CN120958136A

CN120958136A - Phage vector

Info

Publication number: CN120958136A
Application number: CN202480007443.5A
Authority: CN
Inventors: 阿明·哈吉图; 基蒂萨克·苏万
Original assignee: Imperial Institute Of Technology Innovation Co ltd
Current assignee: Imperial Institute Of Technology Innovation Co ltd
Priority date: 2023-01-10
Filing date: 2024-01-10
Publication date: 2025-11-14
Also published as: WO2024149989A1; EP4649161A1; KR20250139307A

Abstract

The present invention relates to phage vectors, and novel phage vectors comprising transgenes, in particular novel phage vectors comprising conventional mammalian transgene cassettes. The invention also extends to the use of such phage vectors as research tools, and their use in a variety of gene therapy applications, DNA and/or peptide vaccine delivery and imaging techniques to deliver transgenes.

Description

Phage vector

Technical Field

The present invention relates to phage vectors, particularly but not limited to novel phage vectors comprising transgenes, particularly conventional mammalian transgene cassettes. The invention extends to the use of such phage vectors as research tools and for delivery of transgenes in a variety of gene therapy applications, DNA and/or peptide vaccine delivery, and imaging techniques.

Background

Phage (phage) are continually becoming safe vectors for targeted delivery of transgenes because they have no inherent chemotaxis for mammalian cell receptors, but can be engineered to display tissue specific ligands on coat proteins, allowing cell entry, without disrupting viral structures (1-6). However, despite the significant advantages over eukaryotic viruses, tissue-targeted phage vectors have shown limited efficacy because phages have evolved to infect only bacteria and do not express the transgene's optimization strategy after entry into eukaryotic cells (2).

Previous studies by the inventors have shown that the gene transfer efficiency of filamentous M13 phage can be improved, and that an effective strategy is to combine phage with animal virus properties. Importantly, over the last few years, the inventors designed various strategies to increase the gene delivery efficiency of filamentous M13 phage-derived vectors. Indeed, as with other viral vectors, successful mediation of gene delivery by the M13 phage vector requires i) efficient diffusion through the extracellular matrix (ECM) to reach the cell surface, ii) cell surface receptor binding to achieve cellular uptake, iii) endosomal escape, and iv) nuclear entry to initiate gene expression. Clearly, phages have been advanced to infect only bacteria, and no optimized strategy has been adopted to accomplish the above steps to express transgenes in mammalian cells. Over the past few years, the inventors have devised a number of approaches to overcome these limitations, including by reducing the size of M13 phage particles to facilitate their diffusion through the extracellular matrix (ECM) (7, 8), and incorporating endosomal escape peptides on the recombinant pVIII major capsid protein to enhance phage escape from the endosomal/lysosomal degradation pathway (9, 10). Furthermore, to improve gene expression in the nucleus, the inventors previously added Inverted Terminal Repeats (ITRs) from adeno-associated virus (AAV 2) on both sides of mammalian transgene expression cassettes, thereby increasing the gene delivery efficiency of phage (6). Furthermore, to enhance transcription of therapeutic genes by vectors in the cancer cell nucleus, the inventors replaced the cytomegalovirus CMV promoter with a tumor activation and chemotherapy-induced promoter of the glucose regulatory protein Grp78 (11, 12). The inventors also bound anticancer drugs to M13 phage vectors to increase nuclear entry of phage in cancer cells (13).

Unlike traditional viral vectors, however, filamentous M13 phage requires additional conversion of its single-stranded DNA (ssDNA) genome to a form of double-stranded DNA (dsDNA) for proper recognition by the cellular transcription machinery (14). Although the ability of M13 phage to enter the nucleus has been successfully addressed, the conversion of single-stranded (ss) genomes to double-stranded (ds) genomes remains a critical issue to be addressed. In mammalian cells, the conversion of ssDNA to dsDNA of M13 phage is dependent on cytokines, a very inefficient process, limiting transduction efficiency (15).

Clearly, conversion of M13 phage to double-stranded DNA (dsDNA) has long been a major challenge for M13 phage-mediated gene delivery to mammalian cells. The need for complementary strand synthesis or recruitment is currently considered to be the rate limiting factor limiting the efficiency of M13 phage vectors. To overcome this limitation, the conversion of ssDNA to dsDNA was promoted by genotoxic treatment of human cell lines pre-incubated with phagemid particles (15). Unfortunately, such treatment methods are not suitable for living organisms.

In addition to the problems described above with filamentous phages, such as M13 phage, there are also significant problems associated with the rate-limiting step of ssDNA conversion to dsDNA in adeno-associated virus (AAV) vectors. Furthermore, although the capsid of AAV may carry two self-complementary ssDNA sequences, each comprising a transgene expression cassette, so as to produce dsAAV DNA upon cell transduction, the maximum length of each ssDNA must not exceed 2.3kb. Thus, packaging problems exist when packaging expression cassettes (i.e., large genomes) of greater than 2.3kb to produce dsDNA AAV in transduced cells and for gene therapy applications.

Thus, there is a need to provide a novel phage vector for delivery of a transgene cassette, for example into mammalian cells.

Disclosure of Invention

Rather than rely on cellular mechanisms that may be mutated to provide complementary strands to a single-stranded genomic phage vector, the inventors have unexpectedly found that this problem can be circumvented by designing a single phage vector carrying the complementary sequences of the transgene expression cassette.

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

As discussed in the examples, the inventors designed a phage vector carrying the complementary sequences of the transgene expression cassette, which could hybridize to form a double-stranded transgene expression cassette. Advantageously, the phage vectors of the present invention overcome the problem of single stranded (ss) DNA to double stranded (ds) DNA conversion of filamentous phage vectors (e.g., M13) and the associated problem of single stranded DNA to double stranded DNA conversion in adeno-associated virus (AAV) vectors. The present invention also addresses the problem of packaging large genomes for the production of double stranded AAV vectors. In order to demonstrate that the phage vector of the present invention has better gene delivery effect than the prior art, the inventors used reporter genes (such as green fluorescent protein GFP and luciferase). Subsequently, the inventors supported their findings with the cytokine TRAIL and further demonstrated that the vectors of the invention show surprising advantages in terms of gene delivery. When genes encoding cytokines such as TRAIL are used in the expression cassette, the inventors also demonstrated death of cancer cells, indicating that the phage vectors of the invention can be used to effectively deliver therapeutic genes.

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

The inventors have performed several in vitro experiments using various cell lines and transgenes and surprisingly, the inventors have observed that the transduction efficiency of the phage vectors of the invention is higher (3-15 fold higher) than that of conventional single-stranded DNA phage vectors. In fact, the phage vectors of the invention advantageously exhibit rapid onset and higher transgene expression levels in all cell lines tested. More importantly, unlike traditional single-stranded phage vectors, inhibitors of DNA replication do not affect transduction of phage vectors of the invention. Furthermore, as discussed in the examples, in vivo studies indicate that the efficiency of gene delivery to solid tumors is significantly improved when the phage vector of the invention is administered systemically to mice as compared to traditional single-stranded DNA phage particles. All of these biological properties demonstrate the generation and nature of a novel class of filamentous phage vectors that are capable of delivering double-stranded DNA, which would make a significant contribution to the continued development of phage-based gene delivery systems.

Since the circular phage genome may affect the formation process of double stranded DNA, the inventors used a phagemid such as that described in WO 2017/077275, the entire contents of which are incorporated herein by reference. This process involves removal of the phage genome, leaving only the f1 origin of replication, to achieve replication and packaging of the transgenic expression cassette in bacteria. The definition of phagemid is a plasmid DNA comprising a phage origin of replication, thus giving the name "phagemid". In this context, the inventors have designed a novel phage genome carrying two transgene expression cassettes using phagemids as DNA backbones. The resulting double-stranded vector is a phage particle.

Thus, preferably, the phage vector is a hybrid phagemid genome encapsulated by phage-derived coat proteins. Such a hybrid phagemid genome may be referred to as a "phagemid genome" (i.e., a genetic construct comprising two origins of replication-one from a phage (e.g., F1) and the other from a bacterium (e.g., pUC 1)).

Preferably, the genome of the phage vector comprises a packaging signal for enabling replication of at least two single-stranded self-complementing transgene expression cassettes which are capable of hybridizing in bacteria and subsequently being packaged into the phage vector as double-stranded transgene expression cassettes within the prokaryotic host. The packaging signal may preferably comprise a phage origin of replication. For example, the origin of replication preferably comprises an F1 origin, more preferably from an F1 phage. The DNA sequence of one embodiment of the F1 origin is denoted herein as SEQ ID No. 1, as follows:

ACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTT

[SEQ ID NO:1]

preferably, the genome of the phage vector comprises an origin of replication for enabling replication of at least two single-stranded self-complementing transgene expression cassettes in a prokaryotic host. Preferably, the origin of replication is capable of achieving high copy number replication of the vector in the host. Preferably, the origin of replication comprises a bacterial origin of replication. Preferably, the origin of replication comprises a pUC origin (for molecular cloning). The DNA sequence of one embodiment of the pUC origin is denoted herein as SEQ ID No. 2, as follows:

TTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA

[SEQ ID NO:2]

Or in another embodiment, the phage vector may be designed to integrate into the genome of the host cell. In this case, nucleic acid sequences are envisaged which facilitate targeted integration (e.g. by homologous recombination) of the vector genome. Thus, the genome of a phage vector may comprise one or more DNA sequences that enable targeted integration into the host genome.

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

In another embodiment, preferably, phage vectors can be used to deliver at least two self-complementing transgene expression cassettes to a tissue-specific target, whether the vector is administered to a subject systemically or locally in vivo, or applied to a cell mixture in vitro, or applied to an organ in an ex vivo case. Preferably, the at least two self-complementing transgene expression cassettes comprise viral transgene expression cassettes. More preferably, the at least two self-complementing transgene expression cassettes comprise mammalian viral transgene expression cassettes. For example, in a preferred embodiment, at least two self-complementing transgene expression cassettes may comprise lentiviral transgene expression cassettes. The at least two self-complementing transgene expression cassettes are preferably adeno-associated virus (AAV) transgene expression cassettes.

