from the source of one of the tail interacting regions, and providing conditions to induce a lytic phase of a bacteriophage lysogen in the bacterial cell to produce a bacteriophage or NRTP with multiple types of tail fibers.

[0035] In one embodiment, the Myoviridae lysogen is from a genus selected from Bcep781-like viruses, Bcepmu-like viruses, FelixO1-like viruses, Hapuna-like viruses, I3-like viruses, Mu-like viruses, Puna-like viruses, Pbuna-like viruses, PhiCD119-like viruses, Phih-like viruses, Phikz-like viruses, Viuna-like viruses, Eucampyvirinae, Cp220-like viruses, Cp8una-like viruses, Peduovirinae, Hpuna-like viruses, P2-like viruses, Spoonavirinae, Spoonavirinae, Twort-like viruses, Tevenvirinae, Schizot4-like viruses, or T4-like viruses. In one embodiment, the Myoviridae lysogen is from the genus Puna-like viruses. In another embodiment, the Myoviridae lysogen is Enterobacteriaceae bacteriophage phage P1 (P1 bacteriophage).

[0036] In one embodiment, the complementary nucleic acid molecule is integrated at the site of the gene disruption of the Myoviridae lysogen. In another embodiment, the complementary nucleic acid molecule is integrated at a site other than the site of the gene disruption of the Myoviridae lysogen. In yet another embodiment, the complementary nucleic acid molecule is integrated into the genome of the bacterial cell. In yet another embodiment, the complementary nucleic acid molecule is integrated into a plasmid molecule.

[0037] In one embodiment, the fusion of two or more receptor binding regions to the tail interacting region is performed by cin recombinase in the presence of multiple cix recombination sites. In one embodiment, the fusion of two or more receptor binding regions to the tail interacting region is performed by cin recombinase or a homolog, ortholog, or paralog of cin recombinase in the presence of multiple associated recombination sites. In one embodiment, the two or more separate tail fiber structural protein encoding genes are expressed in an operon. In another embodiment, the two or more separate tail fiber structural protein encoding genes are expressed from independent promoters. In yet another embodiment, the two or more separate tail fiber structural protein encoding genes are expressed from independent genomic locations.

[0038] In another aspect, the invention provides a bacterial cell packaging system for packaging a reporter or therapeutic nucleic acid molecule into a non-replicating transducing particle (NRTP) for introduction into a cell, comprising: a host bacterial cell; a bacteriophage lysogen containing a gene disruption that prevents expression of one or more genes essential for production of a native tail fiber, said one or more genes encoding a tail fiber structural protein involved in binding to a receptor, a chaperone required for folding of one or more regions of the tail fiber structural protein, a protein required for attachment of the tail fiber structural protein to a tail structure, or any combination of these genes; a bacteriophage lysogen that also contains a first bacteriophage gene containing a non-functional packaging initiation site sequence that prevents preferential packaging of the bacteriophage genome into the NRTP; a tail fiber structural protein involved in binding to a receptor, a chaperone required for folding of one or more regions of the tail fiber structural protein, a protein required for attachment of the tail fiber structural protein to a tail structure, or any combination of these genes; and one or more complementary nucleic acid molecules comprising one or more genes selected from a fiber structural protein, a chaperone required for folding of one or more regions of the tail fiber structural protein, a gene encoding a protein required for attachment of the tail fiber structural protein to a tail structure, or any combination of these genes, which complement a genetic disruption in a bacteriophage lysogen, thereby producing a functional tail fiber; and a reporter or therapeutic nucleic acid molecule, and a second bacteriophage gene containing a functional packaging initiation site sequence to promote packaging of a reporter nucleic acid molecule into a replicon, wherein the functional packaging initiation site sequence on the reporter nucleic acid molecule complements a non-functional packaging initiation site sequence in the bacteriophage lysogen.

[0039] In one embodiment, the reporter gene is luxAB. In another embodiment, the reporter gene is a fluorescent reporter protein. In some embodiments, the fluorescent reporter protein includes, but is not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), and red fluorescent protein (RFP). In one embodiment, the first bacteriophage gene and the second bacteriophage gene are pacA terminase genes. In another embodiment, the first bacteriophage gene and the second bacteriophage gene are terS terminase genes.

[0040] In another aspect, the invention provides a method for making a Myoviridae bacteriophage or Myoviridae-derived NRTP capable of recognizing and transducing a bacterial cell for diagnostic or therapeutic use, comprising the steps of providing a bacterial cell containing a bacteriophage tail fiber exchange platform of the invention, wherein the complementary nucleic acid molecule is not native to Myoviridae lysogens but is derived from Myoviridae, Siphoviridae (represented by SEQ ID NOs: 5, 10, 32, 33), or Polyviridae. and providing conditions to induce the lytic phase of a bacteriophage lysogen in a bacterial cell to produce a Myoviridae bacteriophage or Myoviridae-derived NRTP capable of recognizing and transducing bacteria previously resistant to naturally occurring Myoviridae bacteriophage or Myoviridae-derived NRTP particles for diagnostic or therapeutic uses.

[0041] In one embodiment, the Myoviridae bacteriophage or Myoviridae-derived NRTP is from a genus selected from Bcep781-like viruses, Bcepmu-like viruses, FelixO1-like viruses, Hapuna-like viruses, I3-like viruses, Mu-like viruses, Puna-like viruses, Pbuna-like viruses, PhiCD119-like viruses, Phih-like viruses, Phikz-like viruses, Viuna-like viruses, Eucampyvirinae, Cp220-like viruses, Cp8una-like viruses, Peduovirinae, Hpuna-like viruses, P2-like viruses, Spoonavirinae, Spoonavirinae-like viruses, Twort-like viruses, Tevenvirinae, Schizot4-like viruses, or T4-like viruses. In one embodiment, the Myoviridae bacteriophage or Myoviridae-derived NRTP is from the genus Puna-like virus. In another embodiment, the Myoviridae bacteriophage or Myoviridae-derived NRTP is Enterobacteriaceae bacteriophage phage P1 (P1 bacteriophage).

[0042] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description and accompanying drawings. [Brief description of the drawings]

[0043] [Figure 1] FIG. 1 shows a diagram illustrating the tail fiber exchange platform. [Diagram 2]FIG. 2 is a diagram of the native P1 tail fiber genomic region. Gene r encodes a tail fiber accessory protein putatively involved in attaching the tail fiber to the tail structure. The cin gene encodes a sequence-specific recombinase that inverts DNA between Cin recombination sites (called cix sites), which allow expression of two separate tail fibers from a single piece of DNA; the sites are represented in the diagram as circles. Genes u and u' encode separate tail fiber accessory proteins that are thought to act as chaperones that fold the binding domains of the S and S' tail fiber proteins, respectively (the binding domains encoded by the sv and sv' coding sequences, respectively). Conserved regions of both the S and S' tail fiber proteins are encoded by the sc coding sequence, which is transcriptionally and translationally fused to either sv or sv' depending on the activity of the Cin recombinase. [Diagram 3] Figure 3 is a diagram of the tail fiber exchange platform. A tail fiber-deficient (Bald) packaging strain carrying a spectinomycin resistance cassette in place of the s and u genes is induced to express the recombineering mechanism (SEQ ID NO: 97), which contains the operon promoter, r gene, spectinomycin resistance promoter, spectinomycin resistance gene, u' gene, sv' coding region, cin gene, and cin promoter. The PCR fragment carrying the new tail fiber, hygromycin resistance cassette, and flanking homology arms is then transformed into the induced strain (in this case, "shamrock" and "shamrock" u). The recombineering mechanism incorporates the new tail fiber operon in place of the spectinomycin resistance cassette-u'-sv' region to create a new chromosomal packaging strain capable of producing tail fiber-endowed smarticles. [Figure 4]Figure 4 is a graph showing improved smarticle performance following chromosomal integration of tail fibers. A diagram comparing the performance of smarticles endowed with tail fibers expressed from a plasmid (TFDV) or from a chromosome. The reactivity of smarticles bearing only the S' (top left), S (top right), or "shamrock" (bottom left) tail fibers on various E. coli strains is shown. Bottom right, improved smarticle yields obtained from S tail fiber packaging strains with genomic integration versus plasmid expression. V3 smarticle (native tail fiber region) serves as a positive control. [Diagram 5] Figure 5 shows improved tail fiber activity upon expression of the native U protein homolog or from the bacteriophage genome. A) Performance of the "Indian" NRTP produced in packaging strains expressing or not expressing the plasmid p15B U protein homolog T. B) Performance of the "Scarlet" NRTP produced from the bacteriophage genome or from another packaging plasmid. [Figure 6] Figure 6 shows the production of one, two, or more tail fibers in a single producer strain. A) Single bacterial packaging strains expressing one, two, three, or more functional tail fibers. S, S', and shamrock tail fibers were added to the packaging strain along with an antibiotic marker. The tail fiber genes can be positioned to have a cix crossover site in a novel location. Genes can also be positioned for recombination-independent expression in large or small operons. B) Lysates were made from packaging strains expressing single tail fibers (denoted as S, S', Smrk) as well as engineered double and triple tail fiber strains (denoted as V3, double, triple). An E. coli strain with specific reactivity to a single tail fiber was used to demonstrate that multiple functional tail fibers are expressed from a single host. [Figure 7]FIG. 7 shows the analysis of bifunctional smarticle packaging strains. Bifunctional P1-derived smarticles were created endowed with both "jazzberry" and "shamrock" tail fibers. A) Schematic depiction of the gene arrangement of the genomic integrated "shamjazz" dual expression system. B) Confirmation of bifunctional activity for strains specific for "jazzberry", "shamrock", or "V3" (S and S') tail fibers. C) Predicted and / or observed activity of selected individual and combined smarticles for a collection of E. coli strains (EcoAD panel). Percent coverage is depicted below and stem-and-whisker plots depict the light distribution on the panel. D) Comparison of predicted and observed activity (purple) of indistinguishable bifunctional smarticles. * denotes cumulative predicted coverage vs. actual experimentally observed coverage. [Figure 8] Figure 8 depicts the establishment of elements required for functional tail fibers. A) Three separate tail fiber Sc regions (see native P1, "plum", and "tangerine") were functionalized on the P1 chassis, expanding our understanding of the determinants required for functional tail fibers. All constructs carried the P1r gene encoding the R protein. Each tail fiber variable region (Sv) was most functional when expressed together with its cognate U accessory protein. B) Protein alignment of the three functional Sc regions indicates that the N-terminal 250 amino acids are important. [Figure 9]FIG. 9 shows various configurations for expression of tail fiber genes from fermentation strains. A) Represents expression of tail fibers and viral chassis from a phage genome. B) Represents viral chassis expression from a phage genome and a tail fiber gene from a packaging plasmid. C) Represents viral chassis from a phage genome and tail fibers from an independent plasmid. D) Represents viral chassis from a phage genome and tail fibers from a bacterial genome. E) Represents viral chassis from a phage genome and one set of tail fibers. A second unique set of tail fibers is expressed from an independent plasmid. F) Represents viral chassis from a phage genome and tail fibers. A second unique set of tail fibers is expressed from a packaging plasmid. G) Represents viral chassis from a phage genome and two unique sets of tail fibers. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0045] As used herein, a "reporter nucleic acid molecule" refers to a nucleotide sequence, including DNA or RNA molecules. Reporter nucleic acid molecules can be naturally occurring or artificial or synthetic molecules. In some embodiments, reporter nucleic acid molecules are foreign to a host cell and can be introduced into the host cell as part of an exogenous nucleic acid molecule, such as a plasmid or vector. In certain embodiments, reporter nucleic acid molecules can be complementary to a target gene in a cell. In other embodiments, reporter nucleic acid molecules include a reporter gene that encodes a reporter molecule (e.g., a reporter enzyme, a protein). In some embodiments, reporter nucleic acid molecules are referred to as "reporter constructs" or "nucleic acid reporter constructs."

