phage vector

Abstract

The present invention relates to phage vectors, and in particular to novel phage vectors containing transgenes, particularly conventional mammalian transgene cassettes. The invention extends to the use of such phage vectors as research tools, as well as for the delivery of transgenes in various gene therapy applications, DNA and / or peptide vaccine delivery, and imaging techniques.

Description

[Technical Field]

[0001] The present invention relates to phage vectors, and in particular, but not exclusively, to novel phage vectors containing transgenes, especially conventional mammalian transgene cassettes. The invention extends to the use of such phage vectors as research tools, as well as for the delivery of transgenes in various gene therapy applications, DNA and / or peptide vaccine delivery, and imaging techniques. [Background technology]

[0002] Bacteriophages (phages) continue to emerge as safe vectors for targeted transgene delivery because, although they lack inherent tropism for mammalian cell receptors, they can be modified to display tissue-specific ligands on their coat proteins to enable cell entry without disruption of viral structure (1-6). However, despite their clear advantages over eukaryotic viruses, tissue-targeted phage vectors have shown limited efficacy because bacteriophages have evolved to infect only bacteria and lack optimized strategies for transgene expression upon entry into eukaryotic cells (2).

[0003] Our previous work demonstrated that the efficacy of gene transfer by filamentous M13 phage is amenable to evolution, and one efficient strategy is to combine bacteriophage with the attributes of animal viruses. Importantly, over the past few years, we have designed various strategies to enhance gene delivery by filamentous M13 phage-derived vectors. Indeed, as with other viral vectors, successful gene delivery mediated by M13 phage vectors requires (i) effective diffusion through the extracellular matrix (ECM) to access the cell surface, (ii) binding to its cell surface receptors to enable cellular uptake, (iii) endosomal escape, and (iv) nuclear translocation to initiate gene expression. It is clear that phages have evolved to infect only bacteria and do not have optimized strategies for undergoing these steps to express transgenes in mammalian cells. Over the past few years, we have designed various approaches to overcome the above limitations, such as reducing the size of M13 phage particles to enhance diffusion through the ECM (7, 8) and incorporating an endosomal escape peptide onto the recombinant rpVIII major coat protein to enhance phage escape from the endosomal / lysosomal degradation pathway (9, 10). Furthermore, to improve gene expression in the nucleus, we previously flanked a mammalian transgene cassette with inverted terminal repeats (ITRs) derived from adeno-associated virus (AAV2), resulting in enhanced gene delivery efficiency of bacteriophage (6). Furthermore, to enhance therapeutic gene transcription from vectors in the nuclei of cancer cells, we replaced the cytomegalovirus (CMV) promoter with the tumor-activated and chemotherapy-inducible promoter of the glucose-regulated protein Grp78 (11, 12). We also combined anticancer drugs with M13 phage vectors to increase the nuclear translocation of phages in cancer cells (13).

[0004] However, in contrast to conventional viral vectors, the filamentous M13 phage requires further conversion of its single-stranded DNA (ssDNA) genome to a double-stranded DNA (dsDNA) form in order to be properly recognized by the cellular transcription machinery (14). Although the ability of M13 phage to reach the cell nucleus can be successfully addressed, the conversion of the single-stranded (ss) genome to a double-stranded (ds) genome remains a significant problem to be solved. In mammalian cells, the conversion of M13 phage from ssDNA to dsDNA depends on cellular factors, and this is a process with very low efficiency, which limits transduction efficiency (15).

[0005] It is clear that the conversion of M13 phage to dsDNA has long been a major challenge for M13 phage-mediated gene delivery into mammalian cells. The need for complementary strand synthesis or recruitment is now believed to be the rate-limiting factor in the efficiency of M13 phage vectors. In an attempt to circumvent this limitation, the conversion of ssDNA to dsDNA was promoted by genotoxic treatment of human cell lines previously incubated with phagemid particles (15). Unfortunately, such treatments are inappropriate for application to living organisms.

[0006] In addition to the above-mentioned problems associated with filamentous bacteriophages such as M13 phage, there is also a significant problem with the rate-limiting step of converting ssDNA to dsDNA in AAV vectors. Furthermore, to generate dsAAV DNA upon cell transduction, the AAV capsid can carry two self-complementary ssDNA sequences, each containing a transgene cassette, but the maximum size of each ssDNA cannot exceed 2.3 kb. Therefore, to generate dsDNA AAV in transduced cells for use in gene therapy applications, packaging cassettes (i.e., large genomes) even slightly larger than 2.3 kb is problematic. [Prior art documents] [Non-patent literature]

[0007]

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[0008] Therefore, there is a need to provide novel phage vectors for the delivery of transgene cassettes into, for example, mammalian cells. [Means for solving the problem]

[0009] Rather than relying on potentially variable cellular mechanisms to provide the complementary strand for a single-stranded genomic phage vector, the present inventors have surprisingly found that this problem can be circumvented by designing a single phage vector that carries the complementary sequence of the transgene expression cassette.

[0010] Thus, according to a first aspect of the present invention there is provided a phage vector comprising at least two single-stranded self-complementary transgene expression cassettes separated by a linker which hybridize to form a double-stranded transgene expression cassette.

[0011] As discussed in the Examples, the inventors designed a phage vector carrying a complementary sequence of a transgene expression cassette that hybridizes to generate a double-stranded transgene expression cassette. Advantageously, the phage vector of the present invention overcomes the problems associated with single-stranded (ss) to double-stranded (ds) DNA conversion in filamentous phage vectors (such as M13) and the problems associated with converting ss to ds DNA in AAV vectors. The present invention also overcomes the problem of packaging large genomes for the generation (production) of double-stranded AAV vectors. To demonstrate that the phage vectors of the present invention provide better gene delivery than the prior art, the inventors used reporter genes such as GFP and luciferase. The inventors then used the cytokine TRAIL to confirm their findings and further demonstrated that the vectors of the present invention function surprisingly well for gene delivery. When a gene encoding a cytokine such as TRAIL was used in the expression cassette, the inventors also demonstrated cancer cell death, indicating that the phage vectors of the present invention can be used to effectively deliver therapeutic genes.

[0012] Thus, the phage vector of the present invention may be a filamentous phage vector such as M13, or may be a hybrid vector of AAV DNA and a filamentous phage capsid.

[0013] The present inventors conducted several in vitro experiments using various cell lines and transgenes and surprisingly observed increased transduction efficiency (3-15-fold) from the phage vectors of the present invention compared with conventional single-stranded DNA phage vectors. Indeed, advantageously, the phage vectors of the present invention exhibited rapid initiation and higher levels of transgene expression in all of the cell lines tested. More importantly, unlike conventional single-stranded phage vectors, inhibitors of DNA replication did not affect transduction from the phage vectors of the present invention. In addition, as discussed in the Examples, in vivo studies demonstrated significantly enhanced gene delivery to solid tumors in mice upon systemic administration of the phage vectors of the present invention compared with conventional single-stranded DNA phage particles. All of these biological attributes support the generation and characterization of a new class of filamentous phage vectors capable of delivering double-stranded DNA, which will contribute significantly to the ongoing development of phage-based gene delivery systems.

[0014] Because circular phage genomes can affect the process of double-stranded DNA formation, the present inventors used phagemids, such as those described in International Publication No. WO 2017 / 077275, the entire contents of which are incorporated by reference. This involved removing the phage genome and retaining only the replication origin f1, allowing for replication and packaging of the transgene cassette in bacteria. A phagemid is defined as a plasmid DNA containing a phage replication origin, hence the name phagemid. Herein, the present inventors used phagemids as a DNA backbone to design a new phage genome carrying two transgene cassettes. The resulting double-stranded vector is a phage particle.

[0015] Thus, preferably, the phage vector is a hybrid phagemid genome encapsulated by a phage-derived coat protein. A hybrid phagemid genome may also be referred to as a "phagemid genome" (i.e., a genetic construct containing two origins of replication, one derived from a bacteriophage (e.g., F1) and one derived from a bacterium (e.g., pUC1)).

[0016] Preferably, the genome of the phage vector comprises a packaging signal to enable replication of at least two single-stranded self-complementary transgene expression cassettes, which can hybridize in bacteria and then be packaged as a double-stranded transgene expression cassette in the phage vector within a prokaryotic host. The packaging signal may preferably comprise a bacteriophage origin of replication. For example, the origin of replication preferably comprises an F1 ori, more preferably derived from an F1 bacteriophage. The DNA sequence of one embodiment of an F1 ori is represented herein as SEQ ID NO: 1, as follows: [ka]

[0017] Preferably, the genome of the phage vector comprises an origin of replication to allow replication of at least two single-stranded self-complementary transgene expression cassettes within a prokaryotic host. Preferably, the origin of replication allows high copy number replication of the vector within the host. Preferably, the origin of replication comprises a bacterial origin of replication. Preferably, the origin of replication comprises a pUC ori (for molecular cloning). The DNA sequence of one embodiment of pUC ori is represented herein as SEQ ID NO: 2, as follows: [ka]

[0018] Alternatively, in another embodiment, the phage vector may be designed to integrate into the genome of the host cell. In this case, nucleic acid sequences that favor targeted integration of the vector's genome (e.g., by homologous recombination) are envisioned. Thus, the genome of the phage vector may contain one or more DNA sequences that allow targeted integration into the host genome.

[0019] In one embodiment, the phage vector may be used as an experimental research tool and may be used ex vivo or in vitro.

[0020] In another embodiment, the phage vector may be used for the delivery of at least two self-complementary transgene expression cassettes to tissue-specific targets, whether the vector is administered systemically or locally to a subject in vivo, applied to a mixture of cells in vitro, or applied to an organ ex vivo. Preferably, the at least two self-complementary transgene expression cassettes comprise viral transgene expression cassettes. More preferably, the at least two self-complementary transgene expression cassettes comprise mammalian viral transgene expression cassettes. For example, in one preferred embodiment, the at least two self-complementary transgene expression cassettes may comprise lentiviral transgene expression cassettes. The at least two self-complementary transgene expression cassettes are preferably adeno-associated viral (AAV) transgene expression cassettes.

[0021] The at least two self-complementary transgene expression cassettes may comprise any nucleic acid encoding an agent that may have therapeutic or industrial utility in a target cell or tissue. In one embodiment of the invention, the nucleic acid may be DNA, which may be genomic DNA or cDNA. In some embodiments, non-naturally occurring cDNA may be preferred. In another embodiment, the nucleic acid may be RNA, such as antisense RNA or shRNA.

[0022] The drug encoded by the nucleic acid may be a polypeptide or a protein. For example, in an embodiment in which the phage vector of the first aspect is used to treat cancer, the transgene may encode a herpes simplex virus thymidine kinase gene, which may then exert a therapeutic effect on target tumor cells. The transgene may also encode a cytokine, such as TRAIL. The vector may be used to treat any cancer, such as bone cancer.

[0023] However, it is understood that the type of cell targeted by a phage vector depends on the type of cell-targeting ligand expressed on the surface of the vector. For example, the cell-targeting ligand may include RGD, such as RGD4C.

[0024] The at least two transgene expression cassettes may comprise one or more functional elements required for the expression of nucleic acid in target cells.For example, preferably, the at least two transgene expression cassettes each comprise a promoter for driving the expression of transgene.Suitable promoters may be CMV promoters.The DNA sequence of one embodiment of CMV promoter is represented herein as SEQ ID NO:3, as follows: [ka]

[0025] In another preferred embodiment, the at least two transgene expression cassettes each comprise a grp78 promoter. The nucleic acid sequence of one embodiment of the grp78 promoter is represented herein as SEQ ID NO: 4, as follows: [ka]

[0026] Alternatively, in another preferred embodiment, each of the at least two transgene expression cassettes comprises a tumor-specific or tissue-specific promoter. Tissue-specific promoters can be used to target transcription and gene expression, and the phage vector displays a ligand for delivery to these specific tissues.

[0027] Preferably, at least two transgene expression cassettes each comprise a nucleic acid for a polyA tail. Preferably, the polyA tail is located at the end of the transgene cassette, i.e., at the 5' or 3' end of the transgene cassette. The DNA sequence of one embodiment of a nucleic acid for encoding a polyA tail is represented herein as SEQ ID NO: 5, as follows: [ka]

[0028] Thus, in a preferred embodiment, each of the at least two single-stranded self-complementary transgene expression cassettes comprises a promoter (preferably CMV), a nucleic acid encoding an agent (eg, a therapeutic agent), and a polyA tail.

[0029] Preferably, the phage vector comprises at least two single-stranded, self-complementary transgene expression cassettes separated by linkers that hybridize to form a double-stranded transgene expression cassette. Alternatively, the phage vector may comprise four single-stranded, complementary transgene expression cassettes separated by linkers (i.e., two pairs of self-complementary cassettes) that hybridize to form two double-stranded transgene expression cassettes.

[0030] As shown in Figure 2, for single-stranded, self-complementary transgene expression cassettes to hybridize to each other, they must be located in opposite orientations in the phage vector, i.e., a first cassette extends in the 5' to 3' direction and a corresponding second cassette extends in the 3' to 5' direction. It will be understood that these cassettes are substantially identical in terms of their sequence but extend in opposite or antiparallel directions on either side of the linker separating them. Thus, in a preferred embodiment, two single-stranded, self-complementary transgene expression cassettes are located in opposite orientations in the phage vector.

[0031] It will be understood that for at least two single-stranded, self-complementary transgene expression cassettes to successfully hybridize to form a double-stranded transgene expression cassette, their sequences, although running in opposite directions, should be similar, if not identical, to each other. However, it is not essential that the sequences be identical; hybridization will occur if there is sufficient sequence identity along a sufficient length of each cassette.

[0032] The percentage sequence identity between the first cassette and the second cassette may be at least 65%, 70%, or 75%. Preferably, the percentage sequence identity between the first cassette and the second cassette is at least 80%, 85%, or 90%. Preferably, the percentage sequence identity between the first cassette and the second cassette is at least 92%, 94%, or 95%. Preferably, the percentage sequence identity between the first cassette and the second cassette is at least 96%, 97%, or 98%. Preferably, the percentage sequence identity between the first cassette and the second cassette is at least 99% or 100%.

[0033] Preferably, the linker separating the at least two self-complementary transgene expression cassettes is an inverted terminal repeat (ITR). Preferably, the phage vector comprises a second ITR. More preferably, this second ITR flanks one of the at least two self-complementary transgene expression cassettes.

[0034] Alternatively, in another preferred embodiment, the linker separating the at least two self-complementary transgene expression cassettes is an unrelated DNA segment. Preferably, the linker or unrelated DNA segment is 60 bp to 300 bp, 80 bp to 280 bp, 100 bp to 260 bp, 120 bp to 240 bp, 140 bp to 220 bp, or 160 bp to 200 bp in length. Most preferably, the linker or unrelated DNA segment is 180 bp in length.

[0035] "Unrelated DNA segment" refers to DNA that has low or no sequence identity with the first and second single-stranded self-complementary cassettes. For example, the sequence identity percentage between the linker and the first and second cassettes is less than 50%, less than 45%, or less than 40%. Preferably, the sequence identity percentage between the linker and the first and second cassettes is less than 35%, less than 30%, or less than 25%. Preferably, the sequence identity percentage between the linker and the first and second cassettes is less than 20%, less than 15%, or less than 10%. Preferably, the sequence identity percentage between the first cassette and the second cassette is at least 8% or 5%.