The at least two self-complementing transgene expression cassettes may comprise any nucleic acid encoding a factor which may have therapeutic or industrial use in the 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 cdnas may be preferred. In another embodiment, the nucleic acid may be RNA, such as antisense RNA or shRNA.

The factor encoded by the nucleic acid may be a polypeptide or a protein. For example, in embodiments of the phage vector of the first aspect for use in treating cancer, the transgene may encode a herpes simplex virus thymidine kinase gene, which may then exert a therapeutic effect on the target tumor cells. The transgene may encode a cytokine, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The vector can be used for treating any cancer, such as bone cancer.

However, it should be understood that the type of cell targeted by the phage vector depends on the type of cell targeting ligand expressed on the surface of the vector. For example, the cell targeting ligand may comprise an arginine-glycine-aspartic acid (RGD) sequence, such as RGD4C.

The at least two transgenic expression cassettes described above may comprise one or more functional elements required for expression of the nucleic acid in the target cell. For example, preferably, the at least two transgene expression cassettes each comprise a promoter for driving expression of the transgene. A suitable promoter may be a Cytomegalovirus (CMV) promoter. Herein, the DNA sequence of one embodiment of the CMV promoter is represented herein as SEQ ID No. 3, as follows:

ACGCGTGGAGCTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCC

[SEQ ID NO:3]

In another preferred embodiment, at least two transgenic expression cassettes each comprise a glucose regulatory protein 78 (grp 78) promoter. The nucleic acid sequence of one embodiment of the grp78 promoter is represented herein as SEQ ID No. 4, as follows:

CCCGGGGGCCCAACGTGAGGGGAGGACCTGGACGGTTACCGGCGGAAACGGTTTCCAGGTGAGAGGTCACCCGAGGGACAGGCAGCTGCTCAACCAATAGGACCAGCTCTCAGGGCGGATGCTGCCTCTCATTGGCGGCCGTTAAGAATGACCAGTAGCCAATGAGTCGGCTGGGGGGCGCGTACCAGTGACGTGAGTTGCGGAGGAGGCCGCTTCGAATCGGCAGCGGCCAGCTTGGTGGCATGAACCAACCAGCGGCCTCCAACGAGTAGCGAGTTCACCAATCGGAGGCCTCCACGACGGGGCTGCGGGGAGGATATATAAGCCGAGTCGGCGACCGGCGCGCTCGATACTGGCTGTGACTACACTGACTTGGAC

[SEQ ID NO:4]

Or in another preferred embodiment, at least two transgenic expression cassettes each comprise a tumor-specific promoter or a tissue-specific promoter. Tissue specific promoters can be used to target transcription and gene expression by phage vectors displaying ligands to deliver them to these specific tissues.

Preferably, at least two transgenic expression cassettes each comprise a nucleic acid encoding a poly a (polyA) tail. Preferably, the polyadenylation tail is located at the end of the transgene expression cassette, i.e., either the 5 'or 3' end of the transgene expression cassette. Herein, a DNA sequence of one embodiment of a nucleic acid encoding a polyadenylation tail is represented herein as SEQ ID No. 5, as follows:

ACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTT

[SEQ ID NO:5]

Thus, in a preferred embodiment, at least two single stranded self-complementing transgene expression cassettes each comprise a promoter (preferably CMV), a nucleic acid encoding a factor (e.g. a therapeutic factor), and a polyA tail.

Preferably, the phage vector comprises at least two single stranded self-complementing transgene expression cassettes separated by a linker, which form a double stranded transgene expression cassette by hybridization. Alternatively, the phage vector may comprise four single-stranded self-complementing transgene expression cassettes (i.e., two pairs of self-complementing expression cassettes) separated by a linker, which upon hybridization form two double-stranded transgene expression cassettes.

As shown in FIG. 2, to allow hybridization of single-stranded self-complementing transgene expression cassettes to each other, they must be arranged 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 appreciated that these expression cassettes are substantially identical in sequence, but extend in opposite or antiparallel directions on either side of the linker separating them. Thus, in a preferred embodiment, the two single-stranded self-complementing transgene expression cassettes are arranged in opposite directions in the phage vector.

It will be appreciated that in order for at least two single stranded self-complementing transgene expression cassettes to hybridize successfully to form a double stranded transgene expression cassette, their sequences should be similar, if not identical, although their extension directions are reversed. However, their sequences do not have to be identical, so long as each expression cassette has sufficient sequence identity over a sufficiently long fragment, hybridization will occur.

The percent sequence identity between the first transfer cassette and the second transfer cassette may be at least 65%, 70% or 75%. Preferably, the percent sequence identity between the first transfer cassette and the second transfer cassette is at least 80%, 85% or 90%. More preferably, the percent sequence identity between the first transfer cassette and the second transfer cassette is at least 92%, 94% or 95%. Even more preferably, the percent sequence identity between the first transfer cassette and the second transfer cassette is at least 96%, 97% or 98%. Most preferably, the percent sequence identity between the first transfer cassette and the second transfer cassette is at least 99% or 100%.

Preferably, the linker separating at least two self-complementing transgene expression cassettes is an Inverted Terminal Repeat (ITR). Preferably, the phage vector comprises a second ITR. More preferably, the second ITR flanks one of the at least two self-complementing transgene expression cassettes.

Or in another preferred embodiment, the linker separating at least two self-complementing transgene expression cassettes is a piece of unrelated DNA. Preferably, the length of the linker or unrelated DNA fragment is between 60bp and 300bp, between 80bp and 280bp, between 100bp and 260bp, between 120bp and 240bp, between 140bp and 220bp, or between 160bp and 200 bp. Most preferably, the length of the linker or unrelated DNA fragment is 180bp.

An "unrelated DNA fragment" refers to DNA that has low or no sequence identity to the first single-stranded self-complementary expression cassette and to the second single-stranded self-complementary expression cassette. For example, the percentage of sequence identity between the linker and the first and second expression cassettes is less than 50%, 45% or 40%. Preferably, the percentage of sequence identity between the linker and the first and second expression cassettes is less than 35%, 30% or 25%. Preferably, the percentage of sequence identity between the linker and the first and second expression cassettes is less than 20%, 15% or 10%. Preferably, the percent sequence identity between the first expression cassette and the second expression cassette is at least 8% or 5%.

Preferably, the first ITR and the second ITR are AAV ITRs. ITRs may be specific for AAV-2 or other AAV serotypes, and may be any sequence, so long as they are capable of forming hairpin loops in the secondary structure. For example, the AAV serotype may be AAV1-9, but is preferably AAV1, AAV2, AAV5, AAV6 or AAV8. One embodiment of the ITR DNA sequence (left inverted terminal repeat from a commercially available AAV plasmid) is represented herein as SEQ ID No. 6, as follows:

CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[SEQ ID NO:6]

The DNA sequence of the ITR of another embodiment (right inverted terminal repeat from a commercially available adeno-associated virus (AAV) plasmid) is represented herein as SEQ ID No. 7, as follows:

AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGG

[SEQ ID NO:7]

Preferably, the phage vector comprises only two ITRs. Preferably, the phage vector comprises less than three ITRs.

Preferably, the genome of the phage vector comprises a selectable marker that will depend on the host cell in which the vector is carried, e.g., for conferring antibiotic (e.g., ampicillin) resistance to the host cell (preferably bacterial). The marker provides a selection pressure during production of the vector in the host cell. Thus, in a preferred embodiment, the phage vector comprises an ampicillin resistance gene.

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, thereby enabling the vector to be delivered 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 an exogenous peptide thereon.

Phage vectors may contain modifications of the capsid structure, for example by treatment or chemical and biochemical coupling means (to make it modified). Examples of suitable modifications may include cross-linking peptide residues to phage particles. In another embodiment, the phage vector may comprise one or more functional peptides attached to its capsid. For example, the functional peptide may comprise a nuclear translocation signal or an endosomal escape peptide. Thus, the phagemid particles may be multifunctional and may utilize the features disclosed in WO 2014/184528, the contents of which are incorporated herein by reference.

In another embodiment, the phage vector may be combined with a cationic polymer to form a complex having a net positive charge, as described in 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), polyethylenimine (PEI), polybrene, protamine sulfate, and cationic lipids. Preferably, the cationic lipid is selected fromAnd DOTAP (N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium methylsulfate). Preferably, the cationic polymer comprises DEAE, more preferably DEAE.

Preferably, the phage vector comprises a genome that is substantially deleted from the phage genome from which the vector is derived. Preferably, the genome of the phage vector lacks at least 60%, more preferably at least 70%, even more preferably at least 80% of the phage genome from which it is derived. More preferably, the genome of the phage vector lacks at least 90%, more preferably at least 95%, even more preferably at least 99% of the phage genome from which it is derived. Preferably, the genome of the phage vector lacks all phage genomes from which it is derived. However, as discussed above, in some embodiments, the genome of the phage vector may comprise a phage origin of replication, such that single-stranded DNA is capable of replication in the host bacterium, i.e., an F1 phage origin of replication.

Preferably, the genome of the phage vector lacks structural genes required for phage formation, packaging, or release of the particle from the prokaryotic host. Such structural genes encode capsid proteins and the like. Thus, preferably, the phage vector lacks a structural gene encoding a phage capsid protein. Preferably, the phage vector comprises a genome that lacks a gene encoding a secondary or primary coat protein of the phage from which the vector is derived. Preferably, the phage vector comprises a genome that lacks a gene encoding a pIII capsid minor coat protein, or lacks a gene encoding a pVIII capsid major coat protein. Most preferably, the phage vector comprises a genome that lacks both the gene encoding the pIII capsid minor coat protein and the gene encoding the pVIII capsid major coat protein.

Thus, the phage vector preferably comprises a replication-defective virus-like particle or virion constructed from and displaying structural components derived from phage, including but not limited to proteins and other conjugates, although the genome of the vector does not comprise structural genes of the phage from which it is derived.