[0046] As used herein, a "therapeutic nucleic acid molecule" refers to a nucleotide sequence that has a therapeutic effect of treating a disease by sequence-specific recognition of a target nucleic acid (e.g., an antisense nucleic acid) associated with the disease, or by encoding a gene product (antimicrobial protein) with therapeutic activity. In some embodiments, a therapeutic nucleic acid molecule can be introduced into a host cell as part of an exogenous nucleic acid molecule, such as a plasmid. As used herein, a "therapeutic expression plasmid" refers to a plasmid that contains a therapeutic nucleic acid molecule that encodes a gene product with therapeutic activity.

[0047] A "reporter molecule" or "reporter" refers to a molecule (e.g., a nucleic acid or a protein) that confers a detectable or selectable phenotype on an organism. The detectable phenotype can be, for example, colorimetric, fluorescent or luminescent. Reporter molecules can be expressed from reporter genes that code for enzymes that mediate fluorescent reactions (luxA, luxB, luxAB, luc, ruc, nluc), genes that code for enzymes that mediate colorimetric reactions (lacZ, HRP), genes that code for fluorescent proteins (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent protein), nucleic acid molecules that code for affinity peptides (His-tag, 3X-FLAG), and genes that code for selectable markers (ampC, tet(M), CAT, erm). Reporter molecules can be used as markers to indicate successful uptake of a nucleic acid molecule or foreign sequence (plasmid) into a cell. Reporter molecules can also be used to indicate the presence of a target gene, a target nucleic acid molecule, a target intracellular molecule, or a cell as described herein. Alternatively, the reporter molecule can be a nucleic acid, such as an aptamer or a ribozyme.

[0048] In some aspects of the present invention, the reporter nucleic acid molecule is operably linked to a promoter. In other aspects of the present invention, the promoter can be selected or designed to contribute to the responsiveness and cross-reactivity of the reporter system based on the activity of the promoter in a specific cell (e.g., a specific species) but not in other cells. In certain aspects, the reporter nucleic acid molecule includes an origin of replication. In other aspects, the selection of the origin of replication can also contribute to the responsiveness and cross-reactivity of the reporter system when the replication of the reporter nucleic acid molecule in the target cell contributes to or is necessary for the reporter signal generation based on the activity of the origin of replication in a specific cell (e.g., a specific species) but not in other cells. In some embodiments, the reporter nucleic acid molecule forms a replicon that can be packaged as concatemeric DNA into progeny virus during viral replication.

[0049] As used herein, "target transcript" refers to a portion of the nucleotide sequence of a DNA sequence or an mRNA molecule naturally formed by a target cell, including an mRNA molecule formed during transcription of a target gene, and an mRNA that is a product of RNA processing of a primary transcript. A target transcript can also be referred to as a cellular transcript or a naturally occurring transcript.

[0050] As used herein, the term "transcript" refers to a length of nucleotide sequence (DNA or RNA) transcribed from a DNA or RNA template sequence or gene. A transcript can be a cDNA sequence transcribed from an RNA template or an mRNA sequence transcribed from a DNA template. A transcript can be protein coding or non-coding. A transcript can also be transcribed from an engineered nucleic acid construct.

[0051] A transcript derived from a reporter nucleic acid molecule can be referred to as a "reporter transcript." A reporter transcript can include a reporter sequence and a cis-repressing sequence. A reporter transcript can have sequences that form regions of complementarity such that the transcript contains two regions that form a duplex (e.g., an intermolecular duplex region). One region can be referred to as the "cis-repressing sequence" and has complementarity to part or all of the target transcript and / or the reporter sequence. A second region of the transcript can be referred to as the "reporter sequence" and has complementarity to the cis-repressing sequence. Complementarity can be full complementarity or substantial complementarity. The presence of the cis-repressing sequence and / or the binding of the cis-repressing sequence to the reporter sequence can form a conformation in the reporter transcript that can block further expression of the reporter molecule. A reporter transcript can form a secondary structure, such as a hairpin structure, such that regions in the reporter transcript that are complementary to each other can hybridize to each other.

[0052] When referring to a nucleic acid molecule or an exogenous sequence (e.g., a plasmid, vector, construct), "introducing into a cell" means facilitating uptake or absorption into the cell, as understood by those skilled in the art. Absorption or uptake of a nucleic acid construct or transcript can occur by auxiliary agents or devices, including through unassisted diffusion or active cellular processes, or through the use of bacteriophages, viruses, and transducing particles. The meaning of the term is not limited to cells in vitro, as a nucleic acid molecule may be "introduced into a cell," which is part of a living organism. In such cases, introduction into a cell would include delivery to the organism. For example, for in vivo delivery, the nucleic acid molecule, construct, or vector of the invention can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Additional techniques are described herein or known in the art.

[0053] A "transduction particle" is a virus capable of delivering a non-viral nucleic acid molecule into a cell. The virus can be a bacteriophage, adenovirus, etc.

[0054] "Phage tail-like particle" or "phage tail-like structure" refers to a high molecular weight phage tail-like protein complex produced by bacteria that resembles a bacteriophage tail in shape but does not encode or deliver a replicating viral genome and is therefore not a virus. Non-limiting examples of phage tail-like particles or structures include type 6 secretion systems (T6SS) and R-type tailocins (also known as pyocins) found in a variety of bacterial types. Further descriptions of these particles can be found in Williams et al., Applied and Environmental Microbiology, 2008, 74:12, 3868-3876, and Scholl et al., Antimicrobial Agents and Chemotherapy, 2009, 53:7, 3074-3080. These example structures do not encode a viral capsid head, but can mediate binding and tail injection into target cells to kill the target cells or deliver protein effectors. Phage tail-like particles or structures can be used that have specificity determining elements, such as tail fibers, and therefore their genetic sequences can be used to target cell binding specificity. An example of a pyocin-derived tail fiber is shown in the nucleotide sequence of SEQ ID NO:11.

[0055] A "non-replicating transducing particle" or "NRTP" is a virus that can deliver non-viral nucleic acid molecules into a cell, but cannot package its own replicated viral genome into the transducing particle. The virus can be a bacteriophage, adenovirus, etc.