[0036] Preferably, the first and / or second ITRs are AAV ITRs. The ITRs may be specific to AAV-2 or another AAV serotype, and may have any sequence as long as they form a hairpin loop in their secondary structure. For example, the AAV serotype may be AAV1 to AAV9, but is preferably AAV1, AAV2, AAV5, AAV6, or AAV8. The DNA sequence of one embodiment of an ITR (the left ITR from a commercially available AAV plasmid) is represented herein as SEQ ID NO: 6, as follows: [ka]

[0037] The DNA sequence of another embodiment of an ITR (the right ITR from a commercially available AAV plasmid) is represented herein as SEQ ID NO: 7, as follows: [ka]

[0038] Preferably, the phage vector contains only two ITRs. Preferably, the phage vector contains fewer than three ITRs.

[0039] Preferably, the genome of the phage vector contains a selectable marker, e.g., to confer antibiotic (e.g., ampicillin) resistance to a host cell, preferably a bacterium, where the selectable marker is dependent on the host cell in which the vector is harbored. The marker provides selection pressure during production of the vector in the host cell. Thus, in a preferred embodiment, the phage vector contains an ampicillin resistance gene.

[0040] Preferably, the phage vector comprises one or more capsid minor coat proteins. The phage vector may comprise a pIII capsid minor coat protein configured to display a cell targeting ligand to enable delivery of the vector to a target cell. Preferably, the phage vector comprises one or more capsid major coat proteins. The phage vector may comprise at least one pVIII capsid major coat protein configured to display a foreign peptide.

[0041] The phage vector may include modifications of the capsid structure, for example, by processing or chemical or biochemical conjugation. Examples of suitable modifications may include cross-linking of peptide residues on the phage particle. In another embodiment, the phage vector may include one or more functional peptides attached to its capsid. For example, the functional peptide may include a nuclear localization signal or an endosomal escape peptide. Thus, the phagemid particle may be multifunctional and may use the features disclosed in WO 2014 / 184528, the contents of which are incorporated herein by reference.

[0042] In another embodiment, the phage vector may be combined with a cationic polymer to form a complex with a net positive charge, as described in International Publication No. WO 2014 / 184529, the contents of which are incorporated herein by reference. The cationic polymer may be selected from the group consisting of chitosan, poly-D-lysine (PDL), diethylaminoethyl (DEAE), diethylaminoethyl-dextran (DEAE.DEX), polyethyleneimine (PEI), polybrene, protamine sulfate, and a cationic lipid. Preferably, the cationic lipid is selected from the group consisting of fugene®, Lipofectamine®, and DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate). Preferably, the cationic polymer comprises DEAE, more preferably DEAE.DEX.

[0043] Preferably, the phage vector comprises a genome that is substantially devoid of the phage genome from which it is derived. Preferably, the genome of the phage vector lacks at least 60%, more preferably at least 70%, and even more preferably at least 80% of the genome of the bacteriophage from which it is derived. More preferably, the genome of the phage vector lacks at least 90%, more preferably at least 95%, and even more preferably at least 99% of the genome of the bacteriophage from which it is derived. Preferably, the genome of the phage vector lacks all of the genome of the bacteriophage from which it is derived. However, as noted above, the genome of the phage vector may, in some embodiments, include a bacteriophage origin of replication, i.e., an F1 bacteriophage ori, to enable replication of single-stranded DNA in a host bacterium.

[0044] Preferably, the phage vector lacks in its genome bacteriophage structural genes necessary for particle formation, packaging, or extrusion from a prokaryotic host. Such structural genes encode capsid proteins, etc. Thus, preferably, the phage vector lacks structural genes encoding bacteriophage capsid proteins. Preferably, the phage vector comprises a genome lacking genes encoding the minor or major coat proteins from which the vector is derived. Preferably, the phage vector comprises a genome lacking the pIII capsid minor coat protein or lacking the pVIII capsid major coat protein. Most preferably, the phage vector comprises a genome lacking both the pIII capsid minor coat protein and the pVIII capsid major coat protein.

[0045] Thus, the phage vector preferably comprises a replication-deficient virus-like particle or virion that is constructed from and displays structural components (including, but not limited to, proteins and other conjugated compounds) derived from bacteriophage, even though the genome of the vector does not contain the structural genes of the bacteriophage from which it is derived.

[0046] Thus, given that the genome of the phage vector of the first aspect lacks many of the structural genes of derivative phage genomes, an alternative system is required to provide the structural (i.e., capsid) genes necessary for packaging the phage vector genome into a bacteriophage capsid to generate the phage vector of the present invention. Accordingly, the present inventors have devised a system for generating the vector of the first aspect that involves the use of a separate so-called "helper virus" vector. Thus, in effect, the phage vector of the first aspect is a hybrid phagemid vector comprising the cis genetic elements of a phagemid and a eukaryotic virus, e.g., the AAV ITRs.

[0047] Thus, in a second aspect, there is provided a system for producing a phage vector from a prokaryotic host, comprising: (i) a first vector configured to persist within a prokaryotic host, the first vector comprising at least two single-stranded, self-complementary transgene expression cassettes separated by a linker that hybridize to form a double-stranded transgene expression cassette, and a packaging signal to enable replication of the at least two single-stranded, self-complementary transgene expression cassettes; (ii) a second vector comprising nucleic acids encoding structural proteins required for packaging the double-stranded transgene expression cassette, resulting in the formation of a phage vector and its release from the prokaryotic host; A system is provided comprising:

[0048] The system of the second aspect is preferably capable of packaging the genome of a eukaryotic virus (such as an AAV or lentivirus) provided by the first vector into a prokaryotic viral capsid (i.e., a bacteriophage) provided by the second vector.

[0049] Advantageously, by separating the reproductive elements of a phage vector into a first "therapeutic" vector carrying the transgene expression cassette and a second, separate "helper" vector carrying the viral packaging structural genes, genome / vector size is substantially reduced, thereby greatly increasing transgene capacity. In embodiments where the phage vector is used therapeutically, this is a particularly useful advantage for gene therapy applications. Consequently, this results in improved production yields, gene transduction efficiency, and flexibility of the vector system for other uses.

[0050] Preferably, the system of the second aspect is used to generate a phage vector according to the first aspect. Preferably, therefore, the first vector comprises a phage vector genome. The packaging signal of the first vector may preferably comprise an origin of replication, preferably a bacteriophage origin of replication. Preferably, the origin of replication in the first vector comprises an F1 ori, more preferably derived from an F1 bacteriophage ori.

[0051] Preferably, the first vector contains a second origin of replication to allow replication of at least two single-stranded, self-complementary transgene expression cassettes within a prokaryotic host for molecular cloning. Preferably, this origin of replication allows high copy number replication of the vector within the host for molecular cloning. Preferably, this origin of replication comprises a pUC ori. Alternatively, the first vector may contain one or more DNA sequences that favor targeted integration into the host genome, thus eliminating the need for an origin of replication.

[0052] The at least two single-stranded self-complementary transgene expression cassettes comprise viral transgene expression cassettes, more preferably mammalian viral transgene expression cassettes. For example, the at least two transgene expression cassettes may comprise AAV transgene expression cassettes or lentiviral transgene expression cassettes. AAV transgene expression cassettes are preferred.

[0053] Preferably, the linker of the first vector is an ITR, more preferably an AAV ITR, preferably an AAV2 ITR. Alternatively, in another preferred embodiment, the linker of the first vector is an unrelated DNA segment. Preferably, the linker is as described above for the phage vector of the first aspect.

[0054] In a preferred embodiment, the first vector comprises a second ITR. Preferably, this second ITR is adjacent to one of at least two self-complementary transgene expression cassettes. Preferably, the first and / or second ITRs are AAV ITRs. Preferably, the first vector comprises only two ITRs. Preferably, the first vector comprises fewer than three ITRs.

[0055] The second vector, or "helper phage," is preferably a bacteriophage specifically engineered to rescue the genome of the first vector from a prokaryotic host. Therefore, the second vector (i.e., the helper phage) is provided to lend its proteins and polypeptides to the first vector or any other DNA entity containing a functional packaging signal and / or single-stranded origin of replication. The second vector is most preferably replication-deficient. Preferably, the second vector contains a disrupted packaging signal, which significantly impairs its ability to package itself into phage particles. Preferably, the second vector contains a disrupted origin of replication. In one embodiment, the disrupted origin of replication is a medium copy number origin, such as p15a. In another embodiment, the disrupted origin of replication is a low copy number origin, such as pMB1. Preferably, the first vector (ie, the genome of the phage vector) is configured to outcompete the second vector (ie, the helper phage) in both replication and packaging.

[0056] The genome of the second vector may be engineered to confer the resulting phage vector targeting properties (or multifunctional properties as described in WO 2014 / 184528). Thus, the genome of the second vector provides the structural capsid proteins for phage vector assembly. Preferably, the second vector contains nucleic acids encoding one or more capsid minor coat proteins or one or more capsid major coat proteins. All capsid proteins may be either wild-type or recombinant, may be present in single or multiple copies, and may be modified to display chimeric or synthetic peptides. This includes the display of antigens of other viruses for peptide vaccine delivery or as adjuvants if a DNA vaccine (delivered by the phagemid particles of the first aspect) is desired.

[0057] Thus, in one embodiment, the second vector may comprise a first nucleic acid sequence encoding a pIII capsid minor coat protein that is configured to present a cell-targeting ligand to enable delivery of the phage vector to a target cell (e.g., a tumor). v β3 and α v It may be desirable to introduce a nine amino acid mutation into the pIII minor coat protein of the recombinant phagemid particle for targeting tumor cells and angiogenic tumor-associated endothelial cells that express β5 integrin. Thus, the genome of the second vector may contain the RGD4C targeting peptide (CDCRGDCFC - SEQ ID NO:8).

[0058] In another embodiment, the second vector may include a second nucleic acid sequence encoding at least one pVIII capsid major coat protein configured to present a foreign peptide. Thus, it may be desirable to mutate the wild-type pVIII major coat protein of the phage vector to present a short peptide, e.g., a peptide less than 10 amino acids in length. This short peptide may be a targeting moiety or may have inherent biological / chemical functionality in vivo or in vitro, such as immune stimulation in vivo via antigen presentation or binding to nanoparticles (e.g., gold) in vitro via presentation of a gold-binding peptide.

[0059] The first vector may be a member of the Retroviridae family or the Orthoretrovirinae subfamily. The first vector may be a member of the Lentivirus genus. Preferably, the first vector is a member of the Parvoviridae family or subfamily. Preferably, the first vector is a member of the Dependoparvovirus genus or the Adeno-associated virus species.

[0060] Once the first vector (i.e., the genome of the phage vector) and the second vector (i.e., the helper phage) are constructed, they are used together to generate the phage vector of the first embodiment in a prokaryotic host. It is understood that the packaging signal (e.g., origin of replication) of the first vector, which is intended to allow replication of the phage vector genome, functions to signal the structural proteins of the second vector (i.e., the helper phage) to package the genome (i.e., cooperate in trans in the host) and generate particles of the first embodiment.

[0061] In a third aspect, there is provided a method for producing a phage vector from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell a first vector configured to persist within the prokaryotic host, the first vector comprising at least two single-stranded, self-complementary transgene expression cassettes separated by a linker that hybridize to form a double-stranded transgene expression cassette, and a packaging signal to enable replication of the at least two single-stranded, self-complementary transgene expression cassettes; (ii) introducing into the host a helper phage containing nucleic acid encoding a bacteriophage structural protein; (iii) culturing the host under conditions that result in the double-stranded transgene expression cassette being packaged by the structural proteins to form and release from the prokaryotic host a phage vector carrying the double-stranded transgene expression cassette; A method is provided that includes:

[0062] Advantageously, this results in a very high yield of phage vector. The first vector (i.e., the genome of the phage vector) may be introduced into a host cell, for example by infection. The host cell may then be transformed with a helper phage, resulting in the production of the phage vector. Preferably, the method includes a purification step after the culturing step. Purification may include centrifugation and / or filtration.

[0063] In a fourth aspect, there is provided a method for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell: (a) a first vector configured to persist within the prokaryotic host, the first vector comprising at least two single-stranded, self-complementary transgene expression cassettes separated by a linker that hybridize to form a double-stranded transgene expression cassette, and a packaging signal to enable replication of the at least two single-stranded, complementary transgene expression cassettes; and (b) a second vector comprising nucleic acids encoding structural proteins necessary for packaging the double-stranded transgene expression cassettes; (ii) culturing the host under conditions that result in the double-stranded transgene expression cassette being packaged by the structural proteins to form a phage vector and be released from the prokaryotic host; A method is provided that includes:

[0064] Advantageously, this results in improved safety. The second vector (i.e., helper phage) may be introduced into the host cell, for example by infection. The host cell may then be transformed with the first vector (i.e., the genome of the phage vector), resulting in the production of the phage vector. Preferably, the method includes a purification step after the culturing step. Purification may include centrifugation and / or filtration.

[0065] In a fifth aspect, there is provided the use of a helper phage comprising nucleic acid encoding a viral vector structural protein for producing a phage vector according to the first aspect from a prokaryotic host.

[0066] In a sixth aspect, there is provided a host cell comprising the first and / or second vector defined in the second aspect.

[0067] The host cell is preferably a prokaryotic cell, more preferably a bacterial cell. Examples of suitable host cells include (i) TG1 (genotype: K-12 supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5, (r K - m K - ), plasmid: F'[traD36 proAB + lacI qlacZΔM15]), (ii) DH5α F'IQ™ (genotype: F-φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17(rk−, mk+) phoA supE44 λ- thi-1 gyrA96 relA1, plasmid: F' proAB+ lacIqZΔM15 zzf::Tn5[KmR]; and (iii) XL1-Blue MRF' (genotype: Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac, plasmid: F' proAB lacIqZΔM15 Tn10(Tetr).

[0068] In another aspect, there is provided a phage vector according to the first aspect, or a system according to the second aspect, for use as an experimental research tool.

[0069] For example, the vector or system can be used ex vivo or in vitro.

[0070] Preferably, however, the vector is used in a therapeutic or diagnostic method, preferably in vivo.

[0071] Thus, in a seventh aspect there is provided a phage vector according to the first aspect or a system according to the second aspect for use in therapy or diagnosis.

[0072] The present invention may be used to treat a wide variety of diseases due to the target-specific nature and transduction efficiency of the phage vectors of the present invention. As a result, the therapeutic opportunities of recombinant bacteriophages used in gene therapy may be greatly increased by the present invention due to the ability of these recombinant bacteriophages to provide the host bacterium with two self-complementary transgene expression cassettes that hybridize to form a double-stranded transgene expression cassette during phage particle production in the host bacterium. The present invention may be used prophylactically to prevent disease, or to ameliorate and / or treat disease after its onset.

[0073] Thus, in an eighth aspect there is provided a phage vector according to the first aspect, or a system according to the second aspect, for use in gene therapy techniques.

[0074] In a ninth aspect, there is provided a method for treating, preventing or ameliorating a disease in a subject using gene therapy techniques, the method comprising administering to a subject in need of such treatment a therapeutically effective amount of a phage vector according to the first aspect or a system according to the second aspect.

[0075] It is understood that the present invention may be used to generate a variety of different phage vectors that can be used to treat and / or diagnose various diseases, depending on the nature of the vector and the foreign protein displayed. For example, in embodiments in which the phage vector contains a tumor-targeting ligand and / or a transgene expressing an anti-tumor gene (e.g., the HSVtk gene), the phage vector may be used to treat cancer in combination with ganciclovir (GCV). Target cells in gene therapy techniques are preferably eukaryotic cells, preferably mammalian cells.

[0076] Therefore, gene therapy techniques are preferably used to treat, prevent, or ameliorate cancer. The tumor may be located in the brain, such as medulloblastoma, glioblastoma, or diffuse intrinsic pontine glioma (DIPG). The phage vector may be used in combination with conventional treatments, such as chemotherapy drugs (i.e., doxorubicin, temozolomide, lomustine), radiation therapy, immune checkpoint inhibitors (i.e., inhibitors of PD-1, PD-L1, or CTLA4), or other drugs / xenobiotic compounds, including, but not limited to, histone deacetylase inhibitors (HDAC inhibitors), proteasome inhibitors, and anticancer products derived from natural and dietary sources (i.e., genistein).