Thus, in view of the fact that the genome of the phage vector of the first aspect lacks a substantial portion of the genome (including structural genes) of its derivative phage, an alternative system is needed to provide the necessary structural (i.e., capsid) genes required to package the phage vector genome into a phage capsid to produce the phage vector of the invention. Accordingly, the inventors devised a system for producing the vector of the first aspect, which involves the use of a separate so-called "helper" vector. Thus, in practice, the phage vector of the first aspect is a hybrid phagemid vector comprising the cis-genetic component of a phagemid and the cis-genetic component of a eukaryotic virus, e.g. AAV ITR.

Thus, in a second aspect, there is provided a system for producing a phage vector from a prokaryotic host, the system comprising:

(i) A first vector configured to persist within the prokaryotic host and comprising at least two single-stranded self-complementing transgene expression cassettes separated by a linker and hybridized to form a double-stranded transgene expression cassette, and a packaging signal for enabling replication of the at least two single-stranded self-complementing transgene expression cassettes, and

(Ii) A second vector comprising a nucleic acid encoding a structural protein required for packaging the double-stranded transgenic expression cassette,

Thereby enabling phage vectors to be formed in and released from the prokaryotic host.

The system of the second aspect is preferably capable of packaging the genome of a eukaryotic virus (e.g.AAV or lentivirus) provided by a first vector into a prokaryotic viral capsid (i.e.phage) provided by a second vector.

Advantageously, the isolation of the reproductive elements of the phage vector into a first "therapeutic" vector carrying the transgene expression cassette and a second, independent "helper" vector carrying the viral packaging structural gene can significantly reduce the size of the genome/vector, thereby greatly increasing transgene capacity. In embodiments where phage vectors are used for therapy, this is a particularly useful advantage for gene therapy applications. Thus, this will increase the production yield of the vector system, the efficiency of gene transduction, and enhance its flexibility in other applications.

Preferably, the system of the second aspect is used for generating a phage vector according to the first aspect. Thus, preferably, the first vector comprises the genome of the phage vector. The packaging signal of the first vector may preferably comprise an origin of replication, preferably a phage origin of replication. Preferably, the origin of replication in the first vector comprises an F1 origin of replication, more preferably from an F1 phage origin of replication.

Preferably, the first vector comprises a second origin of replication for enabling replication of at least two single-stranded self-complementing transgene expression cassettes in a prokaryotic host for molecular cloning. Preferably, the origin of replication enables the vector to make high copy number replications in a host for molecular cloning. Preferably, the origin of replication comprises a pUC origin of replication. Alternatively, the first vector may comprise one or more DNA sequences that facilitate targeted integration into the host genome, thereby eliminating the need for any origin of replication.

The at least two single stranded self-complementing transgene expression cassettes comprise a viral transgene expression cassette, more preferably a mammalian viral transgene expression cassette. For example, the at least two transgene expression cassettes may comprise an AAV transgene expression cassette or a lentiviral transgene expression cassette, with AAV transgene expression cassettes being preferred.

Preferably, the linker of the first vector is an ITR, more preferably an AAV ITR, even more preferably an adeno-associated virus type 2 (AAV 2) ITR. Or in another preferred embodiment, the linker of the first vector is an unrelated DNA fragment. Preferably, the linker is as described above for the phage vector of the first aspect.

In a preferred embodiment, the first vector comprises the second ITR. Preferably, the second ITR flanks one of the at least two self-complementing transgene expression cassettes. Preferably, the first ITR and/or the second ITR is an AAV ITR. Preferably, the first vector comprises only two ITRs. Preferably, the first vector comprises less than three ITRs.

The second vector or "helper phage" is preferably a phage specifically engineered to rescue the first vector genome from the prokaryotic host. Thus, the second vector (i.e., helper phage) is provided to provide its proteins and polypeptides to the first vector, or to any other DNA entity comprising a functional packaging signal and/or a single-stranded replication origin. The second vector is most preferably replication defective. Preferably, the second vector comprises a disrupted packaging signal, which significantly impairs its ability to package itself into phage particles. Preferably, the second vector comprises a disrupted replication origin. 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 (i.e., the genome of the phage vector) is configured to outperform the second vector (i.e., the helper phage) both in terms of replication and packaging.

The genome of the second vector may be engineered such that the resulting phage vector possesses targeting properties (or multifunctional properties as described in WO 2014/184528). Thus, the second vector provides the structural capsid protein for the assembly of the phage vector. Preferably, the second vector comprises nucleic acid encoding one or more capsid minor coat proteins, or encoding one or more capsid major coat proteins. All capsid proteins can be wild-type or recombinant, exist in single or multiple copies, and can be modified to display chimeric or synthetic peptides. This includes displaying antigens of other viruses for peptide vaccine delivery, or acting as an adjuvant in the case where a DNA vaccine (delivered by the phagemid particles of the first aspect) is required.

Thus, in one embodiment, the second vector may comprise a first nucleic acid sequence encoding a pIII capsid minor coat protein configured to display a cell targeting ligand to enable delivery of the phage vector to a target cell (e.g., a tumor cell). Thus, it may be desirable to introduce a 9 amino acid mutation in the pIII minor coat protein of the recombinant phagemid particles to render it specific for tumor cells expressing the α _νβ₃ and α _νβ₅ integrins as well as angiogenic tumor-associated endothelial cells. Thus, the genome of the second vector may comprise an RGD4C targeting peptide (sequence CDCRGDCFC-SEQ ID No: 8).

In another embodiment, the second vector may comprise a second nucleic acid sequence encoding at least one pVIII capsid major coat protein configured to display an exogenous peptide thereon. Thus, it may be desirable to introduce mutations in the wild-type pVIII major coat protein of the phage vector to reveal a short peptide, e.g., less than 10 amino acids in length. Such short peptides may be targeting moieties or have inherent biological/chemical functions in vivo or in vitro. For example, immune stimulation is shown to occur in vivo by antigen, or binding to nanoparticles (e.g., gold) in vitro by a gold binding peptide.

The first vector may be a member of the retrovirus family or the antiretroviridae subfamily. The first vector may also be a member of the lentivirus genus. Preferably, the first vector is a member of the parvoviridae family or subfamily thereof. More preferably, the first vector is a member of the genus dependoviridae or adeno-associated virus.

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 produce the phage vector of the first aspect in a prokaryotic host. It will be appreciated that the role of the packaging signal (e.g. replication origin) in the first vector to enable replication of the phage vector genome is to signal the structural proteins of the second vector (i.e. helper phage) to package the genome (i.e. they act synergistically in the host in a trans-acting manner) to form the particles of the first aspect.

In a third aspect, a method for producing a phage vector from a prokaryotic host is provided, the method comprising

(I) Introducing a first vector into a prokaryotic host cell, the first vector configured to persist within the prokaryotic host and comprising at least two single-stranded self-complementing transgene expression cassettes separated by a linker and hybridized to form a double-stranded transgene expression cassette, and a packaging signal for enabling replication of the at least two single-stranded self-complementing transgene expression cassettes;

(ii) Introducing a helper phage into the host, the helper phage comprising nucleic acid encoding a phage structural protein, and

(Iii) The host is cultured under conditions that produce a double-stranded transgene expression cassette, which is packaged with structural proteins, such that the phage vector carrying the double-stranded transgene expression cassette is formed in and released from the prokaryotic host.

Advantageously, this results in extremely high yields of phage vectors. The first vector (i.e., the genome of the phage vector) may be introduced into the host cell by, for example, infection. The host cell may then be transformed with the helper phage, thereby producing a phage vector. Preferably, the method comprises a purification step after the culturing step. Purification may include centrifugation and/or filtration.

In a fourth aspect, a method for producing phage particles from a prokaryotic host is provided, the method comprising

(I) Introducing into a prokaryotic host cell (a) a first vector configured to persist within the prokaryotic host and comprising at least two single-stranded self-complementing transgene expression cassettes separated by a linker and hybridized to form a double-stranded transgene expression cassette, and a packaging signal for enabling replication of the at least two single-stranded self-complementing transgene expression cassettes, and (b) a second vector comprising a structural protein encoded by a nucleic acid required for packaging the double-stranded transgene expression cassette, and

(Ii) The host is cultured under conditions that produce a double-stranded transgene expression cassette packaged by the structural protein such that the phage vector is formed in and released from the prokaryotic host.

Advantageously, this improves safety. The second vector (i.e., helper phage) may be introduced into the host cell by, for example, infection. The host cell may then be transformed with the first vector (i.e., the genome of the phage vector) to produce the phage vector. Preferably, the method comprises a purification step after the culturing step. Purification may include centrifugation and/or filtration.

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

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

The host cell is preferably a prokaryotic cell, more preferably a bacterial cell. Examples of suitable host cells include (i) TG1 (genotype: K-12supE thi-1. DELTA. (lac-proAB) DELTA. (mcrB-hsdSM) 5, (r _K-m_K ^-), plasmid F' [ traD36 proAB ⁺lacI^q lacZΔM15]);(ii)DH5αF′IQ^TM (genotype: Delta (lacZYA-argF) U169 recA1endA1 hsdR17 (rk ^-,mk⁺) phoA supE44 lambda-thi-1 gyrA96 relA1, plasmid F ' proAB ⁺ lacIqZ DeltaM 15 zzf::: tn5[ KmR ]), and (iii) XL1-Blue MRF ' (genotype: delta (mcrA) 183 delta (mcrCB-hsdSMR-mrr) 173endA1 supE44 thi-1recA1 gyrA96 relA1 lac, plasmid F ' proAB lacIqZ DeltaM 15 Tn10 (Tetr)).

In a further aspect, a phage vector according to the first aspect or a system according to the second aspect is provided for use as an experimental research tool.

For example, the vector or system may be used ex vivo or in vitro.

Preferably, however, the vector is for use in a therapeutic or diagnostic method, preferably in vivo.

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.

The phage vector of the present invention has targeting specificity and transduction efficiency, and thus can be used for treating a wide variety of diseases. Therefore, the invention can provide two self-complementary transgene expression cassettes for host bacteria, and the expression cassettes are hybridized to form double-chain transgene expression cassettes in the process of producing phage particles in the host bacteria, and the characteristic can remarkably expand the treatment application prospect of recombinant phage in gene therapy. The invention can be used prophylactically for the prevention of diseases, and also for the amelioration and/or treatment of diseases after they have occurred.