[0056] A "plasmid" is a small DNA molecule that is physically separate from the chromosomal DNA in a cell and can replicate independently of the chromosomal DNA. Although most commonly found in bacteria as small circular double-stranded DNA molecules, plasmids can also be found in archaea and eukaryotes. A plasmid is considered a replicon that can replicate autonomously in a suitable host. As used herein, the term "packaging plasmid" refers to a plasmid that encodes both the elements required to be packaged in a given viral capsid (i.e., packaging genes) and a reporter gene that can be expressed. As used herein, the term "tail fiber display vector" and "TFDV" for short refers to a plasmid that encodes a tail fiber coding sequence that produces the elements required to be packaged in a given viral capsid, a reporter gene that can be expressed, and a binding protein that can bind to a given capsid. As used herein, the term "secondary tail fiber expression plasmid" refers to a plasmid that encodes a functional tail fiber that can be bound to a given capsid, but does not necessarily have a reporter molecule or a packaging site / mechanism.

[0057] A "vector" is a nucleic acid molecule used as a vehicle to artificially transport exogenous genetic material into another cell where it can be replicated and / or expressed.

[0058] A "virus" is a small infectious agent that replicates only inside living cells of other organisms. A virus particle (known as a virion) contains two or three parts: i) genetic material made from DNA or RNA molecules that carry the genetic information; ii) a proteinaceous membrane that protects these genes; and, in some cases, iii) a lipid envelope.

[0059] As used herein, the term "complement" refers to a non-disrupted sequence in the presence of an identical sequence that is disrupted, or the relationship of a non-disrupted sequence to a disrupted sequence. In one embodiment, a complement comprises a gene that is encoded on a polynucleotide in a cell, is functional and expressible, and expresses a protein with the same function as the disrupted gene on the bacteriophage prior to disruption. In some embodiments, the complementing gene has greater than 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the bacteriophage gene to be disrupted prior to disruption, i.e., the native bacteriophage gene. In some embodiments, the complementing gene is identical to the bacteriophage gene to be disrupted prior to disruption, i.e., the native bacteriophage gene. In some embodiments, the complementing gene comprises a polynucleotide sequence that is deleted from the bacteriophage. In some embodiments, the complementing gene refers to a gene encoding the packaging machinery of a bacteriophage on a plasmid, the same gene being disrupted in the bacteriophage, and thus the plasmid is required in the presence of a bacteriophage with a mutated packaging machinery gene to provide the essential packaging machinery required to package the polynucleotide into a transduction particle.

[0060] As used herein, the term "packaging-associated enzyme activity" refers to one or more polypeptides that are critical for interaction with a packaging initiation site sequence to package a polynucleotide into a transduction particle. In some embodiments, a pair of terminase genes is required for such interaction, with each terminase encoding a packaging-associated enzyme activity. In some embodiments, the enzyme activity is encoded by the terS and / or terL genes from S. aureus bacteriophage φ11 or φ80α, the terA and terB genes from E. faecalis bacteriophage φEf11, or the pacA and pacB genes of Enterobacteriaceae bacteriophage P1. In these embodiments, each of the pair of terminase genes expresses a packaging-associated enzyme activity, and both functional versions are required for packaging of a polynucleotide with a packaging initiation site. In some embodiments, disruption of one of the genes in the plurality of genes associated with packaging-associated enzyme activity eliminates the packaging-associated enzyme activity. In some embodiments, both of the pair of terminase genes are disrupted on the bacteriophage, thus disrupting the entire set of genes encoding packaging-related enzyme activities on the bacteriophage.

[0061] The term "in situ" refers to a process that occurs in living cells grown apart from a living organism, for example, in tissue culture.

[0062] The term "in vivo" refers to a process that occurs in a living organism.

[0063] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0064] "G", "C", "A" and "U" generally represent nucleotides containing guanine, cytosine, adenine and uracil as their respective bases. "T" and "dT" are used interchangeably herein and refer to deoxyribonucleotides, e.g., deoxyribothymine, whose nucleobase is thymine. However, it is understood that the term "ribonucleotide" or "nucleotide" or "deoxyribonucleotide" can also refer to modified nucleotides, or surrogate replacement moieties, as further detailed below. Those skilled in the art will appreciate that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially altering the base pairing properties of an oligonucleotide that includes a nucleotide with such replacement moieties. For example, and without limitation, a nucleotide containing inosine as its base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine can be replaced with a nucleotide containing, for example, inosine in the nucleotide sequences of the present invention. Sequences containing such exchange moieties are an embodiment of the present invention.

[0065] As used herein, the term "complementary" when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, as understood by those skilled in the art, refers to the ability of the oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize under certain conditions with the oligonucleotide or polynucleotide comprising the second nucleotide sequence to form a duplex structure. Complementary sequences are also described as binding to each other and are characterized by their binding affinity.

[0066] For example, a first nucleotide sequence can be described as complementary to a second nucleotide sequence if the two sequences hybridize (anneal) under stringent hybridization conditions. Hybridization conditions include temperature, ionic strength, pH, and organic solvent concentration for the annealing and / or washing steps. The term "stringent hybridization conditions" refers to conditions under which a first nucleotide sequence hybridizes preferentially to its target sequence, e.g., a second nucleotide sequence, and hybridizes relatively less to, or not at all to, other sequences. Stringent hybridization conditions are sequence-dependent and are different under different environmental parameters. In general, stringent hybridization conditions are defined as the thermal melting point (T) for a nucleotide sequence at a defined ionic strength and pH. m ) is chosen to be approximately 5°C lower than T m is the temperature at which 50% of a first nucleotide sequence hybridizes to a perfectly matched target sequence (under defined ionic strength and pH). An extensive guide to nucleic acid hybridization can be found, for example, in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes part I, chap.2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays," Elsevier, NY ("Tijssen"). Other conditions can be applied, such as physiologically relevant conditions that may be encountered inside an organism. Those skilled in the art can determine the set of conditions most suitable for testing the complementarity of two sequences according to the final application of the hybridized nucleotides.

[0067] This includes base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of the first and second nucleotide sequences. Such sequences may be referred to herein as "fully complementary" with respect to each other. However, when a first sequence is referred to herein as "substantially complementary" with respect to a second sequence, the two sequences may be fully complementary, or the first and second sequences may form one or more, but generally no more than four, three or two mismatched base pairs upon hybridization while retaining the ability to hybridize under conditions most appropriate for the end application. However, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs shall not be considered mismatches for purposes of determining complementarity. For example, a dsRNA containing one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, where the longer oligonucleotide is perfectly complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for the purposes described herein.

[0068] A "complementary" sequence, as used herein, may contain or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, provided that the above requirements regarding its ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble or Hoogsteen base pairing.

[0069] As used herein, the terms "complementary," "fully complementary," and "substantially complementary" may be used in reference to base matches between the two strands of a dsRNA, or between the antisense strand of a dsRNA and a target sequence, the complementary strand of a single-stranded RNA sequence or a single-stranded DNA sequence, as understood from the context of their use.

[0070] As used herein, a "duplex structure" comprises two antiparallel, substantially complementary nucleic acid sequences. Complementary sequences in a nucleic acid construct between two transcripts, between two regions within a transcript, or between a transcript and a target sequence can form a "duplex structure". Generally, the majority of nucleotides in each strand are ribonucleotides, but as described in detail herein, each or both strands can also contain at least one non-ribonucleotide, e.g., deoxyribonucleotides and / or modified nucleotides. The two strands forming the duplex structure can be different parts of a larger RNA molecule, or the two strands can be separate RNA molecules. When the two strands are parts of a larger molecule and are therefore linked by an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands forming the duplex structure, the linked RNA strands are called "hairpin loops". When two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3' end of one strand and the 5' end of each of the other strands forming the duplex structure, the linking structure is called a "linker." The RNA strands may have the same or different numbers of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the duplex minus any overhangs present in the duplex. In general, the duplex structure is 15-30 or 25-30, or 18-25, or 19-24, or 19-21, or 19, 20, or 21 base pairs long. In one embodiment, the duplex is 19 base pairs long. In another embodiment, the duplex is 21 base pairs long. When two different siRNAs are used in combination, the duplex lengths can be the same or different.

[0071] As used herein, the term "region of complementarity" refers to the region on the antisense strand that is substantially complementary to a sequence defined herein, for example, a target sequence.If the region of complementarity is not completely complementary to the target sequence, mismatches are most tolerated at the terminal regions, and when present, are generally within the terminal region(s), for example, 6, 5, 4, 3, or 2 nucleotides of the 5' and / or 3' end.

[0072] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that have a specified percentage of nucleotide or amino acid residues that are the same when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the full length of the two sequences being compared.

[0073] In sequence comparison, typically one sequence serves as a reference sequence to which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, and if necessary, coordinates are subsequently designated, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity for test sequence(s) compared to the reference sequence based on the designated program parameters.

[0074] Optimal alignment of sequences for comparison can be carried out, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search approach of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0075] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described by Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0076] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to produce a detectable signal from a cell.

[0077] The term "therapeutically effective amount" is an amount that is effective for ameliorating symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" since prevention can be considered a treatment.