[0077] The inventors believe that the phage vectors of the present invention will have significant commercial value in the delivery of peptide and / or DNA and / or adjuvant vaccines.

[0078] Thus, in a tenth aspect there is provided a vaccine comprising a phage vector according to the first aspect or a system according to the second aspect.

[0079] In an eleventh aspect, there is provided a phage vector according to the first aspect, or a system according to the second aspect, for use in delivering a vaccine to a subject.

[0080] Preferably, the vaccine is a peptide vaccine. The vaccine is preferably a DNA vaccine. The vaccine preferably contains a suitable adjuvant. In one embodiment, the phage vector may be used to carry a transgene or DNA cassette (i.e., at least two single-stranded self-complementary transgene expression cassettes that hybridize to form one double-stranded transgene expression cassette) encoding an antigen for stimulating the body's immune system. The phage vector may also be used to directly display and express the antigen of interest on the major pVIII coat protein, thus providing an efficient platform for simultaneous delivery of multiple antigens, or proteins that are easily expressed on the phage surface, or adjuvants, by a single phage particle as a vaccine DNA vaccine. The subject may be a mammal, preferably a human.

[0081] Thus, in a twelfth aspect there is provided a phage vector according to the first aspect, or a system according to the second aspect, for use in delivering and targeting foreign antigens to tumours in vaccinated subjects.

[0082] Animals will first be vaccinated against the foreign antigen or will have already been vaccinated against the antigen used, and then the tumor-targeting vector will be administered to the vaccinated animals to deliver the foreign antigen to the tumors in order to induce an immune attack against these tumors.

[0083] The inventors also believe that the phage vectors of the present invention can be used in a variety of different genetic-molecular imaging techniques, such as positron emission tomography (PET), ultrasound (US), SPECT imaging, functional magnetic resonance imaging, or bioluminescence imaging.

[0084] Thus, in a thirteenth aspect there is provided the use of a phage vector according to the first aspect or a system according to the second aspect in gene-molecular imaging techniques.

[0085] The transgene carried by the phagemid particle may encode HSVtk and / or sodium / iodide symporter (NIS), and the particle is preferably used in combination with a radiolabeled substrate. For example, the human sodium / iodide symporter (NIS) imaging gene is preferably radiolabeled for clinically applicable positron emission tomography (PET) imaging. 124 and for clinically applicable SPECT imaging I 125 / 99m Used in combination with Tc-pertechnetate.

[0086] Alternatively, the HSVtk gene is preferably used in combination with a radiolabeled nucleoside analogue such as 20-[18F]-fluoro-20-deoxy-1-bD-arabino-furanosyl-5-ethyluracil ([18F]FEAU).

[0087] It is understood that the phage vectors and systems of the present invention (i.e., hereinafter referred to as "drugs") may be used in medicines that may be used in monotherapy or as an adjunct to or in combination with known therapies for treating, ameliorating, or preventing diseases such as cancer. For example, a combination treatment approach using the phage particles and systems of the present invention together with existing chemotherapeutic agents such as temozolomide, doxorubicin, or genistein is preferred.

[0088] In another preferred embodiment, therapy may involve the combination of the phage vectors and systems of the present invention with an extracellular matrix degrading agent, such as an enzyme or losartan, which the inventors believe should enhance phage vector spread in treated subjects, particularly in solid tumors.

[0089] The agent according to the present invention (i.e., the phage vector of the first aspect or the system according to the second aspect) may be combined into a composition having several different forms, depending in particular on the manner in which the composition is to be used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, etc., or any other suitable form that may be administered to a person or animal in need of treatment. It will be understood that the pharmaceutical vehicle according to the present invention should be one that is well tolerated by the subject to which it is given.

[0090] A medicament containing the agent of the present invention may be used in many ways. For example, oral administration may be required, in which case the agent may be contained in a composition that can be taken orally, for example, in the form of a tablet, capsule, or liquid. A composition containing the agent of the present invention may be administered by inhalation (e.g., intranasally). A composition may also be formulated for topical use. For example, a cream or ointment may be applied to the skin.

[0091] The agents of the present invention may be incorporated into a sustained- or delayed-release device. Such a device may, for example, be inserted above or below the skin, and the medication may be released over a period of weeks or months. The device may be positioned at least adjacent to the treatment site. Such a device may be particularly advantageous when long-term treatment with the agents used in accordance with the present invention is required, which would normally require frequent administration (e.g., at least daily injections).

[0092] In a preferred embodiment, the agents and compositions of the present invention may be administered to a subject by injection into the bloodstream or directly to the site in need of treatment. The injection may be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intraperitoneal, or may be enhanced by convection (convection-enhanced drug delivery - relating to local injection at the disease site).

[0093] It will be understood that the amount of agent required will be determined by its biological activity and bioavailability, which will depend on the mode of administration, the physiochemical properties of the agent (i.e., the phage vector or system), and whether the agent is used as a monotherapy or in a combination therapy. The frequency of administration will also be affected by the half-life of the agent in the subject being treated. The optimal dosage to be administered may be determined by one skilled in the art and will vary depending on the particular agent used, the strength of the pharmaceutical composition, the mode of administration, and the progression of the disease. The need to adjust the dosage will arise due to additional factors depending on the particular subject being treated, including the subject's age, weight, sex, diet, and time of administration.

[0094] Generally, a daily dose of between 0.01 μg / kg body weight and 500 mg / kg body weight of the agent of the present invention may be used, more preferably the daily dose is between 0.01 mg / kg body weight and 400 mg / kg body weight, more preferably between 0.1 mg / kg and 200 mg / kg body weight.

[0095] The drug may be administered before, during, or after the onset of the disease. For example, the drug may be administered shortly after the subject develops the disease. The daily dose may be given systemically as a single administration (e.g., a single daily injection). Alternatively, the drug may require two or more administrations per day. By way of example, the drug may be administered as two (or more depending on the severity of the disease being treated) daily doses of between 25 mg and 7000 mg (i.e., assuming a body weight of 70 kg). The patient receiving treatment may take a first dose upon waking, then a second dose in the evening (in the case of a two-dose regimen), or at three- or four-hour intervals thereafter. Alternatively, a sustained-release device may be used to provide the patient with an optimal dose of the drug of the present invention without the need for repeated doses.

[0096] Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to formulate specific formulations and precise treatment regimens (such as daily doses and administration frequency of drugs) containing the vectors or systems of the present invention.

[0097] Thus, in a fourteenth aspect of the present invention there is provided a pharmaceutical composition comprising a phage vector according to the first aspect or a system according to the second aspect and a pharmaceutically acceptable vehicle.

[0098] The compositions can be used for the therapeutic amelioration, prevention or treatment of any disease in a subject that is treatable with gene therapy, such as cancer.

[0099] In a fifteenth aspect, the present invention also provides a process for producing a pharmaceutical composition according to the twelfth aspect, the process comprising the step of contacting a therapeutically effective amount of a phage vector according to the first aspect or a system according to the second aspect with a pharmaceutically acceptable vehicle.

[0100] A "subject" may be a vertebrate, a mammal, or a domestic animal. Thus, the agents, compositions, and medicaments of the present invention may be used to treat any mammal, such as domestic animals (e.g., horses or dogs), companion animals, or in other veterinary applications. Most preferably, however, the subject is a human.

[0101] A "therapeutically effective amount" of a drug (i.e., a phage vector) is any amount that, when administered to a subject, is the amount of drug necessary to treat a target disease or produce a desired effect, e.g., to effect effective delivery of a transgene to a target cell or tissue, e.g., to effect tumor killing.

[0102] For example, the therapeutically effective amount of the drug used may be about 0.01 mg to about 800 mg, preferably about 0.01 mg to about 500 mg.

[0103] A "pharmaceutically acceptable vehicle" as referred to herein is any known compound or combination of known compounds known to those skilled in the art to be useful in formulating pharmaceutical compositions.

[0104] In one embodiment, the pharmaceutically acceptable vehicle may be solid, and the composition may be in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may contain one or more substances that may also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet disintegrants. The vehicle may also be an encapsulating material. In powders, the vehicle is a finely divided solid mixed with the finely divided active agent of the present invention. In tablets, the active agent (e.g., the particles or system of the present invention) may be mixed with a vehicle having the necessary compression properties in appropriate proportions and compressed into the desired shape and size. Powders and tablets preferably contain up to 99% of the active agent. Suitable solid vehicles include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low-melting waxes, and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.

[0105] However, the pharmaceutical vehicle may also be liquid, and the pharmaceutical composition may be in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The particles or systems of the present invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle, such as water, an organic solvent, a mixture of both, or a pharmaceutically acceptable oil or fat. The liquid vehicle may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colorants, viscosity regulators, stabilizers, or osmolality regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing the above-mentioned additives, such as cellulose derivatives, preferably carboxymethylcellulose sodium solution), alcohols (including monohydric and polyhydric alcohols, such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and peanut oil). For parenteral administration, the vehicle can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

[0106] Liquid pharmaceutical compositions that are sterile solutions or suspensions can be utilized by injection, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous, and especially subcutaneous injection. The vector or system may also be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

[0107] The phage vectors, systems, and pharmaceutical compositions of the present invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monooleate, polysorbate 80 (oleic acid ester of sorbitol and its anhydrides copolymerized with ethylene oxide), etc. The particles and systems of the present invention can also be administered orally in either liquid or solid composition form. Compositions suitable for oral administration include solid forms such as pills, capsules, granules, tablets, and powders, and liquid forms such as solutions, syrups, elixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

[0108] It will be appreciated that adeno-associated viruses (AAVs) are often the vectors of choice for gene therapy. As gene delivery vectors, lentiviral vectors also have several important advantages over other systems. First, they have a large packaging capacity of at least 8 kb of DNA, an important feature when packaging large expression cassettes for tissue-specific promoters and transgenes. Second, they differ from simpler retroviruses in that they can transduce not only genomic tissues but also non-dividing cells, a very useful quality when considering their application as gene therapy vectors in non-proliferating tissues such as muscle, neurons, and hematopoietic stem cells. In addition, lentivectors have lower immunogenicity compared to adenoviral vectors, which allows for the consideration of systemic delivery routes. However, barriers to the use of AAVs or lentiviruses for laboratory and clinical research include their extremely high production costs and low yields.

[0109] In addition to showing useful applications in gene therapy, imaging, and vaccine delivery, the phage vectors of the present invention can also be used to generate recombinant viral vectors, such as AAV or lentivirus, in vitro or in vivo (including in situ). Phage-guided AAV production utilizes the ability of phage vectors to package large amounts of single-stranded ssDNA. A typical AAV production system consists of three major elements: rAAV, rep-cap, and adeno-helper genes, which function together to generate rAAV particles.

[0110] Thus, in a sixteenth aspect there is provided the use of a phage vector according to the first aspect or a system according to the second aspect to generate a recombinant viral vector comprising or derived from a viral genome within the genome of the phage vector.

[0111] In a seventeenth aspect, there is provided a method for producing a recombinant viral vector, the method comprising introducing into a eukaryotic host cell a phage vector according to the first aspect, or a system according to the second aspect, and allowing the host cell to produce the recombinant viral vector.

[0112] Preferably, the recombinant viral vector is a recombinant mammalian virus, rAAV, recombinant self-complementary AAV vector, or recombinant lentiviral vector. In other words, the recombinant viral vector may be a conventional AAV vector or the self-complementary AAV vector of the first aspect. Preferably, the phage vector of the first aspect or the system of the second aspect is used in cis and / or trans, with the delivery and / or presence inside the eukaryotic host cell of other genetic elements required for the generation of mammalian viruses as determined by the genome of the phage vector. Methods used to assist or enhance gene transfer into host cells by phagemid particles include those described in WO 2014 / 184528 (i.e., multifunctional) and WO 2014 / 184529 (i.e., in combination with a cationic polymer to form a complex with a net positive charge).

[0113] Eukaryotic host cells may be mammalian cells. Host cells may include or be derived from human embryonic kidney cells (HEK293), Spodoptera frugiperda pupal ovary tissue (Sf9), or Chinese hamster ovary (CHO). Insect cells are also contemplated.

[0114] In one embodiment, a host cell may be transformed with one or more phage vector genomes carrying genes selected from the group consisting of genes encoding rAAV, lentivirus, capsid, replication, helper proteins, and any other genes necessary for mammalian viral expression and packaging.

[0115] For example, in phage vector-guided rAAV / scAAV production, the rAAV genes or self-complementary AAV sequences may be carried by the phage vector according to the first aspect, and the adeno-helper genes and rep-cap genes may be carried on separate vectors or integrated into the eukaryotic host genome. Any combination of rAAV, rep-cap, and adeno-helper genes may be carried on one or more vectors, i.e., in cis or trans configuration. Alternatively, the rep-cap or adeno-helper proteins may also be integrated or introduced into the eukaryotic host as stably expressed accessory DNA (e.g., a plasmid) in the context of rAAV production, whereby the phage vector supplies the recombinant viral genome for packaging into recombinant virus, as determined by a transgene cassette within the phage vector's genome.

[0116] The method may be carried out in vivo, in vitro, ex vivo, or in situ. For in situ production, the phage vector preferably includes a targeting moiety for a target eukaryotic cell, which is a selected eukaryotic host. Preferably, in the context of in situ, ex vivo, and in vivo virus production, the cell type of the selected eukaryotic host is a diseased cell. Preferably, the diseased cell is a malignant or benign tumor. In the context of in vitro virus production, the eukaryotic host is preferably a derivative of any of the eukaryotic hosts listed above. Application of the phage vector and the genetic elements required for the generation of recombinant virus (determined by the transgene cassette in the phage vector) within the eukaryotic host cell may be by any of the modes indicated above, in either a cis-acting or trans-acting combination.

[0117] It will be understood that the present invention extends to any nucleic acid or peptide, or variant, derivative or analogue thereof, that substantially comprises the amino acid or nucleic acid sequence of any of the sequences referred to herein (including functional variants or functional fragments thereof). The terms "substantially an amino acid / polynucleotide / polypeptide sequence", "functional variant" and "functional fragment" can be a sequence that has at least 40% sequence identity with the amino acid / polynucleotide / polypeptide sequence of any one of the sequences referred to herein, for example a sequence that has 40% identity to a nucleic acid specified herein.

[0118] Also contemplated are amino acid / polynucleotide / polypeptide sequences that have greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and even more preferably greater than 80% sequence identity to any of the referenced sequences. Preferably, the amino acid / polynucleotide / polypeptide sequence has at least 85% identity to any of the referenced sequences, more preferably at least 90% identity to any of the sequences referenced herein, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity, and most preferably at least 99% identity.

[0119] Those skilled in the art will understand how to calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences. To calculate the identity percentage between two amino acid / polynucleotide / polypeptide sequences, the two sequences must first be aligned, and then the sequence identity value must be calculated. The identity percentage between two sequences can vary depending on (i) the method used to align the sequences, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented by different programs), or structural alignment from 3D comparison, and (ii) the parameters used by the alignment method, such as local versus global alignment, the pair score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and gap penalties, such as function form and constant.

[0120] After alignment, there are many different ways to calculate the percentage identity between two sequences. For example, the number of identical portions may be divided by (i) the length of the shortest sequence, (ii) the length of the alignment, (iii) the average length of the sequences, (iv) the number of non-gap positions, or (v) the number of equivalent positions excluding overhangs. It will be further understood that the percentage identity is also strongly dependent on length. Therefore, the shorter the sequence pair, the higher the expected sequence identity will be by chance.