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.

In a ninth aspect, there is provided a method of 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.

It will be appreciated that the present invention can be used to construct a variety of different phage vectors that can be used to treat and/or diagnose a variety of diseases, depending on their own nature and the foreign proteins shown. For example, in embodiments where the phage vector comprises a tumor targeting ligand and/or comprises a transgene that expresses an anti-tumor gene, such as the herpes simplex virus thymidine kinase (HSVtk) gene, the vector may be used in combination with Ganciclovir (GCV) to treat cancer. The target cells in the gene therapy technology are preferably eukaryotic cells, more preferably mammalian cells.

Thus, the gene therapy technology is preferably used for treating, preventing or ameliorating cancer. The tumor may be located in the brain, such as a medulloblastoma, glioblastoma, or diffuse endogenous pontic glioma (DIPG). The phage vector may be used in combination with conventional therapies, such as chemotherapeutic drugs (i.e., doxorubicin, temozolomide, lomustine), radiation therapy, immune checkpoint inhibitors (i.e., PD-1, PD-L1 or CTLA4 inhibitors), or other drugs/exogenous compounds, including but not limited to histone deacetylase inhibitors (HDAC inhibitors), proteasome inhibitors, and anticancer products from natural and dietary sources (e.g., genistein).

The inventors believe that the phage vectors of the invention are of great commercial value in the delivery of peptide and/or DNA and/or adjuvant vaccines.

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.

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

Preferably, the vaccine is a peptide vaccine. The vaccine is preferably a DNA vaccine. The vaccine preferably comprises a suitable adjuvant. In one embodiment, the phage vector may be used to carry a transgene or DNA cassette encoding an antigen (i.e., at least two single-stranded self-complementing transgene expression cassettes that hybridize to form a double-stranded transgene expression cassette) to stimulate the immune system of the organism. Phage vectors can also be used to directly display and express the antigen of interest on the primary pVIII coat protein, thereby providing a highly efficient platform for simultaneous delivery, by means of a single phage particle to deliver multiple antigens simultaneously (as vaccine DNA vaccines), proteins or adjuvants that are readily expressed on phage surfaces. The subject may be a mammal, and preferably a human.

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 targeted delivery of an exogenous antigen to a tumor of a vaccine subject.

The animals are first vaccinated with foreign antigens or the animals have been vaccinated with the antigens used, and then tumor targeting vectors are administered to the vaccinated animals to deliver the foreign antigens to the tumor sites, thereby inducing immune attacks against these tumors.

The inventors also believe that the phage vectors of the invention may also be used in a variety of different genetic-molecular imaging techniques, such as Positron Emission Tomography (PET), ultrasound (US), single Photon Emission Computed Tomography (SPECT), functional magnetic resonance imaging, or bioluminescence imaging.

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 genetic molecular imaging techniques.

The transgene carried by the phagemid particle may encode herpes simplex virus thymidine kinase (HSVtk) and/or sodium/iodine co-transporter (NIS), and the particle is preferably used in combination with a radiolabeled substrate. For example, the human sodium/iodine co-transporter (NIS) imaging gene is preferably used in combination with I ¹²⁴ for clinically useful Positron Emission Tomography (PET) imaging, or with I ^125/99m Tc pertechnetate for clinically useful SPECT imaging.

Alternatively, the HSVtk gene is preferably used in combination with a radiolabeled nucleoside analogue, for example 20- [18F ] -fluoro-20-deoxy-1-b-D-arabinofuranosyl 5-ethyluracil ([ 18F ] FEAU).

It will be appreciated that phage vectors and systems according to the invention (hereinafter "agents") can be used in medicaments that can be used in monotherapy, or as an adjunct to, or in combination with, known therapies for the treatment, amelioration or prophylaxis of a disease such as cancer. For example, a therapeutic approach that combines the phage particles and systems of the present invention with existing chemotherapeutic agents (e.g., temozolomide, doxorubicin, or genistein) is preferred.

In another preferred embodiment, the treatment may comprise the use of the phage vectors and systems of the invention in combination with an extracellular matrix degrading agent, such as an enzyme or losartan. The inventors believe that extracellular matrix degrading agents should enhance the spread of phage vectors in a subject being treated, particularly in solid tumors.

The agent of the invention (i.e. the phage vector of the first aspect or the system of the second aspect) may be formulated into a number of different forms of the composition, depending on the manner in which the composition is used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, or the like, or any other suitable form that may be administered to a human or animal in need of treatment. It will be appreciated that the carrier of the medicament of the invention should be one that is well tolerated by the subject being treated.

Medicaments comprising the agents of the invention can be used in a variety of ways. For example, oral administration may be desired, in which case the agent may be included in a composition that may be ingested orally, e.g., in tablet, capsule or liquid form. Compositions comprising the agents of the invention may be administered by inhalation (e.g., nasal inhalation). The compositions may also be formulated as topical preparations, for example, creams or ointments that may be applied to the skin.

The agents of the invention may also be incorporated into slow or delayed release devices. For example, such devices may be implanted on the skin surface or subcutaneously, and the drug may be released continuously over weeks or even months. The device is positionable at least adjacent to the treatment site. Such devices may be particularly advantageous when long-term use of the agents of the invention is required for treatment, and frequent administration (e.g., at least daily injection) is often required.

In a preferred embodiment, the agents and compositions of the invention may be administered to a subject by injection into the bloodstream or directly into a site in need of treatment. The injection mode includes intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), intraperitoneal injection, or administration by convection enhancement (local injection suitable for the disease site).

It will be appreciated that the required amount of agent depends on its biological activity and bioavailability, which in turn depends on the mode of administration, the physicochemical properties of the agent (i.e. phage vector or system), and whether it is for monotherapy or combination therapy. The frequency of administration may 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 with the particular agent used, the concentration of the pharmaceutical composition, the mode of administration, and the advancement of the disease. Dosages may also need to be adjusted depending on other factors of the particular subject being treated, including the age, weight, sex, diet and time of administration of the subject.

Typically, the daily dosage of the agent of the invention may be between 0.01 μg/kg body weight and 500 μg/kg body weight. More preferably, the daily dose is between 0.01 μg/kg body weight and 400 μg/kg body weight, still more preferably between 0.1 μg/kg body weight and 200 μg/kg body weight.

The agent may be administered before, during or after the onset of the disease. For example, the agent may be administered immediately after the onset of the disease in the subject. Daily doses may be administered by systemic administration once (e.g., once daily injection). Or the agent may need to be administered twice or more times a day. For example, the agent may be administered at a dose of 25mg to 7000mg twice daily (or an increase in number depending on the severity of the disease being treated) (i.e. assuming a weight of 70 kg). The patient receiving treatment may receive a first dose at wake-up and then a second dose at night (if a twice daily dosing regimen), or once every 3 or 4 hours after the first dose. Furthermore, sustained release devices can be used to provide the patient with optimal dosages of the agents of the present invention without the need for repeated administration.

Known protocols, such as those routinely employed in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.), may be used to prepare specific formulations containing the vectors or systems of the invention, as well as to formulate accurate treatment regimens (e.g., daily doses of the agent and frequency of administration).

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 carrier.

The composition can be used for therapeutic amelioration, prevention or treatment of any disease (e.g., cancer) in a subject that can be treated by gene therapy.

The present invention also provides in a fifteenth aspect a method of preparing a pharmaceutical composition according to the twelfth aspect, the method comprising 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 carrier.

The "subject" may be a vertebrate, mammal, or livestock. Thus, the agents, compositions and medicaments of the invention may be used to treat any mammal, such as livestock (e.g., horses or dogs), pets, or may be used in other veterinary applications. Most preferably, however, the subject is a human.

A "therapeutically effective amount" of an agent (i.e., a phage vector) refers to the amount of drug required to treat a disease of interest or to produce a desired effect (e.g., to effect efficient delivery of a transgene to a target cell or tissue, to effect tumor killing, etc.) when administered to a subject.

For example, a therapeutically effective amount of the agent used may be from about 0.01mg to about 800mg, preferably from about 0.01mg to about 500mg.

Reference herein to a "pharmaceutically acceptable carrier" is to any known compound or combination of known compounds known to those skilled in the art that can be used to formulate a pharmaceutical composition.

In one embodiment, the pharmaceutically acceptable carrier may be a solid and the corresponding composition may be in the form of a powder or tablet. The solid pharmaceutically acceptable carrier may comprise one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, coating agents or tablet disintegrating agents. The carrier may also be an encapsulating material. In powders, the carrier is a finely divided solid which is admixed with the finely divided active agent according to the invention. In tablets, the active agent (e.g., particles or systems of the invention) may be mixed in a suitable ratio with a carrier having the necessary compression properties and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active agent. Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutically acceptable carrier may be a gel and the composition may be in the form of a cream or the like.

However, the pharmaceutically acceptable carrier may also be a liquid and the corresponding pharmaceutical composition in solution. Liquid carriers can be used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions. The particles or systems according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, an organic solvent, a mixture of both or a pharmaceutically acceptable oil. The liquid carrier may contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators. Examples of liquid carriers suitable for oral and injectable use include water (partially containing additives as described above, such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, such as glycols) and their derivatives, and oils (e.g., fractionated coconut oil and arachis oil). For injection, the carrier may also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid carriers are useful in preparing sterile liquid compositions for injection. The liquid carrier of the pressurized composition may be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions in the form of sterile solutions or suspensions may be used by means of, for example, intramuscular injection, intrathecal injection, epidural injection, intraperitoneal injection, intravenous injection, in particular subcutaneous injection. The carrier or system may be prepared as a sterile solid composition which may be dissolved or suspended at the time of administration using sterile water, physiological saline or other suitable sterile injection medium.