[0078] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0079] Viral lysogenization and lytic cycle Viruses undergo lysogeny and lytic cycles in host cells. If they undergo the lysogeny cycle, the phage chromosome can be integrated into the bacterial chromosome, or the phage chromosome can become established as a stable plasmid in the host, where it can remain dormant for long periods of time. If lysogens are induced, the phage genome is excised from the bacterial chromosome, initiating the lytic cycle, resulting in cell lysis and release of phage particles. The lytic cycle produces new phage particles, which are released upon lysis of the host.

[0080] Furthermore, virus-based reporter assays, such as phage-based reporters, can suffer from limited reactivity (i.e., analytical coverage) due to the restricted phage host range caused by host-based and prophage-derived phage resistance mechanisms. These resistance mechanisms can target native phage nucleic acids, resulting in degradation or otherwise inhibition of phage DNA and function. Such resistance mechanisms include restriction systems that cleave phage DNA and CRISPR systems that inhibit phage-derived transcripts.

[0081] Both lytic activity and phage resistance can be inhibitory to reporter phage-based assays. Lytic activity can inhibit the signal by destroying or otherwise inhibiting the cell's ability to produce a detectable signal, thus affecting the detection limit by reducing the amount of detectable signal or preventing the development of a detectable signal. Phage resistance mechanisms can limit the host range of the phage and limit the inclusiveness of the phage-based reporter, similarly affecting the detection limit by reducing the amount of detectable signal or preventing the development of a detectable signal. Both lytic activity and phage resistance caused by the integration of phage DNA in reporter phage can result in false negative results for assays incorporating these phage reporters.

[0082] III. Methods for Producing Non-replicating Transducing Particles (NRTPs) Disruption / complementation-based method for producing non-replicating transducing particles Disclosed herein is a non-replicating transducing particle packaging system based on disruption of a component of the viral genome that is recognized by the viral packaging machinery as an element from which genome packaging is initiated during virus production. In embodiments, the disruption disrupts the packaging initiation site from the bacteriophage and also disrupts terminase function. Examples of disrupted elements include the pac site sequence of pac-type bacteriophages and the cos site sequence of cos-type bacteriophages. If the packaging initiation site sequence in the phage is disrupted, the phage will not be able to produce a functional terminase. As an example, the pac site is encoded within the pacA gene sequence, and terminase function requires both functional PacA and PacB. Plasmid DNA is packaged into the phage capsid by including a recognizable packaging initiation site on the plasmid DNA that complements the disrupted terminase. The bacteriophage can be any bacteriophage, such as Enterobacteriaceae bacteriophage P1 or φEF11, or S. aureus bacteriophage φ80α or bacteriophage φ11.

[0083] Packaging initiation sites are often found within the coding regions of genes that are essential for virus production. Regions of bacteriophage genomes can be disrupted by insertion, substitution, deletion, or mutation that disrupts the packaging initiation site. Examples of disruptions that achieve this include, but are not limited to, allelic exchange events that exchange the sequence on the bacteriophage genome that contains the packaging initiation site sequence with another sequence, such as the sequence of an antibiotic resistance gene, or complete deletion of the small and large terminase genes. In an example using the terminase genes pacA and pacB, pacA can be disrupted in a manner that causes a polar effect that also disrupts pacB expression and / or the overall terminase function mediated by PacA and PacB. Other examples that can include terminase genes can also include the terS and terL genes from S. aureus bacteriophage φ11 or φ80α, or the terS and terL genes from E. faecalis bacteriophage φEf11.

[0084] In one example, the cell genome is lysogenized with a viral genome in which the packaging initiation site has been disrupted. In some embodiments, the cell can be an E. coli cell, a S. aureus cell, or an E. faecalis cell. The cell can be gram-negative or gram-positive. A complementing plasmid (or reporter nucleic acid molecule) is introduced into the cell, the plasmid DNA containing at least the gene disrupted in the bacteriophage, as well as a packaging initiation site sequence, and optionally additional bacteriophage genes and a reporter gene, where the reporter gene can encode a detectable and / or selectable marker. The plasmid can be constructed using methods found in U.S. Pat. No. 9,388,453 and in U.S. Patent Application Publication No. 2017 / 0166907. One or more genes of the plasmid can be operably linked to a promoter, such as an inducible promoter (which can be induced when packaging is initiated by inducing the bacteriophage). In some embodiments, the promoter can be the native promoter of the small or large terminase gene, which can be controlled by the bacteriophage and thus effectively functions as a conditional promoter that is induced during packaging.

[0085] In some cases, the disruption / complementation is preferably designed such that there is no homology between the mutant viral DNA and the complementary foreign DNA. This is because the lack of homology between the mutant viral DNA and the complementary foreign DNA avoids the possibility of homologous recombination between the two DNA molecules that can result in the reintroduction of the packaging sequence into the viral genome. To achieve the lack of homology, one strategy is to delete the entire gene(s) containing the packaging initiation site sequence from the viral genome, and then complement this gene with a foreign DNA molecule that preferably contains only the exact DNA sequence deleted from the virus. In this strategy, the complementary DNA molecule is designed to express the gene deleted from the virus. Another example of such a system is provided using bacteriophage φ80α, a pac-type phage. The phage genome is lysogenized in a host bacterial cell, and the phage genome contains a small terminase gene in which the pac site of the pac-type prophage φ80α has been deleted. A plasmid containing a complementary small terminase gene with a natural pac site is transformed into the cell. Upon induction of the lytic cycle of the lysogenized prophage, the bacteriophage packaging system packages the plasmid DNA into progeny bacteriophage structural components rather than packaging the native bacteriophage DNA, thus producing non-replicating transducing particles carrying the plasmid DNA.

[0086] A reporter gene encodes a detectable or selectable marker. In examples, the reporter gene is selected from the group consisting of an enzyme that mediates a luminescent reaction (luxA, luxB, luxAB, luc, ruc, nluc), an enzyme that mediates a colorimetric reaction (lacZ, HRP), a fluorescent protein (GFP, eGFP, YFP, RFP, CFP, BFP, mCherry, near-infrared fluorescent protein), an affinity peptide (His-tag, 3X-FLAG), and a selectable marker (ampC, tet(M), CAT, erm). In embodiments, the reporter gene is luxA. In some embodiments, the resistance marker comprises an antibiotic resistance gene. In some embodiments, the resistance marker is a kanamycin resistance gene (kan). In some embodiments, the constitutive promoter comprises Pblast. In some embodiments, the bacteriophage genome disruption is achieved by an allelic exchange event that replaces or disrupts a sequence on the bacteriophage genome that contains a packaging initiation site sequence.

[0087] In an example, a pair of terminase genes on a bacteriophage genome, e.g., pacA and pacB, terA and terB, or terS and terL, can be disrupted to cause a polar effect that disrupts expression of one of the terminase genes and / or the overall terminase function mediated by the terminase genes. The disrupted bacteriophage can be complemented with a plasmid containing the terminase genes of the bacteriophage genome, e.g., pacA and pacB, terA and terB, or terS and terL. When the mutated virus undergoes a lytic cycle, viral packaging proteins produced from the bacteriophage genome or the complementing plasmid (if disrupted) package the plasmid DNA replicon into packaging units because the plasmid DNA contains the packaging initiation site and non-replicating transducing particles are produced carrying the replicated plasmid DNA.

[0088] Reporter In some embodiments, the NRTPs and constructs of the invention include a reporter nucleic acid molecule that includes a reporter gene. The reporter gene can code for a reporter molecule, which can be a detectable marker or a selectable marker. In certain embodiments, the reporter gene codes for a reporter molecule that produces a detectable signal when expressed in a cell.

[0089] In certain embodiments, the reporter molecule can be a fluorescent reporter molecule, such as, but not limited to, green fluorescent protein (GFP), enhanced GFP, yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), red fluorescent protein (RFP) or mCherry, as well as near-infrared fluorescent proteins.

[0090] In other embodiments, the reporter molecule can be an enzyme that mediates a luminescent reaction (LuxA, LuxB, LuxAB, Luc, Ruc, nLuc, etc.). Reporter molecules can include bacterial luciferase, eukaryotic luciferase, enzymes suitable for colorimetric reactions (LacZ, HRP), proteins suitable for immunodetection such as affinity peptides (His-tag, 3X-FLAG), nucleic acids that function as aptamers or exhibit enzymatic activity (ribozymes), or selection markers such as antibiotic resistance genes (ampC, tet(M), CAT, erm). Other reporter molecules known in the art can be used to provide a signal for detecting target nucleic acids or cells.

[0091] In other embodiments, the reporter molecule comprises a nucleic acid molecule. In some embodiments, the reporter molecule is an aptamer (e.g., an aptazyme, DNAzyme, ribozyme) that has specific binding activity or exhibits enzymatic activity.

[0092] Reporters and reporter assays are further described in Section V herein.

[0093] V. NRTP and Reporter Assays Inducer reporter assay In some embodiments, the invention includes methods for the use of NRTPs as reporter molecules for use with exogenous or natural inducers that target gene promoters in living cells. The NRTPs of the invention can be engineered using the methods described in Section III and Examples 1-2 below.

[0094] In some embodiments, the method includes using an NRTP as a reporter, the NRTP comprising a reporter gene operably linked to an inducible promoter that controls expression of a target gene in a target cell, the reporter nucleic acid molecule allowing expression of the reporter gene via induction of the target gene promoter when the NRTP comprising the reporter gene is introduced into the target cell.