[0121] It will therefore be appreciated that accurate alignment of protein or DNA sequences is a complex process. The commonly used multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating protein or DNA multiple alignments according to the present invention. Suitable parameters for ClustalW may be as follows: for DNA alignments: gap open penalty = 15.0, gap extension penalty = 6.66, and Matrix = Identity; for protein alignments: gap open penalty = 10.0, gap extension penalty = 0.2, and Matrix = Gonnet; for DNA and protein alignments: ENDGAP = -1 and GAPDIST = 4. Those skilled in the art will recognize that it may be necessary to vary these and other parameters for optimal sequence alignment.

[0122] Preferably, the percentage identity between two amino acid / polynucleotide / polypeptide sequences is then calculated from such an alignment as (N / T) x 100, where N is the number of positions where the sequences share identical residues, and T is the total number of positions being compared, including gaps, and either including or excluding overhangs. Preferably, overhangs are included in the calculation. Thus, the most preferred method for calculating the relative percentage identity between two sequences comprises (i) preparing a sequence alignment using the ClustalW program, for example, using an appropriate set of parameters as described above, and (ii) substituting the values ​​of N and T into the following formula: sequence identity = (N / T) x 100.

[0123] Alternative methods for identifying similar sequences are known to those of skill in the art. For example, a substantially similar nucleotide sequence is encoded by a sequence that hybridizes under stringent conditions to the nucleic acid sequences described herein or their complements. By stringent conditions, we mean that the nucleotides hybridize to filter-bound DNA or RNA in 3× sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least one wash in 0.2× SSC / 0.1% SDS at about 20-65°C. Alternatively, a substantially similar polypeptide may differ from the sequences described herein by at least one, but fewer than 5, 10, 20, 50, or 100 amino acids.

[0124] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence can be altered or modified without substantially affecting the sequence of the protein encoded thereby to provide functional variants thereof. Suitable nucleotide variants are those having a sequence that is altered by the substitution of different codons that encode the same amino acid within the sequence, thus resulting in a silent (synonymous) change. Other suitable variants are those that contain all or portions of a sequence that have a homologous nucleotide sequence but are altered by the substitution of different codons that encode amino acids with side chains with similar biophysical properties to the amino acid being replaced, resulting in a conservative change. For example, small nonpolar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar, hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is understood which amino acids may be substituted with amino acids having similar biophysical properties, and one of skill in the art would know the nucleotide sequences encoding these amino acids.

[0125] All of the features described in this specification (including any accompanying claims, abstract, and drawings), and / or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. [Brief explanation of the drawings]

[0126] For a better understanding of the present invention and to show how embodiments thereof may be carried into effect, reference will now be made, by way of example, to the accompanying drawings in which:

[0127] [Figure 1]FIG. 1 shows a schematic diagram of the DNA constructs of prior art M13 phage-derived single-stranded DNA vectors, i.e., adeno-associated virus / phage ("AAVP") and prior art phagemid / adeno-associated virus ("PAAV") vectors, compared with the novel self-complementary phage particle ("self-complementary phage," hereinafter referred to as "self-complementary phage particle" or "sc phagemid") of the present invention, which may be M13 or AAV. In the present invention, a phagemid carrying two single-stranded, self-complementary transgene expression cassettes is used as a DNA backbone to induce hybridization of the transgene cassettes in a host bacterium, resulting in the generation of a double-stranded transgene cassette that is subsequently packaged by the phage capsid. The definition of "phagemid" is plasmid DNA containing a phage origin of replication, hence the name phagemid. In the present invention, the inventors have designed a new phage genome carrying two transgene cassettes using a phagemid as a DNA backbone. The resulting double-stranded vectors are phage particles. Prior art AAVPs contain a complete phage genome and a single mammalian transgene cassette flanked by ITR sequences derived from the AAV2 virus (6). Prior art PAAV particles, on the other hand, are based on a phagemid design, containing a single transgene cassette and requiring a helper phage to provide the structural gene during production (8). In contrast, the latest generation phage vectors of the present invention (i.e., "self-complementary phage particles" or "sc-phagemids") carry an additional transgene cassette compared to AAVP and PAAV. The two cassettes are identical and separated from the AAV by an inverted terminal repeat (ITR) linker, but are positioned in opposite orientations, i.e., the first cassette extends in the 5' to 3' direction and the second cassette extends in the 3' to 5' direction, as shown in Figure 1. As can be seen, the cassettes are identical but extend in opposite or antiparallel directions on either side of the ITR that separates them. A second AAV ITR is included to flank one of the transgene cassettes. As with PAAV, a helper phage provides the structural genes for the "single-stranded complementing phage" to replicate. [Figure 2]FIG. 2 shows how the single-stranded self-complementary phagemid "sc phagemid" or "scPP" of the present invention allows hybridization between two complementary transgene expression cassettes on either side of an ITR linker, thereby creating a dsDNA of the transgene expression cassette resembling a hairpin loop structure. [Figure 3] Figure 3 shows the cloning strategy used to generate a phagemid backbone carrying complementary transgene expression cassettes for the generation of scPPs delivering green fluorescent protein (GFP). The entire GFP transgene expression cassette, from the promoter to the poly(A) signal, was amplified from the phagemid by PCR using primers containing PciI restriction sites. The insert was then cloned into the PciI site of the same phagemid. The final phagemid contains two complementary GFP transgene expression cassettes and two AAV2 ITRs, with one left ITR linking the two transgene cassettes and a second right ITR flanking one cassette. As can be seen, the cassettes are identical but extend in opposite or antiparallel directions on either side of the left ITR, allowing hybridization to occur between the two complementary transgene expression cassettes on either side of the ITR linker, thereby creating dsDNA. [Figure 4] Figure 4 shows GFP expression in B16-F1 cells 5 days after transduction with the targeted RGD4C.scPP (i.e., a phage vector according to the present invention that formed a dsDNA hairpin loop structure) or the RGD4C.PAAV vector (i.e., a single-stranded phage vector used as a control for comparison). A) Microscopic imaging of cells using a fluorescent microscope; B) Quantification of GFP-positive cells by FACS analysis. [Figure 5]Figure 5 summarizes the constructs used to evaluate gene delivery efficacy: PAAV, scPP (a phage vector of the present invention), and cwPP. PAAV exemplifies a phage vector with one copy of the expression cassette flanked by AAV ITRs; scPP exemplifies a phage particle vector of the present invention with two expression cassettes in opposite orientations that will form dsDNA; and cwPP exemplifies a control phage particle carrying two Lucia transgene cassettes in the same orientation, i.e., clockwise (cw), so that the cassettes cannot hybridize to form dsDNA. Particles carrying the reporter genes Lucia or GFP and the therapeutic genes TNFα, IL15, and TRAIL were used. [Figure 6] Figure 6 compares Lucia gene expression between targeted RGD4C.scPP-Lucia (i.e., a phage vector of the present invention) and targeted RGD4C.PAAV.-Lucia in B16-F1 melanoma cells. Cells were transduced with various vector doses: 25,000, 50,000, 100,000, 500,000, and 106 TU / cell. Error bars represent the standard error of the mean (SEM). One-way ANOVA was used for statistical analysis for each day and each construct, followed by multiple comparisons selected: pairwise comparisons of double-stranded vector versus single-stranded vector. Cells treated with a non-targeted vector lacking RGD4C (scPP-Lucia or PAAV-Lucia) and untreated cells were also included in the experiment. Data are presented as relative luminescence units (RLU). Experiments were repeated several times with at least n = 5 biological replicates and n = 3 technical replicates each. [Figure 7] FIG. 7 shows confirmation of the superiority of the scPP vector (i.e., the phage vector of the present invention) in B16-F1 cells at 100,000 TU / cell compared to single-chain PAAV batches prepared by two different researchers. [Figure 8] FIG. 8 shows example data for B16-F1 cells from four experiments using vectors at a dose of 10 6 TU / cell. [Figure 9]Figure 9 shows a comparison of Lucia gene expression in B16-F1 melanoma cells at various doses as in Figure 6. Error bars represent SEM. One-way ANOVA was used for statistical analysis for each day and each construct, followed by selected multiple comparisons: ds vector vs. ss vector pairwise comparisons. Cells treated with the non-targeting vector scPP or PAAV lacking RGD4C, and untreated cells were also included in the experiment. Data are shown as RLU. Experiments were repeated several times with at least n = 5 biological replicates and n = 3 technical replicates each. [Figure 10] Figure 10 shows a comparison of Lucia gene expression in human HEK293 cells at various doses: 100,000, 500,000, and 106 TU / cell. Error bars represent SEM. One-way ANOVA was used for statistical analysis for each day and each construct, followed by selected multiple comparisons: ds vector vs. ss vector pairwise comparisons. Cells treated with the non-targeting vector scPP or PAAV lacking RGD4C, and untreated cells, were also included in the experiment. Data are shown as RLU. Experiments were repeated several times with at least n = 5 biological replicates and n = 3 technical replicates each. [Figure 11] Figure 11 shows a comparison of Lucia gene expression in RMS metastatic cancer cells at 100,000, 500,000, and 106 TU / cell. Error bars represent SEM. One-way ANOVA was used for statistical analysis for each day and each construct, followed by selected multiple comparisons: ds vector vs. ss vector pairwise comparisons. Cells treated with scPP or PAAV lacking RGD4C, as well as untreated cells, were also included in the experiment. Data are shown as RLU. Experiments were repeated several times with at least n = 5 biological replicates and n = 3 technical replicates each. [Figure 12]Figure 12 shows a comparison of Lucia gene expression in human A549 lung cancer cells at 500,000 and 106 TU / cell. Error bars represent SEM. One-way ANOVA was used for statistical analysis for each day and each construct, followed by selected multiple comparisons: ds vector vs. ss vector pairwise comparisons. Cells treated with the non-targeting vector scPP or PAAV lacking RGD4C, and untreated cells were also included in the experiment. Data are shown as RLU. Experiments were repeated several times with at least n = 5 biological replicates and n = 3 technical replicates each. [Figure 13] FIG. 13 shows confirmation of data by different investigators on Lucia gene expression in human A549 lung cancer cells at 100,000 and 10 6 TU / cell. [Figure 14] Figure 14 compares Lucia gene expression in human MCF7 breast cancer cells at 100,000 TU / cell. Error bars represent SEM. One-way ANOVA was used for statistical analysis for each day and each construct, followed by selected multiple comparisons: ds vector vs. ss vector pairwise comparisons. Cells treated with the non-targeting vector scPP or PAAV lacking RGD4C, and untreated cells were also included in the experiment. Data are shown as RLU. Experiments were repeated several times with at least n=5 biological replicates and n=3 technical replicates each. [Figure 15] Figure 15 shows ELISA quantification of secreted TNFα on days 4 (D4) and 6 (D6) after transduction of B16-F1 cells with scPP (i.e., a phage vector according to the present invention) or PAAV carrying the TNFα gene at 500,000 (500k), 1x10 (1M), or 4x10 (4M) TU / cell. One-way ANOVA was used for statistical analysis for each day and each construct, followed by multiple comparisons as selected: ds vector vs. ss vector pairwise comparisons. Cells treated with the non-targeting vector scPP or PAAV lacking RGD4C, and untreated cells, were also included in the experiment. [Figure 16]Figure 16 shows ELISA quantification of secreted IL15 4 days after transduction of B16-F1 cells with RGD4C.scPP (i.e., a phage vector according to the present invention) or RGD4C.PAAV carrying the IL15 gene at 500,000 (500k), 10 (1M), and 4 x 10 (4M) TU / cell. One-way ANOVA was used for statistical analysis for each day and each construct, followed by multiple comparisons as selected: ds vector vs. ss vector pairwise comparisons. Cells treated with a non-targeting vector (M13) and untreated cells were also included in the experiment. [Figure 17] Figure 17 shows ELISA quantification of secreted IL15 4 days after transduction of B16-F10 melanoma cells with RGD4C.scPP (i.e., a phage vector according to the present invention) or RGD4C.PAAV carrying the IL15 gene at 500,000 (500k), 10 (1M), and 4 x 10 (4M) TU / cell. One-way ANOVA was used for statistical analysis for each day and each construct, followed by multiple comparisons as selected: ds vector vs. ss vector pairwise comparisons. Cells treated with the non-targeting vector scPP or PAAV lacking RGD4C, and untreated cells, were also included in the experiment. [Figure 18] Figure 18 shows ELISA quantification of secreted TRAIL 4 days after transduction of human osteosarcoma cells with RGD4C.scPP (i.e., a phage vector according to the present invention) or RGD4C.PAAV carrying a secreted form of the TRAIL gene at 500,000 TU / cell. Cells treated with the empty RGD4C.scPP vector (no TRAIL or mock) and untreated cells were also included in the experiment. The RGD4C.scPP vector carrying the transmembrane form of TRAIL (RGD4C.scPP-TRAIL) was also used to transduce cells. [Figure 19]Figure 19 compares gene delivery to a subcutaneous solid tumor, human osteosarcoma, in immunodeficient mice after intravenous administration of RGD4C.scPP (i.e., a phage vector of the present invention) and the RGD4C.PAAV vector at 5 x 10 TU / mouse. Tumor and healthy tissues were harvested 7 days after vector administration. Non-targeted vectors lacking RGD4C, scPP, or PAAV, and untreated mice were included in the experiment. [Figure 20] Figure 20 shows phage diffusion in Matrigel. scPP and PAAV vectors were labeled with FITC and inoculated into Matrigel at 5 mg / ml. Images were taken with a fluorescence microscope at the time of inoculation (t=0) and 18 hours later (t=18). [Figure 21] Figure 21 shows the internalization of phage particles in B16-F1 cells. A) FACS analysis of cells using anti-phage antibodies; B) qPCR using primers for the ampicillin gene located outside the transgene cassette in the vector. [Figure 22] Figure 22 compares transduction efficiency between scPP (i.e., a phage vector according to the invention) and the control cwPP and awPP vectors. A) Schematic diagram of the three vectors used. ITRs are shown in blue, with the transgene in their orientation. B) B16F1 cells were transduced with the Lucia-encoding vector at 10 TU per cell. The graph shows a representative experiment (n=3) of two replicates, with luminescence measurements corresponding to day 4 post-transduction shown. One-way ANOVA was used, and statistical differences were reported assuming α=0.05. [Figure 23]Figure 23 compares the transduction efficiency between scPP (i.e., a phage vector according to the invention) and a mixture of cwPP and awPP. A) Schematic of transduction and possible hybridization. ITRs are shown in blue, and transgenes are indicated in their orientation. B) B16F1 cells were transduced with 10 TU per cell of Lucia-encoding vector or with a mixture of 5 x 10 TU / cell of cwPP + 5 x 10 TU / cell of awPP. The graph shows a representative experiment (n = 3) of two replicates, with luminescence measurements corresponding to day 4 post-transduction shown. One-way ANOVA was used, and statistical differences were reported assuming α = 0.05. [Figure 24] Figure 24 shows phage particle measurements from TEM images. A) TEM images of different phage particles. B) Size quantification of phage particles. Two different stocks of each type of particle were imaged, and a total of 100 particles were quantified for each phage. Statistical differences were calculated using one-way ANOVA between the four samples. Only pairs without statistical differences are shown. [Figure 25] Figure 25 shows the analysis of scPP, PAAV, and helper phage vectors to determine their particle size. A) Two different preparations were analyzed for each type of vector, and a total of 100 particles were measured from the TEM images acquired. B) Whole phage particles were loaded onto an agarose gel under native conditions. [Figure 26]Figure 26 shows the self-hybridization of a transgene cassette during the generation of an scPP (i.e., a phage vector of the present invention) in bacteria. A) Schematic presentation of the hypothesis. The hypothesized intramolecular hybridization pattern of the scPP genome is presented. This creates a dsDNA target (orange box) for BamHI digestion within the transgene. The digested genome should generate a 1,898 bp dsDNA fragment, which, when denatured, will result in a 3,796 bp ssDNA band. B) Comparison of BamHI-digested and undigested scP-Lucia genome samples. C) Migration analysis of a denatured 1 kb Plus DNA ladder. Aliquots of a 1 kb Plus DNA ladder and a linearized pAAV GFP plasmid (5,378 bp) were run on a 1 M urea denaturing agarose gel in their native and denatured forms. The 5,000 bp DNA ladder band is converted to a doublet when the ladder sample is denatured. D) Evidence of the ability of the scPP-Lucia phage genome to form dsDNA fragments. The 1,898 bp digest fragment obtained after BamHI digestion of the phage genome was run in either its native (left) or denatured (right) form alongside a 1 kb Plus DNA ladder treated in the same way. The denatured sample migrated at the same speed as the 4000 bp DNA ladder band. [Figure 27] Figure 27 shows how hydroxyurea suppresses gene expression from PAAV vectors. A) Lucia gene expression over time in vector-transduced B16-F1 cells in the presence of hydroxyurea (HU). B) A control experiment was also performed in parallel in which cells received water instead of HU. C) The graph shows Lucia gene expression data 6 days after transduction of B16-F1 in the presence of HU. D) The graph shows Lucia gene expression 6 days after transduction in the absence of HU. [Figure 28]Figure 28 shows a comparison of Lucia reporter gene delivery into metastatic human osteosarcoma 143B cells between PAAV and scPP over a time course from day 1 to day 3 after treatment with increasing doses of vector. Lucia expression is presented as relative luminescence units (RLU). The vector was targeted to tumor cells using the RGD4C ligand. A non-targeting vector (NT) was used as a control. [Figure 29] Figure 29 shows a comparison of the delivery of the secreted cytokine TRAIL (i.e., soluble TRAIL (sTRAIL)) to metastatic human osteosarcoma 143B cells between PAAV and scPP. ELISA data used to quantify the release of sTRAIL protein into the culture medium of cancer cells upon treatment with the vectors is shown. [Figure 30] FIG. 30 shows the induction of osteosarcoma cell death in vitro after treatment with scPP-sTRAIL, which encodes a secreted form of sTRAIL. [Figure 31] Figure 31 shows toxicity assessment. There is no increase in the toxicity biomarker LDH (lactate dehydrogenase) in mice bearing established osteosarcoma after administration of the RGD4C.scPP vector encoding sTRAIL. [Figure 32] FIG. 32 shows the biodistribution of sTRAIL delivery in tumor-bearing mice with established osteosarcoma after systemic treatment with PAAV and scPP encoding sTRAIL. [Figure 33] Figure 33 shows immunofluorescence staining of tumors showing sTRAIL protein expression after treatment with RGD4C.PAAV and RGD4C.scPP encoding sTRAIL. Higher sTRAIL production was detected in tumors from mice that received RGD4C.scPP. [Figure 34] FIG. 34 shows hematoxylin and eosin staining of tumors showing extensive tumor damage after systemic treatment with RGD4C.scPP-sTRAIL compared to untreated mice or mice injected with the non-targeted NT vector. [Example]