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

It will be appreciated that adeno-associated virus (AAV) is generally the vector of choice for gene therapy. As a gene delivery vector, lentiviral vectors also have several key advantages over other systems. First, it has a large packaging capacity, at least to accommodate 8Kb of DNA, an important property when packaging large expression cassettes comprising tissue specific promoters and transgenes. Secondly, lentiviral vectors are not only structurally different from retroviruses of relatively simple construction, but also are capable of transducing non-dividing cells, a property which is very useful when it is considered to be applied as a gene therapy vector to non-proliferating tissues such as muscle, neuron and hematopoietic stem cells. Furthermore, lentiviral vectors are less immunogenic than adenoviral vectors, which makes systemic routes of administration viable. However, the hurdles of AAV or lentivirus use in laboratory and clinical studies include its extremely high production costs and lower yields.

In addition to exhibiting practical value in gene therapy, imaging and vaccine delivery, the phage vectors of the invention can also be used to produce recombinant viral vectors, such as AAV or lentiviruses, in vitro or in vivo (including in situ). Phage-mediated AAV production takes advantage of the ability of phage vectors to package large amounts of single-stranded DNA (ssDNA). Typical AAV production systems contain three major elements, recombinant adeno-associated virus (rAAV), rep-cap genes, and adenovirus helper genes, which interact to produce rAAV particles.

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 for the production of a recombinant viral vector comprising or derived from a viral genome within the genome of a phage vector.

In a seventeenth aspect, there is provided a method of producing a recombinant viral vector, the method comprising introducing a phage vector according to the first aspect or a system according to the second aspect into a eukaryotic host cell, enabling the host cell to produce the recombinant viral vector.

Preferably, the recombinant viral vector is a recombinant mammalian virus, a rAAV, a recombinant self-complementing AAV vector or a recombinant lentiviral vector. In other words, the recombinant viral vector may be a conventional AAV vector or an auto-mutual AAV vector according to the first aspect. Preferably, the phage vector according to the first aspect or the system according to the second aspect is used to function in cis and/or trans with the delivery and/or presence of other genetic elements determined by the genome of the phage vector, required for production of a mammalian virus in a eukaryotic host cell. Methods for aiding or enhancing gene transfer of phagemid particles to host cells include those described in WO 2014/184528 (i.e. multifunctional) and WO 2014/184529 (i.e. combined with a cationic polymer to form a complex with a net positive charge).

The eukaryotic host cell may be a mammalian cell. The host cell may comprise or be derived from a human embryonic kidney cell (HEK 293), spodoptera frugiperda pupa ovarian tissue cell (Sf 9) or chinese hamster ovary Cell (CHO). Insect cells are also contemplated.

In one embodiment, the host cell may be transformed with one or more phage vector genomes carrying genes selected from the group consisting of rAAV genes, lentiviral genes, capsid genes, replication genes, helper protein encoding genes, and any other genes required for mammalian viral expression and packaging.

For example, in phage vector mediated production of rAAV/scAAV, the rAAV gene or scAAV sequence may be carried by a phage vector according to the first aspect, while the adenovirus helper gene and rep-cap gene may be carried by separate vectors, or integrated into the eukaryotic host genome. Any combination of rAAV, rep-cap, and adenovirus helper genes may be carried by one or more vectors, i.e., in cis or trans configuration. Alternatively, in the production of rAAV, the rep-cap protein or adenovirus helper protein may be integrated into or introduced into a eukaryotic host as a stably expressed helper DNA (e.g., a plasmid), where the phage vector provides a recombinant viral genome for packaging into a recombinant virus, as determined by a transgene expression cassette within the phage vector genome.

The method can be performed in vivo, in vitro, ex vivo, or in situ. For in situ generation, the phage vector preferably comprises a targeting moiety to a target eukaryotic cell as a designated eukaryotic host. Preferably, in situ, ex vivo and in vivo virus production, the eukaryotic host cell type specified is a diseased cell. Preferably, the diseased cells are malignant tumor cells or benign tumor cells. In the in vitro viral production, the eukaryotic host is preferably a derivative of any of the eukaryotic hosts listed above. The phage vectors and the genetic elements required to produce the recombinant virus (as determined by the transgene expression cassette in the phage vector) can be used in eukaryotic host cells in the manner described above, either in cis-acting or trans-acting combinations.

It is to be understood that the invention extends to any nucleic acid, peptide or variant, derivative or analogue thereof comprising essentially the amino acid or nucleic acid sequence of any one of the sequences mentioned herein. The terms "substantially the amino acid/polynucleotide/polypeptide sequence", "functional variant" and "functional fragment" refer to a sequence having at least 40% sequence identity to an amino acid/polynucleotide/polypeptide sequence of any of the sequences mentioned herein, e.g., 40% sequence identity to a nucleic acid as defined herein.

The invention also encompasses amino acid/polynucleotide/polypeptide sequences having a higher sequence identity to any of the sequences mentioned herein, in particular a sequence identity of more than 65%, more preferably more than 70%, even more preferably more than 75%, even more preferably more than 80%. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, yet even more preferably at least 97% identity, more preferably at least 98% identity, most preferably at least 99% identity to any of the sequences mentioned herein.

Those skilled in the art will understand how to calculate the percent sequence identity between two amino acid/polynucleotide/polypeptide sequences. To calculate the percent sequence identity of two amino acid/polynucleotide/polypeptide sequences, the two sequences must first be aligned and then the sequence identity value calculated. The percentage identity of two sequences may take on different values due to (i) the sequence alignment method used, e.g., clustalW, BLAST, FASTA, smith-Waterman (implemented in different programs), or structural alignment based on three-dimensional structural comparisons, (ii) parameters used by the alignment method, e.g., local alignment versus global alignment, pairing scoring matrices used (e.g., BLOSUM62, PAM250, gonnet, etc.), and gap penalties, e.g., functional forms and constants.

After alignment of the sequences is completed, there are many different ways to calculate the percent identity between the two sequences. For example, the number of identical sites can be divided by any of (i) the length of the shorter sequence, (ii) the length of the alignment, (iii) the average length of the two sequences, (iv) the number of non-vacant sites, or (v) the number of equivalent sites after the overhang is excluded. Furthermore, it is noted that the percent identity is also closely related to the sequence length. Thus, the shorter a pair of sequences, the more likely the sequence identity due to contingencies.

Thus, it should be appreciated that precise alignment of protein or DNA sequences is a complex process. The commonly used multiple sequence alignment program ClustalW (Thompson et al, 1994, nucleic ACIDS RESEARCH, vol. 22, pages 4673-4680; thompson et al, 1997, nucleic ACIDS RESEARCH, vol. 24, pages 4876-4882) is a preferred method of generating multiple sequence alignments of proteins or DNA in the present invention. Parameters applicable to ClustalW are as follows: gap Open Penalty = 15.0,Gap Extension Penalty =6.66, matrix=identity for DNA alignment. For protein alignment Gap Open Penalty = 10.0,Gap Extension Penalty =0.2, matrix=gonnet. For DNA and protein alignment ENDGAP = -1, gapdi st = 4. Those skilled in the art will appreciate that these and other parameters may need to be adjusted in order to obtain optimal sequence alignment results.

Preferably, the percent identity between two amino acid/polynucleotide/polypeptide sequences is calculated from such alignment by (N/T) ×100, where N is the number of positions where the two sequences have the same residue, T is the total number of positions compared, including gaps, and may or may not include overhangs. Preferably, the calculation should include an overhang. Thus, one most preferred method of calculating the relative percent identity between two sequences includes (i) using the ClustalW program, sequence alignment using a suitable set of parameters, such as those described above, and (ii) substituting the values of N and T into the formula sequence identity = (N/T). Times.100.

Alternative methods for identifying similar sequences will be well known to those skilled in the art. For example, a highly similar nucleotide sequence may be encoded by a sequence that hybridizes under stringent conditions to a nucleic acid sequence described herein or its complement. Stringent conditions mean that the DNA or RNA to which the filter binds at about 45℃hybridizes in 3 times sodium chloride/sodium citrate (SSC) and then washed at least once with 0.2 times SSC/0.1% Sodium Dodecyl Sulfate (SDS) at about 20-65 ℃. Or a highly similar polypeptide may differ by at least 1 but less than 5, 10, 20, 50 or 100 amino acids from the sequences set forth herein.

Due to the degeneracy of the genetic code, it is apparent that any nucleic acid sequence may be altered or changed without substantially affecting the protein sequence it encodes, thereby producing a functional variant thereof. Suitable nucleotide variants are those variants in the sequence that have been altered by the substitution of different codons encoding the same amino acid, thereby producing synonymous mutations. Other suitable variants are those having homologous nucleotide sequences, but comprising all or part of the sequence, which are altered by different codon substitutions encoding amino acids, and the substituted amino acids have similar biophysical side chains as the substituted amino acids, resulting in conservative mutations. For example, small non-polar, 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. It will thus be appreciated which amino acids may be replaced by amino acids having similar biophysical properties, and the skilled person will be aware of the nucleotide sequences encoding these amino acids.

All of the features described herein (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 combination of the above aspects, except combinations where at least some of such features and/or steps are mutually exclusive.

Drawings

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

FIG. 1 shows a schematic representation of DNA constructs of single-stranded DNA vectors derived from M13 phage of the prior art, namely adeno-associated virus/phage ("AAVP") and phagemid adeno-associated virus ("PAAV") vectors of the prior art, in comparison to novel self-complementing phage particles of the invention ("self-complementing phage", hereinafter "self-complementing phage particles" or "SCPHAGEMID"), which may be M13 or AAV. In the present invention, phagemids carrying two single-stranded self-complementing transgene expression cassettes are used as DNA scaffold to induce hybridization of the transgene cassette in the host bacteria, followed by the generation of a double-stranded transgene cassette for packaging by the phage capsid. The definition of "phagemid" is a plasmid DNA comprising a phage origin of replication, thus giving rise to the name phagemid. Here, the inventors used phagemids as DNA backbones to design a novel phage genome carrying two transgene cassettes. The resulting double-stranded vector is a phage particle. The AAVP of the prior art comprises a complete phage genome and a single mammalian transgene cassette flanked by ITR sequences derived from AAV2 virus (6). On the other hand, PAAV particles in the prior art are based on phagemid designs, which contain a single transgene cassette, and require helper phages to provide the structural gene during production (8). In contrast, the latest generation phage vectors according to the present invention (i.e. "self-complementing phage particles" or "SCPHAGEMID") carry an additional transgene cassette compared to AAVP and PAAV. The two cassettes are identical, separated by an ITR linker from AAV, but 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 fig. 1. It can be seen that these expression cassettes are identical, but extend in opposite or antiparallel directions on either side of the ITR separating them. Also included is a second AAV ITR flanking one of the transgenic expression cassettes. As in the case of PAAV, the helper phage provides a structural gene for the "single-stranded complementary phage" that enables replication.