[0095] Transcripts As stated above, a transcript is a series of nucleotide sequences (DNA or RNA) transcribed from a DNA or RNA template sequence or gene. A transcript can be a cDNA sequence transcribed from an RNA template or an mRNA sequence transcribed from a DNA template. A transcript can be transcribed from an engineered nucleic acid construct. A transcript can have a region of complementarity within itself such that the transcript contains two regions capable of forming an intramolecular duplex. One region can be referred to as a "cis-repressing sequence" that binds to the reporter sequence and blocks translation of the reporter sequence. The second region of the transcript is referred to as a "reporter sequence" that codes for a reporter molecule such as a detectable or selectable marker.

[0096] The transcripts of the present invention can have transcript sequences that can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In other embodiments, the transcripts can be at least 25, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 1500, 2000, 3000, 4000, 5000, or more nucleotides in length. The cis repression sequence and the reporter sequence can be the same length or different lengths.

[0097] In some embodiments, the cis repression sequence is separated from the reporter sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 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, 55, 60 or more spacer nucleotides.

[0098] vector In another embodiment, the transcripts of the invention (including antisense and sense sequences) are expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A. et al., TIG. (1996), 12:5-10; Skillern, A. et al., International PCT Publication No. 00 / 22113, Conrad, International PCT Publication No. 00 / 22114, and Conrad, U.S. Patent No. 6,054,299). These sequences can be introduced as linear constructs, circular plasmids, or viral vectors, including bacteriophage-based vectors, and these sequences can be incorporated and inherited as transgenes integrated into the host genome. The transcript can also be constructed so that it can be inherited as an extrachromosomal plasmid (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

[0099] The transcript sequence can be transcribed by a promoter located on an expression plasmid. In one embodiment, the cis repression and reporter sequences are expressed as inverted repeats linked by a linker polynucleotide sequence such that the transcript has a stem and loop structure.

[0100] The transcripts of the present invention can be expressed using recombinant expression vectors. Recombinant expression vectors are generally DNA plasmids or viral vectors. The viral vectors expressing the transcripts can be constructed based on, but are not limited to, adeno-associated viruses (for review, see Muzyczka et al., Curr. Topics Micro. Immunol. (1992) 158:97-129), adenoviruses (see, for example, Berkner et al., BioTechniques (1998) 6:616), Rosenfeld et al., (1991, Science 252:431-434), and Rosenfeld et al., (1992), Cell 68:143-155), or alphaviruses, as well as viruses known in the art.Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and / or in vivo (see, e.g., Eglitis et al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA 87:6141-6145). 88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Natl. Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA (See, e.g., Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; WO 89 / 07136; WO 89 / 02468; WO 89 / 05345; and WO 92 / 07573.) Recombinant retroviral vectors capable of transducing genes into the genome of cells to express genes inserted into the genome can be generated by transfecting recombinant retroviral genomes into suitable packaging cell lines such as PA317 and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349).Recombinant adenovirus vectors can be used to infect a wide range of cells and tissues in susceptible hosts (e.g., rats, hamsters, dogs, and chimpanzees) (Hsu et al., 1992, J. Infectious Disease, 166:769), with the added advantage that they do not require mitotically active cells for infection.

[0101] Any viral vector capable of accepting the coding sequence of the transcript(s) to be expressed can be used, for example, vectors derived from adenovirus (AV); adeno-associated virus (AAV); retrovirus (e.g., lentivirus (LV), rhabdovirus, murine leukemia virus); herpes virus, and the like. The tropism of the viral vector can be modified by pseudotyping the vector with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins as needed.

[0102] For example, lentiviral vectors featured in the present invention can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors featured in the present invention can be targeted to different cells by engineering the vector to express different capsid protein serotypes. Techniques for constructing AAV vectors that express different capsid protein serotypes are within the skill of the art, see, for example, Rabinowitz JE et al. (2002), J Virol 76:791-801.

[0103] The selection of recombinant viral vectors suitable for use in the present invention, the method for inserting the nucleic acid sequence for expressing the transcript into the vector, and the method for delivering the viral vector to the cell of interest are within the scope of the art.See, for example, Dornburg R (1995), Gene Therap.2:301-310; Eglitis MA (1988), Biotechniques 6:608-614; Miller AD (1990), Hum Gene Therap.1:5-14; Anderson WF (1998), Nature 392:25-30; and Rubinson DA et al., Nat.Genet.33:401-406.

[0104] Viral vectors can be derived from AV and AAV. AV vectors suitable for expressing the transcripts featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Xia H et al. (2002), Nat. Biotech. 20:1006-1010. AAV vectors suitable for expressing the transcripts featured in the present invention, methods for constructing recombinant AV vectors, and methods for delivering the vectors into target cells are described in Samulski R et al., (1987), J. Virol. 61:3096-3101; Fisher KJ et al., (1996), J. Virol. 70:520-532; Samulski R et al., (1989), J. Virol. 63:3822-3826; U.S. Patent No. 5,252,479; U.S. Patent No. 5,139,941; WO 94 / 13788; and WO 93 / 24641.

[0105] The promoter driving transcript expression in the DNA plasmid or viral vector featured in the invention may be a eukaryotic RNA polymerase I (e.g., a ribosomal RNA promoter), an RNA polymerase II (e.g., a CMV early promoter or an actin promoter or an U1 snRNA promoter), or generally an RNA polymerase III promoter (e.g., a U6 snRNA or 7SK RNA promoter) or a prokaryotic promoter, e.g., a T7 promoter, provided that the expression plasmid also encodes the T7 RNA polymerase required for transcription from the T7 promoter. The promoter may also direct transgene expression to the pancreas (see, e.g., insulin regulatory sequences directed to the pancreas (Bucchini et al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).

[0106] Furthermore, the expression of the transcript can be precisely regulated by using inducible regulatory sequences and expression systems, such as regulatory sequences that are sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems are suitable for controlling transgene expression in cells or in mammals, and include regulation by ecdysone, estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D-1-thiogalactopyranoside (IPTG). Those skilled in the art will be able to select the appropriate regulatory / promoter sequence based on the intended use of the dsRNA transgene.

[0107] In general, recombinant vectors capable of expressing transcript molecules are delivered as described below and remain in target cells. Alternatively, viral vectors can be used that provide transient expression of transcript molecules. Such vectors can be administered repeatedly as necessary. Once expressed, transcripts bind to target RNA and regulate its function or expression. Transcript expression vectors can be delivered systemically, such as by intravenous or intramuscular administration, by administration to target cells explanted from the patient and then reintroduced into the patient, or by any other means that allows introduction into desired target cells.

[0108] Transcript expressing DNA plasmids are typically transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for dsRNA-mediated knockdown targeting different regions of a single PROC gene or multiple PROC genes over a period of a week or longer are also contemplated by the present invention. Successful introduction of the vector into the host cells can be monitored using a variety of known methods. For example, transient transfection can be signaled using a reporter such as a fluorescent marker, e.g., green fluorescent protein (GFP). Stable transfection of cells ex vivo can be confirmed using a marker that confers resistance to certain environmental factors (e.g., antibiotics and drugs) to the transfected cells, such as hygromycin B resistance.

[0109] Delivery of vectors containing recombinant DNA can be achieved by non-living or biological systems, including, but not limited to, liposomes, virus-like particles, phage- or virus-derived transducing particles, and conjugation.

[0110] Reporters for transcript assays In some embodiments, the nucleic acid construct comprises a reporter sequence (e.g., a reporter gene sequence). The reporter gene encodes a reporter molecule that produces a signal when expressed in a cell. In some embodiments, the reporter molecule can be a detectable marker or a selectable marker. In certain embodiments, the reporter molecule can be a fluorescent reporter molecule, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP), blue fluorescent protein (BFP), or red fluorescent protein (RFP). In other embodiments, the reporter molecule can be a chemiluminescent protein.

[0111] The reporter molecule can be a bacterial luciferase, a eukaryotic luciferase, a fluorescent protein, an enzyme suitable for colorimetric detection, a protein suitable for immunodetection, a peptide suitable for immunodetection or a nucleic acid that functions as an aptamer or exhibits enzymatic activity.

[0112] Selection markers can also be used as reporters and can be, for example, antibiotic resistance genes. EXAMPLES

[0113] Example 1: Disruption / complementation-based method to generate non-replicating transducing particles with non-native tail fibers A diagram of the tail fiber exchange platform is shown in Figure 1. Binding of NRTPs to their target bacterial cells is mediated by bacteriophage tail fibers. A "Bald" chassis was created to test the functionality of heterologous tail fibers by removing the native P1 NRTP tail fiber gene. The new tail fibers were then expressed in the "Bald" background using a tail fiber display vector (TFDV). The P1-based smarticle genome was modified using the λ-RED recombineering method from Murphy et al. (1998) to create deletions in the genes encoding the S-structure tail fiber protein and the U-tail fiber assembly chaperone protein. This disruption created a platform to express any desired tail fiber in trans. Bacterial strains were grown in Luria-Bertani (LB) broth and on LB agar with the following antibiotic concentrations: gentamicin 15 μg / mL, cabinicillin 100 μg / mL, spectinomycin 50 μg / mL, zeocin 100 μg / mL, and hygromycin 150 μg / mL. Plasmids were propagated and stored using New England Biolabs (NEB) cloning strain NEB5α. DNA assembly for plasmids as well as linear DNA products for recombineering were produced using NEB Gibson Assembly Master Mix. EMD Millipore KOD Xtreme Hot Start Polymerase was used for DNA amplification in all PCR reactions.