[0128] The present inventors set out to provide a novel phage vector containing a self-complementary sequence of a transgene expression cassette to achieve hybridization and subsequent delivery of the double-stranded DNA of the mammalian transgene cassette during production in host bacteria or upon transduction of mammalian cells. This novel phage vector addresses the problems associated with working with double-stranded phage due to their capsid and large genome, and also overcomes the problems associated with AAV. This novel phage vector is referred to throughout the examples as self-complementary phage particles, or scPPs. To demonstrate that scPPs provide better gene delivery than prior art techniques, the inventors used reporter genes such as GFP and luciferase. The inventors then confirmed their findings using TRAIL or soluble TRAIL (sTRAIL), further demonstrating that scPP particles function surprisingly well for gene delivery. When using genes such as TRAIL, the inventors also demonstrated cancer cell death, indicating that the vector can be used to deliver therapeutic genes.

[0129] Materials and Methods Molecular cloning of constructs The green fluorescent protein (GFP) transgene cassette (flanked by the AAV2 ITRs), from the promoter to the polyadenylation signal, was amplified by PCR from the pAAV-GFP plasmid (Cell Biolabs) using primers containing PciI restriction sites. The plasmid backbone and PCR insert were then digested with PciI (NEB, UK) and ligated overnight with T4 ligase (NEB, UK). The construct was then transformed into DH5α Escherichia coli (E. coli). Plasmids were then extracted from different bacterial colonies by Miniprep (Qiagen) and verified by restriction enzyme digestion and DNA sequencing (Eurofins). Correct clones were then transformed into TG1 Mix&Go Competent E. coli (Zymo Research, USA) for phage vector generation. A schematic diagram of the cloning strategy is shown in Figure 3. To generate phage particles carrying TNFα, TRAIL, or IL15 transgenes, GFP was replaced with the corresponding DNA coding sequence.

[0130] Phage production TG1 Mix&Go (Zymo research, USA) was transformed with the backbone DNA construct of the vector. Double tandem vectors (control phage particles encoding both transgene copies in the same orientation, either clockwise (cw) or counterclockwise (aw)) were transfected at OD 600nmThe bacteria were grown in 2xYT broth until the rho value reached 0.3–0.6, indicating the bacterial exponential growth phase. At this point, the bacterial culture was infected with the appropriate helper phage (either targeted (RGD4C-displaying) or nontargeted (NT) M13 phage), and the culture was incubated at 37°C for 15 minutes. After this, the culture was added to 2xYT broth supplemented with 50 μg / ml kanamycin and 100 μg / ml carbenicillin antibiotics and grown overnight at 32°C and 160 rpm. The next day, the culture was spun down at 6,000 g for 15 minutes at 4°C. The supernatant was collected and mixed with 0.4 volumes of 21 mM PEG (MW 8000) / 3.36 M NaCl / 1% Triton X-100 and left overnight at 4°C. This solution was then centrifuged at 10,000 g for 30 minutes at 4°C. The pellet was resuspended in PBS, mixed with 0.5 volumes of 21 mM PEG / 3.36 M NaCl, and left overnight at 4 °C. A new centrifugation step was performed at 10,000 g for 30 min at 4 °C, and the pellet was resuspended in a small volume of phosphate-buffered saline (PBS) by gentle shaking at 120 rpm for 3 h at 37 °C. Residual bacterial contaminants were removed from the dissolved pellet by a 10-min centrifugation step at 10,000 g at room temperature, and the resulting supernatant was filtered through a 0.45 μm filter cartridge. The targeting purity of the generated phages was then checked by PCR to confirm the presence of the RGD4C coding sequence in the pIII capsid protein gene and further analyzed on a 2% agarose gel.

[0131] Phage particle titration Phage particles were quantified in prokaryotic hosts. Serial dilutions of phage were made in PBS and used to infect TG1 E. coli grown to logarithmic phase in 2xYT medium, followed by incubation at 37°C. After 20 minutes of incubation at 37°C in a water bath, the particle / bacteria mixture was again thoroughly mixed and plated onto solid agar media containing selective antibiotics. Because phage particles contain an ampicillin resistance gene, TYE top agar containing 100 μg / ml ampicillin was used. On the other hand, because helper phage contain a kanamycin resistance gene, TYE top agar containing 50 μg / ml kanamycin was used. Bacteria were plated on TYE top agar in the presence of ampicillin to determine the concentrations of scPP particles and kanamycin, and the concentration of helper phage present in the sample was determined by colony counting. Phage particles are expressed as bacterial transducing units (TU / μl).

[0132] Intramolecular self-hybridization of transgene cassettes in self-complementary phage particles (scPPs) The scPP was first treated with DNAse-I, and then its genome was extracted. Briefly, the sample was treated with 100 mM Tris-HCl 25 mM EDTA pH 8, and the phage capsid was dissolved with 4% SDS at 70 °C for 10 minutes. The sample was then mixed with 3 M potassium acetate, pH 5.5, and centrifuged at 12,000 g for 10 minutes at room temperature to precipitate the phage capsid protein. An anion exchange column (Midiprep Qiagen kit) was equilibrated with 0.1 M sodium acetate, pH 5.0, 0.6 M NaCl, 0.15% (v / v) Triton X-100. The supernatant from the centrifugation step was then loaded onto the column, and the solution was allowed to drain by gravity flow. The column was then washed twice with 0.1 M sodium acetate, pH 5.0, 825 mM NaCl. The sample was eluted with QF elution buffer (Qiagen). The resulting sample was further purified by isopropanol-ethanol precipitation and resuspended in TE buffer (Qiagen).

[0133] The concentration of the extracted phage genome was determined by the ssDNA sample (1 OD260 The DNA was measured assuming a concentration of 33 μg / ml ssDNA. The genome was then digested with BamHI (NEB, UK) and run on an agarose gel. The 2,000 bp band was gel extracted using a gel extraction kit (Qiagen) and precipitated with isopropanol-ethanol. Aliquots of the extracted DNA band and 1 kb Plus DNA ladder (Thermo Fisher) were then mixed with 0.5 mg / ml bromophenol blue, 8 M urea, 1% (v / v) Triton X-100, and 1 mM Tris pH 8. Half of each sample (extracted band and ladder) was then denatured at 80°C for 5 minutes. The denatured and undenatured versions of both the DNA ladder and extracted band were then loaded onto a denaturing 1 M urea 1.2% agarose gel and run on ice at 55 V for 4 hours. The gel was stained in a solution of 0.5 μg / μl ethidium bromide in TAE buffer for 2 hours at room temperature.

[0134] Because the DNA ladder size reference bands migrate differently under native and denatured conditions, an additional control was used to confirm the migration of these reference bands: To this end, linearized scPP-GFP plasmid (5,378 bp) and denatured samples of the ladder were run side-by-side in a urea denaturing gel.

[0135] Cell transduction and Lucia expression Adherent cells were seeded into well plates / tissue culture dishes of the desired size to achieve 70-80% confluence 48 hours after seeding. On the day of transduction, the average number of cells per well / dish culture was determined and used to calculate the amount of phage particles to add to the cells. The transduction mixture was then prepared by diluting the appropriate amount of particle stock solution into serum-free medium and then thoroughly mixing. The recommended amount of transduction mixture used per well / dish is the minimum amount required to completely cover the cell monolayer. To transduce the cells, the medium was discarded and the transduction mixture was added to the cells for 6-12 hours at 37°C in 5% CO2, followed by replenishment with an equal volume of complete medium. After 24 hours, the entire medium was discarded and replaced with fresh medium. The transduced cells were maintained in culture until analysis.

[0136] Quantification of secreted luciferase expression in culture medium At specific time points after transduction with phage particles carrying the Lucia DNA sequence, 10 μL of culture medium was collected from the wells and transferred to an opaque 96-microwell plate. Luciferase activity was quantified using QUANTI-Luc. Luciferase substrate was prepared according to the manufacturer's protocol (Invivogen, France) and added to the microwell plate. Luciferase activity was measured using a GloMax Discover Microplate Luminometer (Promega, UK). In these experiments, the culture medium was not changed at any time point.

[0137] GFP expression in transduced cells The cells transduced with scPP-GFP and the percentage of transduced cells were analyzed by either FACS or fluorescence microscopy.

[0138] Transmission electron microscopy (TEM) Carbon film-coated copper mesh grids were subjected to glow discharge to induce hydrophilicity. Phage particles were applied to the grid, allowed to incubate for 10–15 min, and then removed by blotting on absorbent paper. The grid was then washed with sterile-filtered deionized water, blotted twice on absorbent paper, and allowed to dry for 15 min. A 1% uranyl acetate solution was applied to the grid to negatively stain the particles for 30 s, followed by washing twice with sterile-filtered deionized water and drying. The grids were imaged using a scanning electron microscope (JEOL JEM-2010, UK) and analyzed using ImageJ software.

[0139] Gel agarose analysis of phage The phage stocks were analyzed by nanodrop to determine 30 μg of phage sample. These were then mixed 1:1 with 2x sample buffer (126 mM Tris-HCl, pH 6.8, 15% Ficoll® Type 400, and 0.002% bromophenol blue) and loaded onto a 0.8% agarose gel. The samples were run at 50 V for 5 hours, and then the gel was fixed overnight with a 10% acetic acid and 50% methanol solution. The next day, the gel was fixed for 3 hours with Coomassie blue staining solution and destained overnight with 20% methanol and 5% acetic acid. Bands were detected using a BioRad gel reader.

[0140] Phage particle fluorescent dye labeling Phage particles were labeled with FITC. 50 mL of particles (5 × 10 11 The phage particles (TU, total) were added to 200 μL of 5 mg / mL FITC (Sigma, UK) and mixed by rotation at room temperature for 1 hour in the dark. The particles were then precipitated by adding PEG / NaCl at a 25-30% total concentration overnight at 4°C. The solution was centrifuged at 13,000 rpm for 15 minutes to obtain a particle pellet. The pellet was resuspended in 250 μL of PBS and reprecipitated with PEG / NaCl until free FITC was completely removed. Finally, the FITC-conjugated phage particles were resuspended in PBS, and the titer was quantified using E. coli bacterial infection and colony counting.

[0141] Matrigel diffusion assay of phage particles 200 μl of Matrigel from 2.5 mg / ml Engelbreth-Holm-Swarm mouse sarcoma (Sigma, UK) was added to a 48-well plate and then transferred at 37°C. Meanwhile, FITC-labeled particles were prepared at a concentration of 5 μg / ml. 5 μl of each particle solution was pipetted into a gel-loading pipette tip, which was inserted into the Matrigel at a fixed position and left to diffuse. Fluorescent images were taken at 0 h and at 18-h intervals thereafter using a fluorescence microscope (Nikon Eclipse TE2000U, Japan) and analyzed using Openlab imaging software.

[0142] Particle internalization 1·10 6 TU / cell or 5·10 5 Cells were transduced using FITC-labeled particles at TU / cell. Six hours after transduction, cells were washed with PBS and detached with 2 mg / ml ice-cold pronase for 10 minutes on ice. The pronase was blocked using 20% ​​FBS, and the cells were centrifuged at 200 g for 5 minutes at room temperature. The pellet was resuspended in 20% FBS, followed by another centrifugation step. The pellet was resuspended in 4% paraformaldehyde and incubated at room temperature for 10 minutes. After incubation, the cells were centrifuged at 300 g for 5 minutes at room temperature and blocked with a solution consisting of 0.1% saponin and 2% BSA for 30 minutes at room temperature. The cells were pelleted at 300 g for 5 minutes at room temperature and stained with rabbit anti-fd phage antibody (Sigma 086k4860; 1:1000 dilution) in 0.1% saponin-1% BSA-PBS for 1 hour at room temperature. After incubation, cells were pelleted under the same conditions and washed with 0.1% saponin in 1% BSA-PBS. This washing step was repeated three times. Cells were labeled with goat anti-rabbit AlexaFluor-647 (Invitrogen 21245; 1:500 dilution) in 0.1% saponin in 1% BSA-PBS and incubated for 1 hour at room temperature protected from light. Cells were washed twice with 0.1% saponin-PBS and resuspended in PBS for the final step.

[0143] Next, flow cytometry analysis of intracellular phage particles was performed. FACS was performed using a BD FACscalibur flow cytometer (BD Biosciences) equipped with an argon ion laser (488 nm) and a red diode laser (635 nm). Mean fluorescence intensity and percentage were measured for at least 10,000 gated cells per triplicate well. Cell populations were gated and analyzed using FACScalibur software.