FIG. 2 shows how a single-stranded autophagy "SCPHAGEMID" or "scPP" of the invention hybridizes between two complementary transgene expression cassettes flanking the ITR linker, resulting in a transgene expression cassette double-stranded DNA (dsDNA) resembling a hairpin loop structure.

FIG. 3 shows a cloning strategy for constructing a phagemid scaffold carrying a complementary transgene expression cassette for the preparation of scPP delivering Green Fluorescent Protein (GFP). The complete GFP transgene expression cassette from the promoter to the polyadenylation (polyA) signal was amplified from one phagemid by Polymerase Chain Reaction (PCR) using primers containing PciI restriction sites. The insert was then cloned into the PciI cleavage site of the same phagemid. The final phagemid contained two complementary GFP transgene cassettes and two AAV 2-ITRs, one left-hand ITR connecting the two transgene cassettes and the other right-hand ITR flanking one cassette. It can be seen that these expression cassettes are identical, but extend in opposite or antiparallel directions on either side of the left ITR, and hybridization can occur between two complementary transgenic expression cassettes on either side of the ITR linker, thereby forming double stranded DNA.

FIG. 4 shows Green Fluorescent Protein (GFP) expression of B16-F1 cells on day 5 after transduction with either a targeting RGD4C.scPP (i.e.phage vector forming a double stranded DNA hairpin loop structure according to the invention) or an RGD4C.PAAV vector (i.e.single stranded phage vector used as a control). A) Microscopic imaging of the cells using a fluorescence microscope, and B) quantification of GFP positive cells by flow cytometry analysis.

FIG. 5 summarizes the constructs used to assess gene delivery efficiency, namely PAAV, scPP (phage vector according to the invention) and cwPP. PAAV shows a phage vector with one copy of the expression cassette flanked by AAV ITRs, scPP shows phage particle vectors of the invention with two opposite-directed expression cassettes that will form double-stranded DNA, and cwPP shows a control phage particle carrying two identical-directed Lucia transgene expression cassettes, i.e., clockwise (cw), such that these expression cassettes cannot hybridize to form double-stranded DNA. The experiments used particles carrying the reporter gene Lucia or Green Fluorescent Protein (GFP), and particles carrying the therapeutic genes tumor necrosis factor alpha (tnfα), interleukin 15 (IL 15) and TRAIL.

FIG. 6 compares the expression of the targeted RGD4C.scpP-Lucia (i.e., phage vector of the invention) and the targeted RGD4C.PAAV-Lucia gene in B16-F1 melanoma cells. Different doses of vector 25000, 50000, 100000, 500000 and 10 ⁶ Transduction Units (TU) per cell were used to transduce cells. Error bars represent mean Standard Error (SEM). Statistical analysis of each day and each construct was performed using One-way ANOVA followed by selected multiple comparisons of double-stranded and single-stranded vectors in pairs. Cells treated with non-targeting vectors lacking RGD4C (scpP-Lucia or PAAV-Lucia) as well as untreated cells were also included in the experiments. Data are expressed in Relative Luminescence Units (RLU). The experiment was repeated multiple times with at least n=5 biological replicates, each biological replicate having n=3 technical replicates.

FIG. 7 demonstrates the superiority of the scPP vector (i.e.phage vector according to the invention) over single stranded PAAV batches prepared by two different researchers in B16-F1 cells under 100000 TU/cell conditions.

FIG. 8 shows an example of data from four experiments on B16-F1 cells using a vector at a dose of 10 ⁶ TU/cells.

FIG. 9 shows a comparison of the expression of the Lucia gene in B16-F1 melanoma cells at different doses, similar to that in FIG. 6. Error bars represent mean Standard Error (SEM). Statistical analysis of day and each construct was performed using one-way anova followed by selected multiple comparisons of double-stranded and single-stranded vectors in pairs. Cells treated with scPP or PAAV, non-targeting vector lacking RGD4C, as well as untreated cells were also included in the experiments. Data are expressed in Relative Luminescence Units (RLU). The experiment was repeated multiple times with at least n=5 biological replicates, each biological replicate having n=3 technical replicates.

FIG. 10 shows a comparison of Lucia gene expression in human HEK293 cells at different doses (100000, 500000 and 10 ⁶ TU/cell). Error bars represent mean Standard Error (SEM). Statistical analysis of each day and each construct was performed using one-way anova followed by selected multiple comparisons of double stranded and single stranded vectors in pairs. Cells treated with scPP or PAAV, non-targeting vector lacking RGD4C, as well as untreated cells were also included in the experiments. Data are expressed in Relative Luminescence Units (RLU). The experiment was repeated multiple times with at least n=5 biological replicates, each biological replicate having n=3 technical replicates.

FIG. 11 shows a comparison of Lucia gene expression in Rhabdomyosarcoma (RMS) metastatic cancer cells at doses of 100000, 500000 and 10 ⁶ TU/cell. Error bars represent mean Standard Error (SEM). Statistical analysis of each day and each construct was performed using one-way anova followed by selected multiple comparisons of double stranded and single stranded vectors in pairs. Cells treated with scPP or PAAV lacking RGD4C and untreated cells were also included in the experiment. Data are expressed in Relative Luminescence Units (RLU). The experiment was repeated multiple times with at least n=5 biological replicates, each biological replicate having n=3 technical replicates.

FIG. 12 shows a comparison of Lucia gene expression in human A549 lung cancer cells at 500000 and 10 ⁶ TU per cell. Error bars represent Standard Errors (SEM). Statistical analysis of each day and each construct was performed using one-way anova followed by selected multiple comparisons of double stranded and single stranded vectors in pairs. Cells treated with scPP or PAAV, non-targeting vector lacking RGD4C, as well as untreated cells were also included in the experiments. Data are expressed in Relative Light Units (RLU). The experiment was repeated multiple times with at least n=5 biological replicates, each biological replicate having n=3 technical replicates.

FIG. 13 shows the results of validation of the expression data of the Lucia gene of human A549 lung cancer cells by another researcher under 100000 and 10 ⁶ TU/cell conditions.

FIG. 14 shows a comparison of the expression of the Lucia gene in human MCF7 breast cancer cells under 100000 TU/cell conditions. Error bars represent Standard Errors (SEM). Statistical analysis of each day and each construct was performed using one-way anova followed by selected multiple comparisons of double stranded versus single stranded vectors in pairs. Cells treated with scPP or PAAV, non-targeting vector lacking RGD4C, as well as untreated cells were also included in the experiments. Data are expressed in Relative Light Units (RLU). The experiment was repeated multiple times with at least n=5 biological replicates, each biological replicate having n=3 technical replicates.

FIG. 15 shows ELISA quantitation results of secreted TNFα on days 4 (D4) and 6 (D6) after B16-F1 cells were transduced with scPP (i.e., phage vector according to the present invention) or PAAV carrying TNFα gene at doses of 500000 (500 k), 1X 10 ⁶ (1M) or 4X 10 ⁶ (4M) TU/cell, respectively. Statistical analysis of each day and each construct was performed using one-way anova followed by selected multiple comparisons of double stranded versus single stranded vectors in pairs. Cells treated with scPP or PAAV, non-targeting vector lacking RGD4C, as well as untreated cells were also included in the experiments.

FIG. 16 shows ELISA quantitation results of secreted IL15 on day 4 after B16-F1 cells were transduced with either RGD4C.scPP carrying the IL15 gene (i.e., phage vector described in the present invention) or RGD4C.PAAV at doses of 500000 (500 k), 10 ⁶ (1M) and 4X 10 ⁶ (4M) TU/cells, respectively. Statistical analysis of each day and each construct was performed using one-way anova followed by selected multiple comparisons of double stranded versus single stranded vectors in pairs. Cells treated with non-targeting vector lacking RGD4C (M13) as well as untreated cells were also included in the experiment.

FIG. 17 shows ELISA quantitation results of secreted IL15 on day 4 after B16-F10 melanoma cells were transduced with RGD4C.scPP carrying the IL15 gene (i.e., phage vector described in the present invention) or RGD4C.PAAV at doses of 500000 (500 k), 10 ⁶ (1M) and 4X 10 ⁶ (4M) TU/cells, respectively. Statistical analysis of each day and each construct was performed using one-way anova followed by selected multiple comparisons of double stranded versus single stranded vectors in pairs. Cells treated with scPP or PAAV, non-targeting vector lacking RGD4C, as well as untreated cells were also included in the experiments.

FIG. 18 shows the ELISA quantification results of human osteosarcoma cells via RGD4C.scPP (i.e., phage vector according to the present invention) or RGD4C.PAAV carrying secreted TRAIL gene at 500000 TU/day 4 post cell transduction. Cells treated with empty rgd4c.scpp vector (no TRAIL gene vector or mock vector) and untreated cells were also included in the experiment. In addition, RGD4C.scPP vector carrying transmembrane TRAIL (RGD 4 C.scPP-TRAIL) was used to transduce cells.

FIG. 19 compares gene delivery to subcutaneous solid tumors (human osteosarcoma) in immunodeficient mice after intravenous injection of 5X 10 ¹⁰ TU/RGD 4C.scPP alone (i.e., phage vector according to the invention) and RGD4C.PAAV vector. Tumors and healthy tissue were collected on day 7 post-vector injection. Non-targeting vector scPP or PAAV treated mice lacking RGD4C were also included in the experiments.

FIG. 20 shows the phage diffusion in Matrigel (Matrigel). The scPP and PAAV vectors were labeled with Fluorescein Isothiocyanate (FITC) and inoculated into matrigel at a concentration of 5 mg/ml. Images were taken by fluorescence microscopy at the time of inoculation (t=0) and 18 hours after inoculation (t=18).