[0114] Example 2: Building the "Baldo" P1 chassis The λ-RED-encoding recombineering plasmid pKM208 was first transformed into the bacterial strain BAA-1001 (pacAB genes replaced with a hygromycin cassette) to facilitate the recombineering reaction. The strain was made electrocompetent and induced with 1 mM IPTG for expression of the recombinant protein. Linear DNA containing the antibiotic resistance marker spectinomycin was then transformed into the strain, which contains homology to the regions surrounding the s and u genes of P1. After recovery at 30°C, transformed cells were plated and patched onto LB agar containing spectinomycin. Colony PCR for removal of the s and u genes followed by Sanger sequencing was used to confirm that the desired modifications had been made. During the construction, u' and s v 'The area was left intact, but s v Since S' does not encode a complete structural protein, the S' tail fiber cannot be produced. This bacterial lysogen represented the "BALDO" P1 chassis, which lacked the ability to express its own functional tail fiber without complementation. The resulting bacterial strain containing the BALDO P1 chassis was named SEG_170 cell line. Functional testing of the chassis consisted of packaging plasmids with and without coding sequences for the native S and U proteins. Loss of smarticle functionality was seen from lysates derived from packaging strains without tail fibers on the packaging plasmid. Activity was restored from lysates derived from packaging strains with tail fibers on the packaging plasmid, indicating successful complementation in trans.

[0115] Example 3: Construction of tail fiber display vector (TFDV) Tail fiber display vectors (TFDV) were utilized to express native and / or novel tail fibers in the Bard bacteriophage P1 chassis. These display vectors utilized gentamicin or zeocin resistance cassettes, pUC / RO1600 origin of replication, and tail fiber genes under the control of the pTac IPTG inducible promoter. All vectors also contained pacAB genes / packaging sites along with a P23-luxAB-TT (luciferase) cassette for transduction and light assays. Tail fiber candidates were PCR amplified from bacterial gDNA or de novo synthesized in IDT, Gibson assembled into PCR amplified vectors, transformed into NEB cells, patched, colony PCR confirmed, and sequence confirmed. Sequence confirmed plasmids were transformed into the well-characterized SEG_170 Bard packaging strain (competent cells purchased from Lucigen). Transformed cells were patched onto selective media and screened in small lysates to determine the best smarticle producing clones. Top clones were carried forward for large-scale lysate production, QC, and reactivity analysis on a bacterial panel.

[0116] Example 4: Construction of genomic expression of tail fibers To facilitate optimal smarticle production with native and / or novel tail fibers, tail fiber and chaperone genes were integrated into the P1 smarticle genome at the native location of the s and u genes. This expression from the genome instead of in trans ensures proper expression and timing with the rest of the P1-based genome, reducing the burden from in trans expression. The tail fibers were first amplified by PCR using primers containing homology to a hygromycin cassette on the C-terminal portion of either the tail fiber structural gene (s gene) or the tail fiber chaperone gene (u gene). PCR was also used to amplify the hygromycin cassette. These two fragments were assembled by Gibson assembly and further amplified using primers containing homology to the P1 genome where s was previously found, as well as homology in the recombinase cin, which is immediately downstream of the native s and u genes (see Figure 2). This recombineering event served the dual function of inserting the tail fiber onto the P1 genome as well as disrupting the cin recombinase, which is responsible for switching the variable region of the s tail fiber gene. This linear DNA product of the tail fiber and hygromycin cassette was transformed into BAA-1001, which contains a spectinomycin resistance cassette. Transformation was followed by recovery and plating at 30°C. Cells were plated on LB agar containing hygromycin and patched for spectinomycin sensitivity. Promising colonies were examined using colony PCR and Sanger sequencing.

[0117] Positive colonies containing the non-native tail fiber and hygromycin resistance were then transformed with the packaging plasmid. Cells were grown overnight in medium containing antibiotics. Lysates were made using 90 min 42°C heat induction to inactivate the P1 master repressor and promote smarticle production. The lysates were then spun down to pellet the bacterial cell debris and then filter sterilized through a 2 μm filter. The lysates were examined for proper smarticle functionality in the new host similar to the method for the TFDV packaging strains described above.

[0118] To construct packaging strains expressing two or more tail fibers from a single smarticle genome, a similar recombineering approach was used as described above. The first tail fiber was similarly positioned on the P1 genome, but instead of the tail fiber being linked to hygromycin resistance, the tail fiber was attached to an alternative antibiotic such as kanamycin or gentamicin. The cin recombinase was also left intact to facilitate the switching of multiple tail fibers. A second recombineering reaction was performed to position the second variable portion of the tail fiber attached to the hygromycin cassette for exchange with the kanamycin (or gentamicin) cassette already on the genome. This could be repeated to add three, four, five, etc. tail fibers to the genome (see Figure 6).

[0119] Example 5: NRTP Assay Conditions The assay involved an initial 2.5 hour pretreatment of bacterial cells at a concentration of 5.0E+05 CFU / mL in assay medium (10 g / L tryptone + 5 g / L yeast extract + 5% PEG8000). Following the pretreatment step, both NRTP and transduction salts (1 M MgCl2 + 0.5 M CaCl2) were added to the reaction and incubated for 2 hours, which allowed for transduction of a reporter molecule containing the luciferase gene, luxAB. Light production from the bacteria was measured by relative light unit (RLU) output using a luminometer.

[0120] Example 6: Performance of NRTP with engineered tail fibers The improved reactivity of the engineered NRTPs against various species and strains can be seen in Table 1. These data clearly demonstrate: 1) the diversity of tail fibers analyzed, 2) the robust altered reactivity observed with different tail fiber protein binding domains, 3) that some tail fibers are highly specific to certain species, and 4) that some tail fibers are highly reactive to many diverse species of bacteria. Both the nucleotide and amino acid sequences of the tail fibers from these engineered NRTPs (e.g., "Sunset," "Indian," "Tangerine," etc.) are listed in the Sequence Listing.

[0121] [Table 1-1]

[0122] [Table 1-2]

[0123] Figure 3 diagrammatically depicts how the BALDO (tail fiber deficient) P1 lysogen was altered to express new tail fibers from the genome. Figure 4 depicts how genomic integration of a given tail fiber can improve performance compared to trans expression from TFDV. Photoactivity is shown for three separate tail fibers on three indicator strains for both TFDV and the genomically expressed version. Additionally, the transduction efficiency of the TFDV and genomic S tail fiber NRTP was also compared to the WT P1 NRTP control (V3).

[0124] Figure 5A depicts a graph showing the improved tail fiber activity as light produced (RLU) of the engineered "Indian" NRTP in a packaging strain expressing the plasmid p15B U protein "t chaperone" compared to a packaging strain not expressing this protein. Figure 5B depicts a graph showing the improved performance of the "Scarlet" NRTP produced from the bacteriophage genome compared to the "Scarlet" NRTP produced from a separate packaging plasmid (TFDV).

[0125] Figure 6A displays the genomic arrangement of production strains with zero, one, two, or three separate tail fiber genes, illustrating how lysogens can be engineered to produce multiple NRTPs from a single fermentation. Tail fiber genes can be positioned with cix crossover sites in novel locations. Genes can also be positioned for recombination-independent expression in large or small operons. Figure 6B depicts indicator strains that are specifically detected by individual tail fibers with a given NRTP (left). The right panel shows that packaging strains expressing multiple tail fibers can detect multiple indicator strains, demonstrating that multiple functional NRTPs with differing specificities can be produced at once.

[0126] Figure 7 compares the performance of NRTPs from a bifunctional (two tail fibers) expression strain to individual NRTPs produced in separate production runs. The top image shows the genomic arrangement of the bifunctional tail fiber regions from jazzberry and shamrock tail fibers. The middle left compares the bifunctional NRTPs to the individually produced NRTPs on indicator strains specific for different tail fibers. The middle right shows how detection and light production was improved on a large panel of E. coli strains by creating a bifunctional production strain in addition to mixing it in a cocktail with other NRTPs (such as the native P1 NRTP called V3). The bottom of Figure 7 directly compares the performance of the bifunctional produced NRTP to the cumulative performance of the individual tail fibers produced separately.

[0127] FIG. 8 shows the results of experiments performed to identify the elements necessary to render the new tail fiber functional on a viral chassis. Panel A shows that the P1r gene is required and that the tail fiber variable region (S v ) should pair with its cognate u homolog accessory protein. Furthermore, panel A shows that three distinct tail fiber conserved regions could enable the functionalization of non-native tail fibers on new chassis. Panel B shows an alignment of the amino acid sequences of three Sc regions from P1 bacteriophage, Kpn type 11 bacteriophage, and p15B plasmid (related to P1), highlighting a highly conserved N-terminal 150 amino acid sequence motif.