[0144] SDS-PAGE Samples were supplemented with loading dye (Laemmli buffer and β-mercaptoethanol) and loaded onto 4-15% mini-PROTEAN TGX Stain-Free™ gels. Tris-glycine-SDS (Sigma) 10x and NEB Protein Colour standard were used as running buffer and protein ladder, respectively.

[0145] Determination of helper phage contamination Phage samples were pretreated with DNAse-I for 30 min at 37°C. DNAse-I was then inactivated with 50 mM EDTA at 65°C for 10 min, and phage capsids were opened in the presence of 1% SDS by heating to 95°C for 10 min. After gradually lowering the temperature to 23°C in 3°C increments, SDS was captured with 1% Triton X-100, and samples were diluted 1:250 in DEPC water. scPAAV and helper phage plasmids were added at 2·10 8 ~2·10 3 Used to generate a standard curve of plasmid / μL.

[0146] [Table 1]

[0147] TNFα ELISA Cells were transduced with phage particles, and 24 hours after transduction, the medium was changed to fresh complete medium. Conditioned medium was collected on day 4 post-transduction, replaced with fresh medium, and collected again on day 6 post-transduction.

[0148] Enzyme-linked immunosorbent assay (ELISA) IL15 production in the supernatant after transduction was quantified using mouse IL15 duoset ELISA (R&D systems, UK).

[0149] TNFα concentrations in conditioned media were quantified using an ELISA MAX™ Standard Set kit according to the manufacturer's instructions.

[0150] For TRAIL ELISA, we coated the plate with a capture antibody. Then, the plate was washed twice with wash buffer (0.05% Tween 20 in PBS) and blocked by adding 1% BSA in PBS at room temperature for 1 hour. The plate was washed twice with wash buffer, and the sample was added to the plate and incubated at room temperature for 2 hours. Next, the detection antibody was incubated at room temperature for 1 hour. Avidin-HRP D was then added to each well, followed by the addition of substrate solution.

[0151] result Example 1 - Construction of a DNA backbone for hybridization of two self-complementary transgene expression cassettes Referring to Figures 1 and 2, schematic diagrams of the known adeno-associated virus / phage ("AAVP") vector (left) and the also known phagemid adeno-associated virus ("PAAV") vector (center) are shown, both of which are derived from the single-stranded M13 filamentous phage. While AAVP contains a complete phage genome and a single mammalian transgene cassette flanked by ITR sequences derived from the AAV2 virus, PAAV is based on a phagemid design, which contains a single transgene cassette flanked by ITRs and requires a helper phage to provide the structural gene during production. The problem with AAVP and PAAV is that upon processing of mammalian cells, these two vectors deliver single-stranded DNA of the transgene cassette, which must be converted to double-stranded DNA for gene expression and transduction to occur. This process, which relies on mammalian cellular factors, is inefficient, resulting in a delayed onset of gene expression and a subsequent slow, inefficient increase in gene delivery over time.

[0152] We previously showed that transducing cells with two phage vectors carrying complementary sequences of a mammalian transgene expression cassette does not enhance gene delivery. Therefore, we sought to provide the complementary sequences of a transgene cassette in a single phage vector (FIG. 1), or in other words, to design a phage vector carrying both the transgene cassette and its complementary sequences to induce hybridization (i) during cell transduction or (ii) during production and manufacturing in a bacterial host (FIG. 2).

[0153] Figures 1 and 2 (right) show the single-stranded (SS) self-complementary phagemid backbone ("self-complementary phage particle or scPP") used for phage generation according to the present invention. Indeed, the self-complementary phagemid backbone provides the ability for two single-stranded, self-complementary transgene cassettes to hybridize and form a double-stranded transgene cassette. While AAVP and PAAV contain only one copy of the expression cassette, the scPP of the present invention carries an additional transgene cassette compared to AAVP and PAAV. The two cassettes are identical and separated by an inverted terminal repeat (ITR) linker, but their sequences are read in opposite directions; i.e., the first cassette extends in the 5' to 3' direction and the second cassette extends in the 3' to 5' direction, as shown in Figure 1. As shown in Figure 2, the phagemid of the present invention allows hybridization between the two self-complementary transgene expression cassettes on either side of the ITR linker, thereby generating a dsDNA that resembles a hairpin loop structure. A second AAV ITR is included to flank one of the transgene cassettes.

[0154] To avoid the circular phage genome, which may affect the process of double-stranded DNA formation, we used phagemids instead of phages, removing the phage genome and retaining only the origin of replication f1 to enable replication and packaging of the transgene cassette in bacteria (Figures 1 and 2). Due to the absence of the phage genome, we used helper phage to infect bacteria and provide the structural genes encoding the packaging coat proteins required for replication (Figures 1 and 2). Phages do not have tropism for mammalian cells, so to enable vector entry into cells, we displayed a double-circular RGD4C ligand on the helper phage, which has been extensively characterized and used for phage-mediated gene delivery (Figures 1 and 2). This ligand enables phage entry into mammalian cells by binding to the αvβ3 integrin heterodimer receptor, which is primarily expressed on the surface of cancer cells. We used RGD4C / αvβ3 as a ligand-receptor system to demonstrate proof-of-concept for our new platform technology.

[0155] In this novel design, two complementary mammalian transgene cassettes were linked using ITRs from AAV2 (Figures 1 and 2). We also included a second ITR flanking the parental transgene cassette to preserve it upon cell transduction and improve its persistence over time (Figures 1 and 2).

[0156] Example 2 - scPP shows a significant increase in gene delivery compared to PAAV In the first set of experiments, we investigated gene delivery using a newly designed phage vector (scPP) to determine whether the scPP vector could function better in mammalian cells than the corresponding single-stranded phage vector control (PAAV). Therefore, we compared gene expression from scPP in parallel with PAAV. To transduce cells, we generated tumor-targeting phage particles displaying a double-circular RGD4C in the pIII gene of filamentous M13KO7 helper phage. The RGD4C ligand binds to the αvβ3 integrin heterodimer receptor overexpressed on tumor cells and tumor vasculature, but is barely detectable in healthy tissues. This ligand is widely used to enable M13 phage vector entry into mammalian cells. A non-targeting vector lacking RGD4C was also included as a negative control and added to cells.

[0157] First, we treated mouse melanoma B16-F1 cells with a vector expressing a green fluorescent protein (GFP) reporter gene (Figure 3), because these cells express the αvβ3 receptor for the RGD4C ligand. Four days after transduction, microscopic analysis of GFP expression revealed widespread GFP production in B16-F1 tumor cells transduced with RGD4C.scPP-GFP, which was significantly higher than that in cells treated with RGD4C.PAAV-GFP (Figure 4A). Furthermore, analysis of GFP expression by FACS revealed that RGD4C.scPP-GFP significantly increased the GFP expression in 10 6 Compared with RGD4C.PAAV-GFP, which generated less than 5% GFP-positive cells in TU / cell, 10 6 We demonstrated that targeted gene delivery produced dose-dependent GFP expression, reaching over 35% GFP-positive cells in TU / cell (Figure 4B). Importantly, no GFP expression was detected in cells treated with non-targeted phage particles (NT) lacking the RGD4C ligand, demonstrating that gene delivery by targeted particles remains selective for integrin-expressing cells and is mediated by the RGD4C ligand (Figure 4B).

[0158] To validate these data, we next performed a comprehensive quantitative analysis of gene delivery using particles carrying a reporter gene encoding secreted Gaussia luciferase (Lucia) (8, 16) (Figure 5). Gene expression was quantified by analyzing luciferase activity in the growth medium. We tested various particle doses and assessed gene expression over a time course of several days. Furthermore, we assembled a panel of tumor cell lines from different species and histological origins to rule out the possibility that the observed gene delivery efficacy of RGD4C.scPP was species- or histologically-specific. Transduction was performed using mouse melanoma B16-F1 and B16-F10, as well as RMS metastatic melanoma cells. We also included human MCF7 breast cancer cells, A549 lung cancer cells, and human osteosarcoma cells. Additionally, we tested the vectors on human embryonic kidney (HEK293) cells because these cells are widely used for general gene delivery, viral and non-viral transduction, and DNA transfection, and have previously been used as a standard in vitro model for phage-mediated gene delivery. The data revealed that gene expression from RGD4C.scPP particles was detected as early as 1–2 days after treatment and gradually increased over time at all doses tested (Figures 6–14). In contrast, the onset of gene expression with RGD4C.PAAV was delayed and consistently significantly lower than that of RGD4C.scPP across all time points, doses, and cell lines tested (Figures 6–14). Control non-targeted particles did not show any gene expression (Figures 6–14). These findings indicate that the observed enhanced gene expression could be the result of earlier induction and a larger number of successfully transduced cells, or perhaps a combination of these two non-mutually exclusive events.

[0159] Finally, to confirm that this advantage of scPP-mediated gene delivery is not limited to the reporter genes GFP or Lucia but may also apply to therapeutic genes, we constructed vectors carrying the cytokines tumor necrosis factor α (TNFα) and interleukin-15 (IL-15), which are used in cancer immunotherapy (8). Expression of these two cytokines was measured at the protein level in the cell culture medium by ELISA after phage transduction. Similarly, the data show that the newly designed scPP particles expressed significantly higher levels of both TNFα and IL-15 in the cell culture medium compared with PAAV (Figures 15-17). We also constructed a vector carrying another cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and again confirmed that scPP produced substantially higher levels of TRAIL in the supernatant of human osteosarcoma cells compared with cells treated with PAAV (Figure 18).

[0160] Example 3 - Comparison of in vivo gene delivery to solid tumors in mice upon systemic administration To translate these findings into in vivo studies, we compared gene delivery between scPP and PAAV after intravenous administration to tumor-bearing mice. We used a vector delivering TRAIL and injected it into immunodeficient mice bearing an established human subcutaneous xenograft, osteosarcoma. To this end, tumor-bearing mice were injected with 5 × 10 vectors, the dose previously used for phage-based vectors. 10Mice were treated with TU / AAV, and then RT-qPCR was applied to identify the expression of TRAIL mRNA transcripts in the tumors (Figure 19). A biodistribution study of scPP was also conducted in parallel with PAAV in tumor-bearing mice to analyze gene delivery in tumors relative to vital organs. These biodistribution experiments confirmed that gene expression was selective to established tumors in mice after intravenous administration of RGD4C.scPP particles, without any expression in healthy tissues, which could lead to off-target effects. Significantly higher expression of TRAIL mRNA transcripts was detected in tumors of mice injected with RGD4C.scPP compared to RGD4C.PssAAV (Figure 19). Furthermore, TRAIL expression in healthy tissues was not significant and was similar to that of the control group, indicating that RGD4C.scPP efficiently and systemically targets tumors while sparing other vital organs. Non-targeted particles showed no significant expression in tumors or any of the organs studied.

[0161] Example 4 - Examination of the mechanism of transgene expression To understand the molecular mechanisms of scPP-mediated transgene expression and its advantages over PAAV, we investigated the extracellular and intracellular fate of the particles after processing of mammalian cells and compared scPP with PAAV in parallel for various steps of gene delivery.

[0162] Diffusion through the ECM The diffusion efficiency was evaluated using FITC-labeled particles and their migration ability in Matrigel support (Figure 20). No obvious differences were detected between both constructs, suggesting a similar diffusion profile through the extracellular matrix (ECM). This is consistent with previous studies reporting that the diffusion of M13 phage vectors is determined by particle size (8). Indeed, ECM analysis of scPP and PAAV indicates that these two particles do not exhibit size differences.

[0163] Internal migration Next, we attempted to examine phage invasion in transduced cells. Therefore, B16-F1 cells were transduced and treated 6 hours after transduction using two different approaches. In the first method, particles were stained with anti-fd phage and quantified by flow cytometry (Fig. 21A). In the second method, DNA was purified and quantified by qPCR using the ampicillin gene present in the phagemid as a target (Fig. 21B). Again, no difference was detected between the two types of particles.

[0164] Example 5 - Increased gene delivery efficacy of scPP is not due to the presence of two transgene expression cassettes Because the two vectors did not show any differences in ECM diffusion and cell entry, and to gain further insight into the mechanisms behind the differences between scPP and PAAV, we investigated whether the gene delivery advantage of scPP arises because this vector carries an additional transgene expression cassette. Therefore, we generated a control vector containing two copies of the transgene cassette but in the same clockwise (cw) orientation to avoid sequence complementarity and hybridization. This vector was named cw phage particle or cwPP (Figure 22A). To account for any potential effects of the specific orientation of the two transgene cassettes, a second control vector was constructed containing both transgene expression cassettes in a counterclockwise (aw) orientation and named awPP (Figure 22A). Parallel transduction with scPP and the control vector then demonstrated improved efficiency of the scPP vector over both the cwPP and awPP controls (Figure 22B). Furthermore, to investigate whether intermolecular hybridization between complementary transgene sequences provided in separate vectors can recapitulate scPP efficiency, we performed cotransduction with cwPP and awPP vectors (Figure 23A). This cotransduction increased efficiency over cwPP and awPP alone, but was still lower than the efficiency of scPP (Figure 23B). Overall, these data suggest that the improved efficiency observed for the scPP vector is related to its ability to achieve successful intramolecular hybridization.

[0165] Example 6 - Particle Size The similarities between scPP and PAAV particles in diffusion and internalization suggest that the particle sizes of the two species are not different, as recently reported (8). Therefore, we hypothesized that scPP should package a compacted genome, resulting in particle sizes similar to PAAV particles. As an initial investigation, we analyzed both scPP and PAAV particles by transmission electron microscopy (TEM) and quantified the length of individual phage particles (Figure 24). To aid in the identification of helper phage populations in scPP and PAAV preparations, we also analyzed helper phage. Importantly, TEM imaging revealed very similar sizes between scPP and PAAV particles (Figure 24). To further support these findings, we also analyzed the size of cwPP particles by EM and found that the cwPP vector was increased in size compared to scPP (Figure 24). These results were confirmed in parallel by agarose gel electrophoresis of whole phage particles (Figure 25).

[0166] Again, these findings indicate that scPP is a more efficient vector than PAAV. Because no differences in spreading and cell entry were detected, the differences are likely related to their distinct genome designs. This is supported by the similar particle size between the two vectors, which could be explained by the more compact genome packaged by self-complementary phage particles (scPPs), which may be the result of self-hybridization between two complementary transgene expression cassettes. Indeed, the fact that control particles encoding two copies of the transgene cassette in the same orientation cannot reproduce scPP transduction efficiency suggests that the presence of dual transgene cassette loads, either individually or in combination, is not directly responsible for the improvement; rather, it supports the self-hybridization formation of double-stranded DNA transgene cassettes during phage production as the mechanism behind the superiority of scPP.

[0167] Example 7 – scPP capsids package double-stranded transgene DNA cassettes To demonstrate the possibility that the scPP genome forms a dsDNA structure and is packaged by phage capsids as dsDNA, we extracted the scPP genome from phage capsids / particles and then digested it with BamHI, a dsDNA-digesting enzyme whose target sequence is located within the transgene cassette. In other words, successful digestion by BamHI can only occur in the presence of dsDNA (Figure 26A). As expected, a 1,898-bp band was detected in the digested sample but was absent in the undigested control (Figure 26B). To further demonstrate that this band was indeed generated by the self-hybridization of a unique ssDNA molecule, we predicted that this molecule would unfold and migrate as a 3,796-kb band under denaturing conditions that cause DNA dehybridization (Figure 26A).

[0168] Under denaturing conditions, the 5,000 bp DNA ladder band generated two distinct bands (due to separation of its complementary strands) (Figure 26C), indicating that the 4,000 bp reference band corresponds to the sixth band in the denatured ladder. As hypothesized, the extracted band in its denatured form, predicted to be 3,796 bp in length, migrated at approximately the same speed as the 4,000 bp band present in the denatured ladder (Figure 26D).