FIG. 21 shows internalization of phage particles in B16-F1 cells. A) Flow cytometry (FACS) analysis of cells using anti-phage antibodies, B) quantitative polymerase chain reaction (qPCR) using primers directed to ampicillin genes in the vector outside the transgene expression cassette.

FIG. 22 compares transduction efficiencies of scPP (i.e., phage vectors according to the invention) with control vectors cwPP and awPP. A) The schematic representation of the three vectors used, ITR in blue, the transgene and its orientation are also noted. B) B16F1 cells were transduced with the vector encoding Lucia at a dose of 10 ⁶ TU/cells. The graph shows representative experimental results (n=3) repeated twice, and the data are luminescence detection values at day 4 after transduction. Statistical differences were analyzed using a one-way anova with a significance level set at α=0.05.

FIG. 23 compares transduction efficiencies of scPP (i.e., phage vectors according to the invention) with the cwPP and awPP hybrid vectors. A) The transduction process and possible hybridization are schematically shown in blue for ITR, and the transgene and its orientation are also noted. B) B16F1 cells were transduced with the vector encoding Lucia at a dose of 10 ⁶ TU/cell, or a mixed vector of cwPP at a dose of 5 x 10 ⁵ TU/cell and awPP at a dose of 5 x 10 ⁵ TU/cell. The graph shows representative experimental results (n=3) repeated twice, and the data are luminescence detection values at day 4 after transduction. Statistical differences were analyzed using a one-way anova with a significance level set at α=0.05.

Fig. 24 shows phage particle measurement results based on Transmission Electron Microscope (TEM) images. A) Transmission electron microscope images of different phage particles. B) The size of phage particles quantified. Two different stock solutions were imaged for each phage particle, and a total of 100 particles were quantified for each phage. Statistical differences between four samples were calculated using one-way anova, only pairwise comparisons without statistical differences are shown.

FIG. 25 shows the particle size determination analysis of the scPP, PAAV and helper phage vectors. A) Two different preparations were analyzed for each support and a total of 100 particles were measured from the Transmission Electron Microscope (TEM) images obtained. B) Whole phage particles were loaded onto agarose gels under non-denaturing conditions.

FIG. 26 shows the self-hybridization of a transgene expression cassette during the production of scPP (i.e.a phage vector according to the invention) in bacteria. A) A schematic diagram of the situation is assumed. The hypothetical intramolecular hybridization format of the scPP genome is shown. This form forms a double stranded DNA target within the transgene (orange box) that can be digested with BamHI. The digested genome should produce a 1898bp double-stranded DNA fragment which, if denatured, forms a 3796bp single-stranded DNA band. B) Comparison between BamHI digested and undigested scP-Lucia genomic samples. C) Migration analysis of denatured 1kb Plus DNA ladder (ladder) 1kb Plus DNA ladder and linearized pAAV GFP plasmid (5378 bp) aliquots were electrophoresed in 1M urea denaturing agarose gel in their native and denatured forms. When the ladder sample is denatured, the 5000bp DNA ladder will be converted to a double-stranded DNA. D) Demonstration of the ability of the scPP-Lucia phage genome to form double-stranded DNA fragments A1898 bp fragment obtained by digestion of the phage genome with BamHI was electrophoresed in native form (left) and denatured form (right) together with the same treated 1kb Plus DNA ladder, respectively. The sample after denaturation was consistent with the migration rate of 4000bp bands in the DNA ladder.

FIG. 27 shows the inhibition of PAAV vector gene expression by hydroxyurea (hydroxyurea, HU). A) Time profile of Lucia gene expression after transduction of B16-F1 cells with the vector in the presence of hydroxyurea. B) Control experiments were also performed, i.e. the cells received water instead of hydroxyurea. C) The graph shows the Lucia gene expression data at day 6 after B16-F1 cell transduction in the presence of hydroxyurea. D) The figure shows the Lucia gene expression data at day 6 post transduction in the absence of hydroxyurea.

Fig. 28 shows a comparison of the effect of PAAV and scPP on delivery of the Lucia reporter gene to metastatic human osteosarcoma 143B cells over a period of 1 day to 3 days as the vector dose increases. Expression of Lucia is expressed in Relative Luminescence Units (RLU). The vector targets tumor cells via RGD4C ligand, non-targeting vector (NT) served as control.

Fig. 29 shows a comparison of the effect of PAAV and scPP on delivery of secreted cytokine TRAIL (i.e. soluble TRAIL, stril) to metastatic human osteosarcoma 143B cells. In the figure, enzyme-linked immunosorbent assay (ELISA) data are shown for quantifying the amount of sTRAIL protein released from the media of cancer cells after treatment with the vector.

FIG. 30 shows induction of osteosarcoma cell death following scPP-sTRAIL treatment with encoded secreted sTRAIL under in vitro conditions.

Fig. 31 shows the toxicity evaluation results. No increase in Lactate Dehydrogenase (LDH), a toxic biomarker in mice with established osteosarcoma, occurred after administration of rgd4c.scpp vector encoding sTRAIL.

Fig. 32 shows the biodistribution of sTRAIL delivery following systemic administration of PAAV encoding sTRAIL and the scPP vector in tumor-bearing mice with established osteosarcomas.

FIG. 33 shows immunofluorescent staining results of sTRAIL protein expression in tumors after RGD4C.PAAV and RGD4C.scPP treatment with sTRAIL encoded. In tumors of mice receiving rgd4c.scpp treatment, higher levels of sTRAIL production were detected.

FIG. 34 shows hematoxylin and eosin staining results of tumors in mice treated with RGD4C.scpP-sTRAIL administered systemically compared to untreated mice or mice injected with non-targeted NT vehicles, showing extensive tumor lesions.

Detailed Description

Examples

The inventors aim to provide a novel phage vector comprising self-complementary sequences of a transgenic expression cassette to effect hybridization during production in host bacteria or upon transduction of mammalian cells to deliver double stranded DNA of the mammalian transgenic expression cassette. The novel phage vector solves the problems associated with double-stranded phage due to capsids and bulky genomes, while also overcoming the problems associated with AAVs. In various embodiments, this novel phage vector is referred to as a self-complementing phage particle or scPP. To demonstrate that the gene delivery effect of scPP is superior to the prior art, the inventors used reporter genes such as Green Fluorescent Protein (GFP) and luciferase. Subsequently, the inventors further validated the results of the study by TRAIL or soluble TRAIL (sTRAIL) to demonstrate that the scPP particles have unexpectedly superior performance in gene delivery. Furthermore, when TRAIL isogenic was used, the inventors also observed cancer cell death, suggesting that the vector may be useful for delivering therapeutic genes.

Materials and methods

Molecular cloning of constructs

A Green Fluorescent Protein (GFP) transgene expression cassette flanked by AAV2 ITRs from the promoter to polyadenylation signals was amplified by PCR from 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 transformed into DH 5. Alpha. E.coli (E.coli). Subsequently, plasmids were extracted from different bacterial colonies by a Miniprep kit (Miniprep, qiagen) and verified by restriction and DNA sequencing (Eurofins). The correct clones were transformed into TG1 Mix & Go competent escherichia coli (Zymo research, usa) to generate phage vectors. A schematic representation of the cloning strategy is shown in FIG. 3. To generate phage particles carrying tnfα, TRAIL or IL15 transgenes GFP was replaced with the corresponding DNA coding sequence.

Phage production

TG1 Mix & Go competent escherichia coli (Zymo Research company, usa) was transformed with the backbone DNA construct of the vector. The double tandem vector (control phage particles encoding two co-rotating copies, either clockwise (cw) or counterclockwise (aw)) was cultured in 2xYT medium until the absorbance at 600nm (OD _600nm) reached 0.3-0.6, which indicates that the bacteria were in exponential growth phase. At this point, the bacterial cultures were infected with the corresponding helper phage, whether the targeted phage displaying RGD4C or the non-targeted (NT) M13 phage, and incubated for 15 min at 37 ℃. After incubation, the cultures were added to 2XYT medium containing 50. Mu.g/ml kanamycin and 100. Mu.g/ml carbenicillin and incubated overnight at 32℃at 160 rpm. The next day, the cultures were centrifuged at 4℃for 15 min at 6000 g. The supernatant was collected, mixed with 0.4 volumes of 21mM polyethylene glycol (PEG, molecular weight 8000)/3.36M sodium chloride/1% triton X-100, and left overnight at 4 ℃. The solution was then centrifuged at 4℃for 30 minutes at 10000 g. The pellet was resuspended in Phosphate Buffer (PBS), mixed with 0.5 volumes of 21mM polyethylene glycol (PEG)/3.36M sodium chloride (NaCl), and left overnight at 4 ℃. Next, the pellet was resuspended in PBS by gentle shaking at 37℃and 120rpm for 3 hours after centrifugation at 10000g at 4℃for another 30 minutes. The residual bacterial contamination in the resuspended pellet was removed by centrifugation at 10000g for 10 min at room temperature and the resulting supernatant was filtered through a 0.45 μm filter. Subsequently, the presence or absence of the RGD4C coding sequence in the pIII capsid protein gene was detected by PCR to verify the targeting purity of the prepared phage and further analyzed by 2% agarose gel.

Titration of phage particles

Phage particles were quantified in prokaryotic hosts. Phage were serially diluted in PBS for infection with TG1 e.coli grown to log phase in 2xYT medium, followed by incubation at 37 ℃. After 20 minutes incubation in a 37 ℃ water bath, the phage/bacteria mixture was thoroughly mixed again and spread on solid agar medium containing the selective antibiotic. Since the phage particles contained the ampicillin resistance gene, a TYE top agar containing 100. Mu.g/ml ampicillin was used. Whereas helper phages contain the kanamycin resistance gene, so a TYE top agar containing 50. Mu.g/ml kanamycin was used. The concentration of the scPP particles in the sample can be determined by colony counting by plating the bacteria on ampicillin-containing TYE top agar, and the concentration of helper phage can be determined by plating on kanamycin-containing medium. The concentration of phage particles is expressed as bacterial transduction unit TU/. Mu.l.