[0128] FIG. 9 displays various configurations for expression of tail fiber genes from fermentation strains. A) shows expression of tail fibers and viral chassis from a phage genome. B) shows viral chassis expression from a phage genome and tail fiber genes from a packaging plasmid. C) shows viral chassis from a phage genome and tail fibers from a separate plasmid. D) shows viral chassis from a phage genome and tail fibers from a bacterial genome. E) shows viral chassis from a phage genome and one set of tail fibers. A second unique set of tail fibers is expressed from a separate plasmid. F) shows viral chassis and tail fibers from a phage genome. A second unique set of tail fibers is expressed from a packaging plasmid. G) shows viral chassis from a phage genome and two unique sets of tail fibers.

[0129] While this disclosure has been particularly illustrated and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein, the true scope of the invention being set forth in the appended claims. [Sequence List Free Text]

[0130] Description of the sequences listed in the sequence listing Nucleotide sequence of tail fibers functionally expressed on the P1 chassis Asparagus tail fiber nucleotide sequence SEQ ID NO:1 Banana tail fiber nucleotide sequence SEQ ID NO:2 Bittersweet tail fiber nucleotide sequence SEQ ID NO:3 Cobalt tail fiber nucleotide sequence SEQ ID NO:4 Cornflower tail fiber nucleotide sequence SEQ ID NO:5 Denim tail fiber nucleotide sequence SEQ ID NO:6 Eggplant tail fiber nucleotide sequence SEQ ID NO:7 Fern tail fiber nucleotide sequence SEQ ID NO:8 Fuchsia tail fiber nucleotide sequence SEQ ID NO:9 Fuzzy tail fiber nucleotide sequence SEQ ID NO:10 Inchworm tail fiber nucleotide sequence SEQ ID NO:11 Indian tail fiber nucleotide sequence SEQ ID NO:12 Indigo tail fiber nucleotide sequence SEQ ID NO:13 Jazzberry tail fiber nucleotide sequence SEQ ID NO:14 Jazzberry-Shamrock double tail fiber nucleotide sequence SEQ ID NO:15 Jungle tail fiber nucleotide sequence SEQ ID NO:16 Mango tail fiber nucleotide sequence SEQ ID NO:17 Maroon tail fiber nucleotide sequence SEQ ID NO:18 Mulberry tail fiber nucleotide sequence SEQ ID NO:19 P1_S' gene nucleotide sequence SEQ ID NO:20 P1S-P1S'-Shamrock double tail fiber nucleotide sequence SEQ ID NO:21 Pine tail fiber nucleotide sequence SEQ ID NO:22 Plum tail fiber nucleotide sequence SEQ ID NO:23 Queen tail fiber nucleotide sequence SEQ ID NO:24 Razzmatazz tail fiber nucleotide sequence SEQ ID NO:25 Salmon tail fiber nucleotide sequence SEQ ID NO:26 Scarlet tail fiber nucleotide sequence SEQ ID NO:27 Shamrock tail fiber nucleotide sequence SEQ ID NO:28 Sunset tail fiber nucleotide sequence SEQ ID NO:29 Tangerine tail fiber nucleotide sequence SEQ ID NO:30 Thistle tail fiber nucleotide sequence SEQ ID NO:31 Tropical_pNS88 tail fiber nucleotide sequence SEQ ID NO:32 Tropical_pNS92 tail fiber nucleotide sequence SEQ ID NO:33 Violet tail fiber nucleotide sequence SEQ ID NO:34 Amino acid sequence of tail fibers functionally expressed on the P1 chassis Asparagus - P1Sc - (P1Sv-MuS hybrid) translation SEQ ID NO: 35 banana - P1Sc(aa1-114)-Ecl112(aa218-endo) translation SEQ ID NO:36 Bittersweet - p15b Sc - p15b SvQ Translation SEQ ID NO: 37 Cobalt_-_p15bSc-Kpn_SCKP_TF4_translation SEQ ID NO:38 Cornflower - P1Sc(aa1-208)-Kpn478 tail fiber (aa131-499) translation SEQ ID NO:39 denim_-_p15bSc-Kpn_SCKP_TF1_translation SEQ ID NO:40 Eggplant_-_D6Sc-D6Sv'_translation SEQ ID NO:41 Fern_-_P1Sc-(P1Sv-D108S_hybrid)_translation SEQ ID NO:42 Fuchsia_-_P1Sc-D6Sv_translation SEQ ID NO:43 Fuzzy_-_P1Sc(aa1-208)-Kpn051_TF(aa40-382)_translation SEQ ID NO:44 Inchworm_-_P1Sc-R2_pyocin_translation SEQ ID NO:45 Indian_-_p15bSc-p15bSvN_translation SEQ ID NO:46 Indigo_-_p15bSc-Kpn_SCKP_TF3_translation SEQ ID NO:47 Jazzberry_-_P1Sc-KpnSv_Translation SEQ ID NO:48 Jungle_-_P1Sc-(P1Sv'-MuS'_hybrid)_translation SEQ ID NO:49 Mango - P1Sc(aa1-214)-Ecl117B(aa214-764) translation SEQ ID NO:50 Maroon_-_p15bSc-p15bSvP_translation SEQ ID NO:51 Mulberry_-_D6Sc-D6Sv_translation SEQ ID NO:52 P1S'-P1Sc-P1Sv' translation SEQ ID NO:53 Pine_-_P1Sc(nt1-987)-P2H_translation SEQ ID NO:54 Plum_-_KpnSc-KpnSv_translation SEQ ID NO:55 Queen_-_S-Queen_Sv_Translation SEQ ID NO:56 Razzmatazz - P1Sc-P7Sv translation SEQ ID NO:57 Salmon_-_P1Sc-P7Sv'_translation SEQ ID NO:58 Scarlett_-_p15bSc-p15bSvI_translation SEQ ID NO:59 Shamrock_-_P1Sc-(P1S-SfMuS'_hybrid)_translation SEQ ID NO:60 Sunset_-_p15bSc-p15bSvR_translation SEQ ID NO:61 Tangerine_-_p15bSc-p15bSv0_translation SEQ ID NO:62 Thistle_-_P1Sc-KpnSv_translation SEQ ID NO:63 Tropical_pNS88_-_P1Sc(aa1-214)-Ecl117A(aa91-437)_translation SEQ ID NO:64 Tropical_pNS92_-_P1Sc(aa1-214)-Ecl117A(aa96-437)_translation SEQ ID NO:65 Violet_-_RCS47Sc-RCS47Sv_Translation SEQ ID NO:66 Chaperone protein amino acid sequence Asparagus - MuU translation SEQ ID NO:67 banana_-_Ecl112_U_chaperone_translation SEQ ID NO:68 Bittersweet - p15b T Translation SEQ ID NO:69 Cobalt_-_p15b_T_translation SEQ ID NO:70 Denim_-_p15b_T_Translation SEQ ID NO:71 Eggplant_-_P1_U_translation SEQ ID NO:72 Fern_-_D108_U_translation SEQ ID NO:73 Fuchsia_-_P1_U'_translation sequence number 74 Fuzzy_-_PROKKA_00043_(Kpn478_chaperone)_translation SEQ ID NO:75 Inchworm_-_PA0621_Translation SEQ ID NO:76 Indian_-_p15b_T_Translation SEQ ID NO:77 Indigo_-_p15b_T_translation SEQ ID NO:78 Jazzberry - Kpn U Translation SEQ ID NO:79 Jungle_-_Mu_U'_Translation sequence number 80 Mango_-_Ecl117B_U_chaperone_translation SEQ ID NO:81 Maroon_-_p15_T_translation SEQ ID NO:82 Mulberry_-_P1_U'_translation SEQ ID NO:83 P1_S'-P1_U' translation sequence number 84 Pine_-_P2_G_translation SEQ ID NO:85 Plum_-_Kpn_U_Translation SEQ ID NO:86 Queen_-_Queen_U_Chaperone_Translation sequence number 87 Razzmatazz - P7 Ua Translation SEQ ID NO:88 Razzmatazz - P7 Ub translation sequence number 89 Salmon_-_P7_U'_translation sequence number 90 Scarlett_-_p15b_T_translation SEQ ID NO:91 Shamrock_-_SfMuU'_translation sequence number 92 Sunset_-_p15b_T_translation SEQ ID NO:93 Tangerine_-_p15_T_translation SEQ ID NO:94 Azami_-_KpnU_translation SEQ ID NO:95 Violet_-_RCS47_U_Translation sequence number 96 Other Arrays Bold_chassis_tail_fiber_region_P1_s-u::aadA SEQ ID NO:97 Rio tail fiber_s_hybrid_nucleotide sequence SEQ ID NO:98 Rio chaperone_u_nucleotide_sequence SEQ ID NO:99 Limon tail fiber_s_hybrid_nucleotide sequence SEQ ID NO:100 Limon chaperone_u_nucleotide_sequence SEQ ID NO:101 Okido tail fiber_s_hybrid_nucleotide sequence SEQ ID NO:102 Orchid Chaperone_u_Nucleotide sequence SEQ ID NO:103 Gold tail fiber s hybrid nucleotide sequence SEQ ID NO:104 Gold Chaperone_u_Nucleotide sequence SEQ ID NO:105 Bluetiful tail fiber_s_hybrid_nucleotide sequence SEQ ID NO:106 Bruteful chaperone_u_nucleotide sequence SEQ ID NO:107 Rio tail fiber S hybrid amino acid sequence SEQ ID NO:108 Limon tail fiber S hybrid amino acid sequence SEQ ID NO:109 Orchid tail fiber S hybrid amino acid sequence SEQ ID NO:110 Gold tail fiber S hybrid amino acid sequence SEQ ID NO:111 Bluetiful tail fiber S hybrid amino acid sequence SEQ ID NO:112 Rio Chaperone_U_amino acid sequence SEQ ID NO:113 Limon Chaperone_U_amino acid sequence SEQ ID NO:114 Orchid Chaperone_U_amino acid sequence SEQ ID NO:115 Gold Chaperone_U_amino acid sequence SEQ ID NO:116 Bruteful Chaperone_U_amino acid sequence SEQ ID NO:117