[0169] These findings provide strong evidence that the phage capsid packages a dsDNA transgene cassette during its production in bacteria, and that this dsDNA transgene cassette arises from the self-hybridization of two complementary transgene cassettes of the scPP during its production in the host bacterium.

[0170] Example 8 - scPP particles deliver double-stranded transgene expression cassettes during transduction of mammalian cells Next, we sought to determine whether scPP vectors deliver double-stranded transgene expression cassettes that do not require host cell synthesis of the complementary strand of the transgene cassette for transduction and gene expression to occur. Indeed, we predicted that if these vectors could deliver double-stranded transgene cassettes upon entry into mammalian cells, they would obviate the role of host cell DNA synthesis in transduction.

[0171] We compared the scPP-Lucia vector and PAAV-Lucia in B16-F1 cells pretreated with hydroxyurea (HU) 24 hours before transduction to inhibit host cell DNA synthesis. Hydroxyurea treatment was continued at the same concentration after transduction and uninterrupted, and cells were maintained until Lucia expression was measured. Importantly, unlike the conventional single-stranded phage vector PAAV, hydroxyurea, an inhibitor of DNA replication, did not affect transduction from the scPP vector (Figure 27). In contrast, hydroxyurea suppressed gene expression from PAAV (Figure 27). These data demonstrate that transduction by scPP is independent of DNA synthesis and subsequent ss-to-ds conversion of the transgene cassette.

[0172] Example 8 – Comparison of Lucia reporter gene delivery Referring to Figure 28, a comparison of Lucia reporter gene delivery into metastatic human osteosarcoma 143B cells between PAAV and scPP is shown over a time course from days 1 to 3 after treatment with increasing doses of the vector. On days 1 and 2 after transduction, no Lucia gene expression was observed in the control groups (untreated and NT). However, in the RGD4C.scPP-treated groups at 500,000 and 1,000,000 TU / cell, Lucia gene expression was observed on day 1 after transduction, but not in the PAAV-treated group. This confirms the effectiveness of the self-complementary scPP vector for immediate transgene expression. On days 2 and 3 after transduction, the RGD4C.scPP-treated groups (100,000 to 1,000,000 TU / cell) showed higher Lucia expression than the RGD4C.PAAV-treated group.

[0173] method: 143B cells were seeded into 96-well culture plates to achieve 60-70% confluence 48 hours after seeding. The average cell number per well or plate culture was calculated on the day of transduction and used to calculate the amount of tumor-targeted RGD4C-PAAV or scPP particles carrying a secreted luciferase (lucia) gene to add to the cultures. Non-targeted (NT) phage carrying the same gene and untreated cells were used as controls. An appropriate amount of particle stock solution was then diluted with 10% DMEM medium and thoroughly mixed to prepare the transduction mixture. The concentration of the transduction mixture ranged from 100,000 to 1,000,000 transducing units (TU) per cell. The minimum volume (50 μl) required to completely cover the cell monolayer is the recommended amount of transduction mixture used per well. 24 hours after transduction, the medium was replenished with 10% DMEM medium to 150 μL. Transduced cells were cultured until analysis (days 1 to 3).

[0174] To assess and quantify gene expression of phage (PAAV or scPP) particles carrying a secreted luciferase reporter gene, 10 μl of medium was collected every day after transduction, mixed with 25 μl of QUANTI-Luc™ (InvivoGen, USA) reagent for 5 minutes, and subjected to measurement of luciferase activity on a GloMax® Navigator Microplate Luminometer (Promega Corporation, USA) with an integration time of 0.1 seconds.

[0175] Example 9 – Comparison of delivery of the secreted cytokine sTRAIL to metastatic human osteosarcoma 143B cells Referring to Figure 29, sTRAIL gene expression in the culture medium of metastatic human osteosarcoma 143B cells transfected with PAAV or scPP DNA constructs is shown. Untreated cells and transfection reagent-treated cells were used as controls. sTRAIL protein levels (pg / ml) were measured by TRAIL ELISA kit. Experiments were performed in three biological replicates. For statistical analysis, unpaired t-test, one-way ANOVA, and Tukey's HSD post-hoc test were used. All results are presented as mean ± SEM. *** P<0.01 and **** *P<0.001. The data show that cells transfected with the scPP-sTRAIL DNA construct expressed higher levels of sTRAIL in the culture medium than the PAAV-sTRAIL DNA construct.

[0176] method: 143B cells were seeded into 6-well culture plates and grown for 24 hours to reach 80% confluence. The culture medium was changed to low-serum medium (Opti-MEM, Thermofisher, UK) 2 hours prior to transfection. The transfection mixture was prepared by adding 2 μg of PAAV-sTRAIL or scPP-sTRAIL DNA construct to 6 μl of FuGENE® HD (Promega, UK) in low-serum medium. The mixture was incubated at room temperature for 20–25 minutes. Next, this mixture was added dropwise to the culture plate containing cells in low-serum medium. The cells were then returned to the incubator for 48 hours. Finally, the culture medium was collected and TRAIL levels were quantified by ELISA. * sTRAIL = secreted TRAIL. Levels of secreted sTRAIL in the supernatant were measured using Human TRAIL / TNFSF10 DuoSet ELISA (R&D systems, UK). The assay was performed according to the manufacturer's protocol.

[0177] Example 10 - Induction of osteosarcoma cell death in vitro after treatment with scPP-sTRAIL encoding a secreted form of sTRAIL Referring to Figure 30, the RGD4C.scPP-sTRAIL particle-treated group exhibited a dose-dependently reduced cell viability. Data are presented as percent cell viability compared to untreated cells. Experiments were performed in three biological replicates. Statistical analysis was performed using one-way ANOVA with Tukey's HSD post-hoc test. All results are presented as mean ± SEM. ** P<0.05.

[0178] method: 143B cells were seeded into 6-well culture plates to achieve 60-70% confluence 48 hours after seeding. The average cell number per well was calculated on the day of transduction and used to calculate the amount of tumor-targeted RGD4C.scPP particles carrying the secreted TRAIL (sTRAIL) gene to add to the cultures. Non-targeting (NT) phage carrying the same gene and untreated cells were used as controls. An appropriate amount of particle stock solution was then diluted with 10% DMEM medium and thoroughly mixed to prepare the transduction mixture. The concentration of the transduction mixture ranged from 500,000 to 1,000,000 transducing units (TU) per cell (shown in the graph as 0.5 and 1.0, respectively). The minimum volume of transduction mixture (1 ml) required to completely cover the cell monolayer is the recommended amount of transduction mixture used per well. 24 hours after transduction, the medium was replenished with 10% DMEM medium to 2 mL. The transduced cells were cultured for an additional 3 days, and a cell viability assay was performed to assess cell death.

[0179] CellTiter-Glo Luminescent Cell Viability Assay: CellTiter-Glo reagent (Promega, UK) was added in an equal volume to the culture medium present in the culture wells containing transduced cells, followed by mixing on an orbital shaker for 2 minutes to induce cell lysis. The mixture was then incubated at room temperature for 10 minutes to stabilize the luminescent signal and transferred to a luminometer for plate reading. The signal was detected using a GloMax Navigator Microplate Luminometer (Promega, UK).

[0180] Example 11 - Toxicity Assessment. No Increase in the Toxicity Biomarker LDH (Lactate Dehydrogenase) in Mice Referring to Figure 31, it is shown that both scPP-sTRAIL and PAAV-sTRAIL particle-treated mice did not have increased serum LDH levels compared to untreated mice. This data suggests that both PAAV and scPP are safe for in vivo treatment. LDH data were shown as relative values ​​compared to the untreated group. The experiment was performed with three biological replicates. For statistical analysis, one-way ANOVA with Tukey's HSD post-hoc test was used. There is no statistical significance in this experiment.

[0181] method: Athymic mice (BALB / c nu / nu, 8–10 weeks old) were obtained from Charles River, UK. Human OS cells were cultured at 2 × 10 per mouse. 6 Tumors were established subcutaneously in athymic mice using 143B cells. On days 3, 5, and 9 of the experiment, tumor-bearing mice were injected with targeted (RGD4C) or non-targeted (NT) phage (PAAV or scPP) particles carrying the sTRAIL gene at 5 × 10 per mouse. 10 The mice were intravenously injected with a dose of TU. At the end of the experiment (day 10), the mice were sacrificed by terminal perfusion through the heart. Whole blood was then collected from the heart, and serum samples were prepared by centrifugation at 1,600 g for 15 minutes. LDH levels in the serum were measured to evaluate the toxicity of the phage treatment. CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, UK) was used in this experiment. The assay was performed according to the manufacturer's protocol.

[0182] Example 12 – Biodistribution of sTRAIL delivery in tumor-bearing mice with established osteosarcoma Referring to Figure 32, the data show the relative expression of the human TRAIL gene (compared to the untreated group) from different organs of mice after treatment with PAAV or scPP particles carrying the sTRAIL gene. RGD4C.scPP-sTRAIL particles most effectively targeted and delivered genes to tumors, followed by RGD4C.PAAV-sTRAIL. Non-targeted particles showed no significant expression in tumors or any other organs. Experiments were performed with three biological replicates. For statistical analysis, a two-way ANOVA with multiple comparison t-test was used. All results are presented as mean ± SEM. *** P<0.01.

[0183] method: Athymic mice (BALB / c nu / nu, 8–10 weeks old) were obtained from Charles River, UK. Human OS cells were cultured at 2 × 10 per mouse. 6 Tumors were established subcutaneously in athymic mice using 143B cells. On days 3, 5, and 9 of the experiment, tumor-bearing mice were injected with targeted (RGD4C) or non-targeted (NT) phage (PAAV or scPP) particles carrying the sTRAIL gene at 5 × 10 per mouse. 10 The mice were intravenously injected with a dose of TU. At the end of the experiment (day 10), the mice were sacrificed by terminal cardiac perfusion. Tumors and normal organs, including lungs, liver, spleen, heart, kidneys, pancreas, and brain, were collected. Total RNA was extracted from these organs, and human TRAIL expression was measured by RT-qPCR.

[0184] Example 13 -Immunofluorescence staining of tumors showing sTRAIL protein expression after treatment with RGD4C.PAAV and RGD4C.scPP encoding sTRAIL Referring to Figure 33, confocal microscopy analysis showed that TRAIL expression (green) was detected only in tumors from the RGD4C.PAAV.sTRAIL-treated and RGD4C.scPP.sTRAIL-treated groups. Higher expression of TRAIL was observed in scPP-treated tumors. These findings demonstrate that RGD4C.scPP.sTRAIL effectively and comprehensively targets tumors. Non-targeting (NT) phage particles did not show appreciable TRAIL expression in tumors.

[0185] method: Athymic mice (BALB / c nu / nu, 8–10 weeks old) were obtained from Charles River, UK. Human OS cells were cultured at 2 × 10 per mouse. 6 Tumors were established subcutaneously in athymic mice using 143B cells. On days 3, 5, and 9 of the experiment, tumor-bearing mice were injected with targeted (RGD4C) or non-targeted (NT) phage (PAAV or scPP) particles carrying the sTRAIL gene at 5 × 10 per mouse. 10At the end of the experiment (day 10), mice were sacrificed by terminal cardiac perfusion. Tumors were harvested and processed for frozen sections. Human TRAIL expression in tumor masses was measured by immunofluorescence staining. TRAIL expression was assessed on 6-μm optimal cutting temperature compound (OCT) frozen sections of tissues using an antibody against human TRAIL. Sections were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) for 15 minutes at room temperature. Sections were then incubated with 5% normal goat serum in TBS (Tris-buffered saline) containing 0.3% Triton-X for 1 hour, followed by incubation with a primary antibody (rabbit anti-human TRAIL polyclonal antibody (Thermo Fisher Scientific, UK)). Subsequently, tissue sections were incubated with Alexa Fluor® 488-conjugated goat anti-rabbit IgG in TBS containing 1% filtered BSA and 0.3% Triton-X for 30 minutes. After three washes with PBS, slides were mounted with Prolong Gold anti-fade mountant (Life Technologies, UK). Cell sections were imaged using a DMi8 Evolve confocal fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

[0186] Example 14 – Hematoxylin and eosin staining of tumors showing extensive tumor damage after systemic treatment Referring to Figure 34, hematoxylin and eosin staining of tumors showing extensive tumor damage after systemic treatment with RGD4C.scPP-sTRAIL compared to untreated mice or mice treated with a non-targeted (NT) vector is shown.

[0187] method: Athymic mice (BALB / c nu / nu, 8–10 weeks old) were obtained from Charles River, UK. Human OS cells were cultured at 2 × 10 per mouse. 6 Tumors were established subcutaneously in athymic mice using 143B cells. On days 3, 5, and 9 of the experiment, tumor-bearing mice were injected with targeted (RGD4C) or non-targeted (NT) phage (PAAV or scPP) particles carrying the sTRAIL gene at 5 × 10 per mouse. 10 At the end of the experiment (day 10), the mice were sacrificed by terminal cardiac perfusion. Tumors were harvested and processed for frozen sectioning. Optimal cutting temperature compound (OCT) frozen sections of 6 μm of the tissue were prepared and subjected to hematoxylin and eosin staining.

[0188] Conclusion We have generated novel phage vectors containing complementary single-stranded sequences of transgene expression cassettes to induce hybridization during cell transduction or during production and manufacturing in bacterial hosts. We conducted in vitro experiments using various cell lines and transgenes and observed a striking increase (3- to 15-fold) in transduction efficiency from our self-complementary phage particles (scPPs) over conventional, exclusively ssDNA phage vectors. Indeed, the self-complementary phage vectors demonstrated rapid initiation and higher levels of transgene expression in all cell lines tested. More importantly, unlike conventional single-stranded phage vectors, inhibitors of DNA replication did not affect transduction from the self-complementary phage vectors. Additionally, in vivo studies demonstrated significantly enhanced gene delivery to solid tumors in mice over ssDNA phage particles upon systemic administration. All of these biological attributes support the generation and characterization of a new class of filamentous phage vectors containing self-complementary single-stranded DNA that hybridizes such that the transgene cassette is delivered as double-stranded DNA, which should contribute significantly to the ongoing evolution of phage-based gene delivery systems.

[0189] The technology described herein has several unique features, including packaging of hybridizable self-complementary ssDNA (i.e., dsDNA) transgene cassettes by M13 phage capsids. This system also allows for packaging of larger genomes by using two ITRs instead of three, compared to existing technologies. Furthermore, this technology allows for rapid initiation of gene expression in mammalian cells by M13 phage compared to existing phage vectors. This is also the first demonstration of hybridization between a hybridizable self-complementary ssDNA (i.e., dsAAV) genome and a phage capsid. In other words, it is the first hybrid vector between a phage capsid and hybridizable complementary ssDNA (i.e., ds rAAV). Furthermore, this is the first report of the ability to package and deliver a hybridizable complementary ssDNA transgene cassette (i.e., a ds transgene cassette) ready for gene expression initiation in mammalian cells. Furthermore, because we used a transgene cassette flanked by AAV ITRs, this is the first report demonstrating packaging of ds AAV DNA (from hybridizable complementary ssDNA) and delivery of ds AAV DNA into mammalian cells using phage capsids (since AAV capsids are absent).

[0190] In addition, the phage vector of the present invention allows for faster initiation of phage-mediated gene expression in mammalian cells than existing phage vectors. Furthermore, while delivery of ds AAV vectors is expensive, using phage capsids to deliver ds AAV DNA, as in the present invention, is highly cost-effective because production of this delivery system occurs in bacteria and utilizes economical production and purification processes for phage vectors in prokaryotic hosts that are compatible with industrial-scale reactors and separation systems. This also allows for scale-up of production, which should directly reduce costs.

[0191] The present inventors have corroborated their findings using soluble TRAIL (sTRAIL), demonstrating that scPP particles work surprisingly well for gene delivery. For example, when using genes such as TRAIL, the present inventors demonstrated cancer cell death, indicating that the scPP vectors can be used to deliver therapeutic genes in vivo or in vitro.

[0192] The inventors believe that this technology will have an impact on the field of phage gene delivery as well as AAV gene therapy and systemic delivery in general. Because the phage capsid has no tropism for human tissues and can therefore be delivered systemically to target diseased tissues via ligands displayed on the phage capsid, enabling the entry and delivery of therapeutic DNA, this delivery platform can be applied to systemic gene therapy for cancer and other human diseases.

[0193] References 1- Ivanenkov V., Felici F., Menon AG “Uptake and intracellular fate of phage display vectors in mammalian cells” Biochim.Biophys.Acta 1999, 1448:450-462. 2- Di Giovine M., Salone B., Martina Y., Amati V., Zambruno G., Cundari E., Failla CM, Saggio I. “Binding properties, cell delivery, and gene transfer of adenoviral penton base displaying bacteriophage” Virology 2004, 282:102-112. 3- Piersanti S.、Cherubini G.、Martina Y.、Salone B.、Avitabile D.、Grosso F.、Cundari E.、Di Zenzo G.、Saggio I “Mammalian cell transduction and internalization properties of lambda phages displaying the full-length adenoviral penton base or its central domain” J.Mol.Med. 2004、82:467-476. 4- Larocca D.、Witte A.、Johnson W.、Pierce G.F.、Baird A. “Targeting bacteriophage to mammalian cell surface receptors for gene delivery” Hum.Gene.Ther. 1998、9:2393-2399 5- Hart S.L.、Knight A.M.、Harbottle R.P.、Mistry A.、Hunger H.D.、Cutler D.F.、Williamson R.、Coutelle C. “Cell binding and internalization by filamentous phage displaying a cyclic Arg-Gly-Asp-containing peptide” J.Biol.Chem. 1994、269:12468-12474. 6- Hajitou A., Trepel M., Lilley CE, Soghomonyan S., Alauddin MM, Marini FC, 3rd, Restel BH, Ozawa MG, Moya CA, Rangel R., Sun Y., Zaoui K., Schmidt M., von Kalle C.、Weitzman MD、Gelovani JG、Pasqualini R.、Arap W. “A hybrid vector for ligand-directed tumor targeting and molecular imaging” Cell 2006、125:385-398. 7- Yata, T., Lee, ELQ, Suwan, K. Syed, N., Asavarut, P. and Hajitou A. “Modulation of Extracellular Matrix in Cancer is Associated with Enhanced Tumor Cell Targeting by Bacteriophage Vectors.” Mol.Cancer 2015, 14:110. 8- Asavarut, A., Waramit, S., Suwan, K., Marais, GJK, Chongchai, A., Benjathummarak, S., Al-Bahrani, M., Vila-Gomez, P., Williams, M., Kongtawelert, P., Yata, T., and Hajitou, A. et al. “Systemically Targeted Cancer Immunotherapy and Gene Delivery using Transmorphic Particles” EMBO Mol.Med. 2022, 14:e1 9 - Stoneham, C.A., Hollinshead, M., and Hajitou A. "Clathrin-Mediated Endocytosis and Subsequent Endo-lysosomal Trafficking of Adeno-associated Virus / phage" J. Biol. Chem. 2012, 287:35849-35859. 10 - Suwan K., Yata T., Waramit S., Przystal J.M., Stoneham C.A., Bentayebi K., Asavarut P., Chongchai A., Pothachareon P., Lee K.Y., Topanurak S., Smith T.L., Gelovani J.G., Sidman R.L., Pasqualini R., Arap W., and Hajitou A. "Next-generation of targeted AAVP vectors for systemic transgene delivery against cancer" Proc Natl. Acad. Sci. USA. 2019, 116:18571-18577. 11 - Kia, A., Przystal, J.M., Nianiaris, N., Mazarakis, N.D., Mintz, P.J., and Hajitou A. "Dual Systemic Tumor Targeting with Ligand-directed Phage and Grp78 Promoter Induces Tumor Regression" Mol. Cancer Ther. 2012, 11:2566-2577. 12- Przystal JM, Waramit S, Pranjol MZI, Yan W, Chu G, Chongchai A, Samarth G, Olaciregui NG, Tabatabai G, Carcaboso AM, Aboagye EO, Suwan K, and Hajitou A. “Efficacy of systemic temozolomide-activated phage-targeted gene therapy in human glioblastoma.” EMBO.Mol.Med. 2019, 11:e8 13- Tsafa E., Bentayebi K., Topanurak S., Yata T., Przystal J., Fongmoon D., Hajji N., Waramit S., Suwan K., and Hajitou A. “Doxorubicin Improves Cancer Cell Targeting by Filamentous Phage Gene Delivery Vectors” . Int.J.Mol.Sci. 2020. 21:7 14- Monaci P.、Urbanelli L.and Fontana L. “Phage as Gene Delivery Vectors” Curr.Opin.Mol.Ther. 2001. 3:159–169. 15- Burg MA、Jensen-Pergakes K.、Gonzalez AM、Ravey P.、Baird A.、Larocca D. “Enhanced phagemid particle gene transfer in camptothecin-treated carcinoma cells.” Cancer Res. 2002. 62:977–981. 16- Wurdinger T.、Badr C.、Pike L.、de Kleine R.、Weissleder R.、Breakefield XO、Tannous BA “A secreted luciferase for ex vivo monitoring of in vivo processes” Nat Methods 2008、17:17.

Claims

1. A phage vector comprising at least two single-stranded, self-complementary transgene expression cassettes separated by a linker that hybridize to form a double-stranded transgene expression cassette.

2. 2. The phage vector of claim 1, wherein the phage vector comprises a packaging signal to enable replication of the at least two single-stranded self-complementary transgene expression cassettes, and the at least two single-stranded self-complementary transgene expression cassettes are capable of hybridizing in bacteria and subsequently being packaged as a double-stranded transgene expression cassette in the phage vector within a prokaryotic host.

3. The phage vector of claim 2 , wherein the packaging signal comprises a bacteriophage origin of replication, which may be an F1 ori.

4. The phage vector of any one of claims 1 to 3, wherein the phage vector comprises a bacterial origin of replication, which may be a pUC ori.

5. The phage vector of any one of claims 1 to 4, wherein the phage vector comprises one or more DNA sequences that allow targeted integration into a host genome.

6. 6. The phage vector of claim 1, wherein the at least two self-complementary transgene expression cassettes comprise viral transgene expression cassettes, preferably mammalian viral transgene expression cassettes.

7. 7. The phage vector of claim 1, wherein the at least two self-complementary transgene expression cassettes comprise a lentiviral transgene expression cassette or an adeno-associated viral (AAV) transgene expression cassette.

8. The phage vector of any one of claims 1 to 7, wherein the at least two self-complementary transgene expression cassettes comprise any nucleic acid encoding a drug that may have therapeutic or industrial utility in a target cell or tissue, and the nucleic acid may be DNA, genomic DNA, cDNA, RNA, antisense RNA or shRNA.

9. The phage vector of claim 8 , wherein the agent encoded by the nucleic acid is a polypeptide or protein.

10. 10. The phage vector of claim 1, wherein the at least two transgene expression cassettes each comprise a promoter, and the promoter may be a CMV promoter, a grp78 promoter, a tumor-specific promoter, or a tissue-specific promoter.

11. 11. The phage vector of claim 1, wherein each of the at least two transgene expression cassettes comprises nucleic acid for a polyA tail.

12. 12. A phage vector according to any one of claims 1 to 11, wherein the phage vector comprises four single-stranded self-complementary transgene expression cassettes separated by linkers that hybridize to form two double-stranded transgene expression cassettes.

13. 13. A phage vector according to any one of claims 1 to 12, wherein the two single-stranded self-complementary transgene expression cassettes are positioned in opposite orientations in the phage vector, preferably with a first transgene expression cassette extending in the 5' to 3' direction and a corresponding second transgene expression cassette extending in the 3' to 5' direction.

14. 14. A phage vector according to any one of claims 1 to 13, wherein the percentage of sequence identity between the first transgene expression cassette and the second transgene expression cassette is at least 65%, 70%, or 75%, or the percentage of sequence identity between the first transgene expression cassette and the second transgene expression cassette is at least 80%, 85%, 90%, or 95%.

15. 15. The phage vector of any one of claims 1 to 14, wherein the linker separating the at least two self-complementary transgene expression cassettes is an inverted terminal repeat (ITR).

16. 16. The phage vector of claim 15, wherein the phage vector comprises a second ITR, the second ITR flanking one of the at least two complementary transgene expression cassettes.

17. 17. The phage vector of claim 15 or claim 16, wherein the first ITR and / or the second ITR is an AAV ITR.

18. 18. The phage vector of any one of claims 15 to 17, wherein the phage vector comprises only two ITRs, preferably the phage vector comprises less than three ITRs.

19. 15. The phage vector of any one of claims 1 to 14, wherein the linker separating the at least two self-complementary transgene expression cassettes is an unrelated DNA segment, and the percentage sequence identity between the linker and the first and second cassettes is less than 50%, less than 45% or less than 40%, preferably less than 35%, less than 30% or less than 25%, and the unrelated DNA segment may be 60 bp to 300 bp, 80 bp to 280 bp, 100 bp to 260 bp, 120 bp to 240 bp, 140 bp to 220 bp, or 160 bp to 200 bp in length.

20. 20. The phage vector of any one of claims 1 to 19, wherein the phage vector comprises a selectable marker, which may be an ampicillin resistance gene.

21. the phage vector comprises one or more capsid minor coat proteins, and the phage vector may comprise a pIII capsid minor coat protein configured to display a cell targeting ligand to enable delivery of the vector to a target cell; and / or The phage vector comprises one or more capsid major coat proteins, and the phage vector may comprise at least one pVIII capsid major coat protein configured to display a foreign peptide.

21. A phage vector according to any one of claims 1 to 20.

22. 22. The phage vector of any one of claims 1 to 21, wherein the phage vector comprises a genome that is substantially devoid of the phage genome from which the vector is derived, and wherein the genome of the phage vector may lack at least 60%, more preferably at least 70%, and even more preferably at least 80% of the bacteriophage genome from which it is derived.

23. 23. The phage vector of any one of claims 1 to 22, wherein the phage vector lacks in its genome bacteriophage structural genes necessary for particle formation, packaging or release from a prokaryotic host.

24. A system for producing phage vectors from prokaryotic hosts, comprising: (i) a first vector configured to persist within a prokaryotic host, the first vector comprising at least two single-stranded, self-complementary transgene expression cassettes separated by a linker that hybridize to form a double-stranded transgene expression cassette, and a packaging signal to enable replication of the at least two single-stranded, self-complementary transgene expression cassettes; (ii) a second vector comprising nucleic acids encoding structural proteins required for packaging the double-stranded transgene expression cassette, resulting in the formation of a phage vector and its release from the prokaryotic host; A system including:

25. 25. A system according to claim 24, used to generate a phage vector according to any one of claims 1 to 23.

26. 26. The system of claim 24 or claim 25, wherein the first vector comprises the genome of the phage vector.

27. 27. A system according to any one of claims 24 to 26, wherein the packaging signal of the first vector comprises a bacteriophage origin of replication, preferably an F1 ori.

28. 28. A system according to any one of claims 24 to 27, wherein the first vector comprises a second origin of replication, preferably a pUC ori.

29. 29. The system of any one of claims 24 to 28, wherein the linker of the first vector is an ITR, which may be an AAV ITR.

30. 30. The system of any one of claims 24 to 29, wherein the second vector is a bacteriophage specifically engineered to rescue the genome of the first vector from a prokaryotic host, preferably wherein the second vector is replication-deficient.

31. 30. The system of any one of claims 24 to 29, wherein the second vector comprises a disrupted packaging signal that significantly impedes its ability to package itself into a phage particle, and preferably the second vector comprises a disrupted origin of replication.

32. 32. The system of claim 31, wherein the disrupted origin of replication is a medium copy number origin, which may be p15a, or a low copy number origin, which may be pMB1.

33. 33. The system of any one of claims 24 to 32, wherein the second vector comprises a first nucleic acid sequence encoding a pIII capsid minor coat protein configured to present a cell targeting ligand to enable delivery of the phage vector to a target cell, and / or a second nucleic acid sequence encoding at least one pVIII capsid major coat protein configured to present a foreign peptide.

34. 1. A method for producing a phage vector from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell a first vector configured to persist within the prokaryotic host, the first vector comprising at least two single-stranded, self-complementary transgene expression cassettes separated by a linker that hybridize to form a double-stranded transgene expression cassette, and a packaging signal to enable replication of the at least two single-stranded, self-complementary transgene expression cassettes; (ii) introducing into the host a helper phage containing nucleic acid encoding a bacteriophage structural protein; (iii) culturing the host under conditions that result in the double-stranded transgene expression cassette being packaged by the structural proteins to form and release from the prokaryotic host a phage vector carrying the double-stranded transgene expression cassette; A method comprising:

35. 1. A method for producing recombinant phagemid particles from a prokaryotic host, comprising: (i) introducing into a prokaryotic host cell: (a) a first vector configured to persist within the prokaryotic host, the first vector comprising at least two single-stranded, self-complementary transgene expression cassettes separated by a linker that hybridize to form a double-stranded transgene expression cassette, and a packaging signal to enable replication of the at least two single-stranded, self-complementary transgene expression cassettes; and (b) a second vector comprising nucleic acids encoding structural proteins necessary for packaging the double-stranded transgene expression cassettes; (ii) culturing the host under conditions that result in the double-stranded transgene expression cassette being packaged by the structural proteins to form a phage vector and be released from the prokaryotic host; A method comprising:

36. 24. Use of a helper phage comprising nucleic acid encoding a viral vector structural protein for producing a phage vector according to any one of claims 1 to 23 from a prokaryotic host.

37. 34. A host cell comprising the first and / or second vector of any one of claims 24 to 33.

38. A phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 for use as an experimental research tool, which may be for ex vivo or in vitro use.

39. A phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 for use in therapy or diagnostics.

40. A phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 for use in gene therapy techniques.

41. 41. A phage vector or system for use according to claim 39 or claim 40, wherein gene therapy techniques are used to treat, prevent or manage cancer.

42. A vaccine comprising a phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33.

43. A phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 for use in delivering a vaccine to a subject.

44. A phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 for use in the delivery and targeting of foreign antigens to tumors in vaccinated subjects.

45. Use of a phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 in gene-molecular imaging technology.

46. A pharmaceutical composition comprising a phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 and a pharmaceutically acceptable vehicle.

47. A method for producing the pharmaceutical composition described in claim 46, comprising the step of contacting a therapeutically effective amount of a phage vector described in any one of claims 1 to 23, or a system described in any one of claims 24 to 33, with a pharmaceutically acceptable vehicle.

48. Use of a phage vector according to any one of claims 1 to 23 or a system according to any one of claims 24 to 33 for producing a recombinant viral vector comprising or derived from a viral genome within the genome of said phage vector.

49. A method for producing a recombinant viral vector, comprising the steps of introducing into a eukaryotic host cell a phage vector described in any one of claims 1 to 23 or a system described in any one of claims 24 to 33, and allowing the host cell to produce the recombinant viral vector.

50. 50. The use of claim 48 or the method of claim 49, wherein the recombinant viral vector is a recombinant mammalian virus, rAAV, a recombinant self-complementary AAV vector, or a recombinant lentiviral vector.

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