Claims

1. 1. A bacteriophage tail fiber exchange platform comprising: - a bacteriophage lysogen from the family Myoviridae obtained from the genus Punalikevirus (Myoviridae lysogen) containing a gene disruption that prevents expression of one or more genes essential for the production of native tail fibers, disruption of a gene encoding a chaperone protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-96 and 113-117, the disruption being necessary for folding of one or more regions of a tail fiber structural protein; and a lysogen comprising a disruption of one or more genes selected from a gene encoding a tail fiber structural protein involved in binding to a bacterial cell receptor, a gene encoding a protein required for attaching the tail fiber structural protein to the tail, and any combination of these genes; and - a complementary nucleic acid molecule, the complementary nucleic acid molecule encoding a chaperone protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 67-96 and 113-117, which is required for folding of one or more regions of the tail fiber structural protein; and one or more genes selected from a gene encoding a tail fiber structural protein involved in binding to a bacterial cell receptor, a gene encoding a protein required for attaching the tail fiber structural protein to the tail, and any combination of these genes; Complementary nucleic acid molecules, which complement a gene disruption of a Myoviridae lysogen, thereby producing a functional tail fiber. It consists of: A bacteriophage tail fiber exchange platform, wherein one or more genes or a combination of genes on said complementary nucleic acid molecule contain a region not derived from one or more genes essential for the production of native tail fibers of Myoviridae lysogens, such that the functional tail fiber produced is not a native tail fiber.

2. 2. The platform of claim 1, wherein the Myoviridae lysogen is Enterobacteria bacteriophage phage P1 (P1 bacteriophage).

3. 3. The platform of claim 2, wherein one or more genes or combinations of genes contain regions that are not derived from P1 bacteriophage, such that the functional tail fiber produced is not a native P1 bacteriophage tail fiber.

4. the complementary nucleic acid molecule is a plasmid molecule; or a complementary nucleic acid molecule is integrated into the site of gene disruption of the Myoviridae lysogen; or a complementary nucleic acid molecule is integrated at a site other than the site of gene disruption in the Myoviridae lysogen; or The platform of claim 1 , wherein the complementary nucleic acid molecule is integrated into the genome of a host bacterial cell containing a Myoviridae lysogen.

5. 5. The platform of any one of claims 1 to 4, wherein the Myoviridae lysogen contains an additional disruption in one or more packaging genes involved in packaging the genome of the Myoviridae lysogen.

6. the complementary nucleic acid molecule is a plasmid molecule, and one or more packaging genes that complement a disruption in one or more packaging genes in a Myoviridae lysogen are contained within said plasmid molecule; or 6. The platform of claim 5, wherein the complementary nucleic acid molecule is a plasmid molecule and one or more packaging genes that complement a disruption in one or more packaging genes in a Myoviridae lysogen are contained in a reporter or therapeutic expression plasmid separate from the plasmid molecule.

7. 1. A method for producing bacteriophage particles having a non-native tail fiber, comprising: - providing a bacterial cell containing a bacteriophage tail fiber exchange platform according to any one of claims 1 to 6, wherein the complementary nucleic acid molecule comprises a gene encoding a chaperone protein and a gene encoding a tail fiber structural protein having one or more regions not derived from one or more genes essential for the production of native tail fibers of Myoviridae lysogens; and - providing conditions in the bacterial cells that induce the lytic phase of Myoviridae lysogens to produce bacteriophage particles having non-native tail fibers; A method comprising:

8. 8. The method of claim 7, wherein the one or more regions not derived from one or more genes native to the Myoviridae lysogen comprise a variable region of a tail fiber structural protein (tail fiber variable region) obtained from a bacterial genome encoding a phage tail-like particle or structure or from a nucleic acid delivery particle.

9. 1. A method for producing non-replicating transducing particles (NRTPs) having non-native tail fibers, comprising: providing a bacterial cell containing the bacteriophage tail fiber exchange platform of any one of claims 1 to 6, wherein the Myoviridae lysogen comprises an additional disruption in one or more packaging genes involved in packaging of the Myoviridae lysogen genome, and the complementary nucleic acid molecule comprises a gene encoding a chaperone protein and a gene encoding a tail fiber structural protein that contains one or more regions not derived from the one or more genes essential for the production of the native tail fiber of the Myoviridae lysogen, and the bacterial cell further comprises a reporter plasmid comprising the one or more packaging genes that have been disrupted in the Myoviridae lysogen; and - providing conditions to the bacterial cells that induce the lytic phase of the Myoviridae lysogen to produce NRTPs having non-native tail fibers; A method comprising:

10. 9. The method of claim 8, wherein the one or more regions not derived from one or more genes native to a Myoviridae lysogen comprise a variable region of a tail fiber structural protein (tail fiber variable region) obtained from a bacterial genome encoding a non-replicating virus-derived structure or from a nucleic acid delivery particle.

11. 1. A method for producing an engineered bacteriophage or non-replicating transducing particle (NRTP) that exhibits a bacterial host cell specificity that differs from that exhibited by naturally occurring bacteriophages, comprising: - fusing a first nucleotide sequence from one tail fiber structural protein-encoding gene with a second nucleotide sequence from a second tail fiber structural protein-encoding gene from a different source to create a chimeric tail fiber structural protein-encoding gene; - providing a chimeric tail fiber structural protein encoding gene as a complementary nucleic acid molecule in a bacterial cell; - expressing a chimeric tail fiber structural protein gene in a bacterial cell containing a bacteriophage tail fiber exchange platform according to any one of claims 1 to 6; and - providing conditions to bacterial cells that induce the lytic phase of Myoviridae lysogens to produce engineered bacteriophages or NRTPs; A method comprising:

12. 1. A method of making an engineered bacteriophage or non-replicating transducing particle (NRTP) having multiple types of tail fibers derived from a single bacterial cell line, wherein at least one of the tail fiber types is a non-native tail fiber, comprising: - providing a bacterial cell containing a bacteriophage tail fiber exchange platform according to any one of claims 1 to 6, wherein the complementary nucleic acid molecule further comprises at least two receptor binding regions from a tail fiber structural protein encoding gene fused to one or more tail interacting regions from a tail fiber structural protein encoding gene, wherein at least one of the receptor binding regions is from a source different from the source of one of the tail interacting regions; and - providing conditions to the bacterial cells that induce a lytic phase of lysogenesis to produce bacteriophages or NRTPs with multiple types of tail fibers; The method includes:

13. 13. The method of claim 12, wherein the complementary nucleic acid molecule is integrated into the site of the Myoviridae lysogen gene disruption; or the complementary nucleic acid molecule is integrated into a site other than the site of the Myoviridae lysogen gene disruption; or the complementary nucleic acid molecule is integrated into the genome of a bacterial cell; or the complementary nucleic acid molecule is integrated into a plasmid molecule.

14. Fusion of two or more receptor binding regions to the tail interacting region is performed by cin recombinase in the presence of multiple cix recombination sites; or The method of claim 12 or 13, wherein the fusion of two or more receptor binding regions to the tail interacting region is performed in the presence of multiple associated recombination sites by cin recombinase or a homolog, ortholog, or paralog of cin recombinase.

15. 1. A method for producing a Myoviridae bacteriophage or a Myoviridae-derived non-replicating transducing particle (NRTP) capable of recognizing and transducing a bacterial cell for diagnostic or therapeutic use, comprising: providing a bacterial cell containing a bacteriophage tail fiber exchange platform according to any one of claims 1 to 6, wherein the complementary nucleic acid molecule comprises a gene encoding a chaperone protein and a tail fiber gene not native to Myoviridae lysogens and derived in part or in whole from a Myoviridae, Siphoviridae, or Podoviridae tail fiber sequence; and - providing conditions that induce a lytic phase of lysogenesis in bacterial cells to produce Myoviridae bacteriophages or Myoviridae-derived NRTPs that are capable of recognizing and transducing bacteria previously resistant to natural Myoviridae bacteriophages or Myoviridae-derived NRTP particles for diagnostic or therapeutic use; A method comprising:

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