Intramolecular self-hybridization of transgene expression cassette in self-complementing phage particles (scPP)

The scPP was first treated with DNAse-I and its genome was then extracted. Briefly, samples were treated with 100mM Tris-HCl, 25mM EDTA pH8, 4% Sodium Dodecyl Sulfate (SDS) was added, and incubated at 70℃for 10 minutes to lyse phage capsids. Subsequently, 3M potassium acetate at pH 5.5 was added to the sample, and the mixture was centrifuged at 12000g at room temperature for 10 minutes to precipitate phage capsid proteins. The anion exchange column (QIAGEN MIDIPREP kit) was equilibrated with 0.1M sodium acetate (pH 5.0), 0.6M sodium chloride, 0.15% (v/v) Triton X-100. The centrifuged supernatant was loaded onto a column and the solution was allowed to flow out by gravity flow. The column was then washed twice with 0.1M sodium acetate (pH 5.0), 825mM sodium chloride and the samples eluted with QF elution buffer (Qiagen). The resulting samples were further purified by isopropanol-ethanol precipitation and finally resuspended in TE buffer (Qiagen).

The concentration of the extracted phage genome was calculated as a single-stranded DNA sample (1 OD ₂₆₀ units = 33 μg/ml single-stranded DNA). Subsequently, the genome was digested with BamHI (NEB Co., UK) and subjected to agarose gel electrophoresis. The 2000bp band was recovered by gel extraction kit (Qiagen Co.) and precipitated with isopropanol-ethanol. An aliquot of the extracted DNA band and 1kb Plus DNA ladder (Thermo Fisher Co.) was mixed with a solution containing 0.5mg/ml bromophenol blue, 8M urea, 1% (v/v) Triton X-100 and 1mM Tris pH 8. Half of each sample (extracted strips and steps) was denatured at 80 ℃ for 5 minutes. Subsequently, the DNA ladder and extracted bands were loaded in denatured 1M urea 1.2% agarose gel in denatured and undenatured form and electrophoresed at 55V for 4 hours under ice bath conditions. The gel was stained in TAE buffer containing 0.5. Mu.g/. Mu.l ethidium bromide for 2 hours at room temperature.

Since the molecular weight of the DNA ladder is different for the migration behavior of the reference bands under native and denaturing conditions, additional controls were used to confirm migration of these reference bands. For this, denatured samples of linearized scPP-GFP plasmid (5378 bp) were electrophoresed alongside DNA ladder in urea denatured gel.

Cell transduction and Lucia expression

The adherent cells are inoculated into a pore plate/tissue culture dish with proper specification, and the cell confluency of 48 hours after inoculation is ensured to reach 70-80%. On the day of transduction, the average number of cells in each well/dish was determined and used to calculate the amount of phage particles to be added to the cells. Subsequently, a transduction mixture was prepared, and an appropriate amount of phage particle stock was diluted into serum-free medium and thoroughly mixed. The recommended volume of transduction mixture used for each well/dish is the minimum volume that just covers the cell monolayer completely. During transduction, the original medium was discarded, the transduction mixture was added to the cells, incubated at 37℃for 6-12 hours with 5% CO ₂, and then an equal volume of complete medium was supplemented. After 24 hours, the whole medium was discarded and replaced with fresh medium. The transduced cells were maintained in culture until analysis.

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 medium was collected from each well and transferred to an opaque 96 well plate. Luciferase substrates were prepared according to the manufacturer's protocol (Invivogen, france) and added to well plates using QUANTI-Luc to quantify luciferase activity. Luciferase activity was measured using GloMax Discover microplate luminometer (Promega, uk). In these experiments, the medium was not changed at each time point.

GFP expression in transduced cells

The scPP-GFP transduced cells and their transduction ratios were analyzed by FACS or fluorescence microscopy.

Transmission Electron Microscope (TEM)

The copper mesh coated with the carbon film was subjected to glow discharge treatment to enhance hydrophilicity. Phage particles were added dropwise to the copper grid and after incubation for 10-15 minutes, excess liquid was removed by blotting with blotting paper. Subsequently, the copper mesh was rinsed with sterile filtered deionized water and blotted dry with blotter paper, repeated twice, and dried for 15 minutes. The particles were negatively stained by dropping a 1% uranyl acetate solution onto the copper grid, and after 30 seconds, rinsed twice with sterile filtered deionized water and dried. Copper grids were imaged using a scanning electron microscope (JEOL JEM-2010, uk) and analyzed by ImageJ software.

Agarose gel analysis of phages

Phage stock was analyzed by nanospectrophotometer (Nanodrop) to determine 30 μg phage samples. These samples were combined with 2 Xloading buffer (126 mM Tris-HCl, pH 6.8,15%Type 400 and 0.002% bromophenol blue) were mixed in a 1:1 ratio and loaded onto a 0.8% agarose gel. Samples were electrophoresed at 50V for 5 hours, followed by fixation of the gel with 10% acetic acid and 50% methanol solution overnight. The next day, the gel was stained with coomassie brilliant blue staining solution for 3 hours and destained with 20% methanol and 5% acetic acid solution overnight. The bands were detected by a BioRad gel imager.

Fluorescent dye labeling of phage particles

Phage particles were labeled with FITC. 50mL of phage particles (total 5X 10 ¹¹ TU) were added to 200. Mu.L of a solution containing 5mg/mL FITC (Sigma Co., UK) and mixed by spinning at room temperature under dark conditions for 1 hour. Subsequently, PEG/NaCl solution was added at a final concentration of 25-30%, and phage particles were precipitated overnight at 4 ℃. The solution was centrifuged at 13000rpm for 15 minutes and the phage particle pellet was collected. After resuspension of the pellet with 250 μl PBS, the pellet was again precipitated with PEG/NaCl and the procedure repeated until the free FITC was completely removed. Finally, FITC-conjugated phage particles were resuspended in PBS and their titers were determined by e.coli infection and colony counting.

Matrigel diffusion of phage particles

200 Μl Engelbreth-Holm-Swarm mouse sarcoma matrigel (Sigma Co., UK) at a concentration of 2.5mg/ml was added to the 48-well plate and subsequently transferred to a 37℃environment. At the same time, FITC-labeled particles were prepared at a concentration of 5. Mu.g/ml. Mu.l of each particle solution was aspirated into a gel loading tip, which was fixedly inserted into the matrigel to allow the particles to diffuse. Thereafter, fluorescence images were taken using a fluorescence microscope (japanese nicol ECLIPSE TE U) at 0 hour and 18 hours, respectively, and analyzed by Openlab imaging software.

Particle internalization

Cells were transduced with FITC-labeled phage particles of 1×10 ⁶ TU/cell or 5×10 ⁵ TU/cell. 6 hours after transduction, cells were washed with PBS and then detached by treatment with 2mg/ml pronase in ice for 10 minutes. The action of pronase was stopped using 20% Fetal Bovine Serum (FBS) and centrifuged at 200g for 5 min at room temperature. The pellet was resuspended in 20% fetal bovine serum and centrifuged again. The pellet was resuspended in 4% paraformaldehyde and incubated for 10 min at room temperature. After incubation, cells were centrifuged at 300g for 5 min at room temperature and blocked with a solution containing 0.1% saponin and 2% Bovine Serum Albumin (BSA) for 30 min at room temperature. Cells were centrifuged at 300g for 5 min at room temperature, the pellet was treated with rabbit anti-fd phage antibodies (sigma 086k4860;1:1000 dilution) diluted with PBS containing 0.1% saponin and 1% bovine serum albumin, and incubated for 1h at room temperature. After incubation, the cells were centrifuged under the same conditions and washed with PBS containing 0.1% saponin and 1% bovine serum albumin, and the washing was repeated 3 times. Subsequently, the cells were labeled with goat anti-rabbit AlexaFluor-647 (Invitrogen 21245;1:500 dilution) diluted with PBS containing 0.1% saponin and 1% bovine serum albumin and incubated at room temperature for 1 hour in the absence of light. Cells were washed twice with PBS containing 0.1% saponin and finally resuspended in PBS.

Next, flow cytometry analysis was performed on phage particles within the cells. FACS detection was performed using BD FACscalibur flow cytometry (BD Biosciences) equipped with an argon ion laser (488 nm) and a red diode laser (635 nm). The average fluorescence intensity and percentage were measured for at least 10000 gated cells in each triplicate well. Cell populations were gated and analyzed using FACScalibur software.

Polyacrylamide gel electrophoresis (SDS-PAGE)

The sample was loaded with the loading dye (Laemmli buffer and beta-mercaptoethanol) and then loaded onto a 4-15% mini-PROTEAN TGX Stain-Free ^TM gel. 10 XTris-glycine-SDS (Sigma) was used as running buffer and NEB protein color standard as protein ladder.

Determination of helper phage contamination

Phage samples were pre-treated with DNAse-I for 30 minutes at 37 ℃. DNAse-I was then inactivated by incubation with 50mM EDTA at 65℃for 10 minutes and heated at 95℃for 10 minutes with 1% SDS to lyse the phage capsids. After the temperature was gradually reduced to 23℃with a gradient of 3℃the SDS was bound using 1% Triton X-100 and the samples were diluted 1:250 with DEPC water. Standard curves were obtained with scPAAV and helper phage plasmids (as standards) at concentrations ranging from 2 x 10 ⁸ to 2 x 10 ³ plasmids/μl.

Tumor necrosis factor alpha (TNF alpha) enzyme-linked immunosorbent assay (ELISA)

Cells were transduced with phage particles and replaced with fresh complete medium 24 hours after transduction. Conditioned medium was collected on day 4 post transduction, replaced with fresh medium, and again collected on day 6 post transduction.

ELISA (ELISA)

The amount of IL15 produced in the supernatant after transduction was quantified using the mouse IL15 DuoSet ELISA kit (British R & D Systems Co.).

Tumor necrosis factor alpha (tnfa) concentrations in conditioned media were quantified using an ELISA MAX ^TM standard kit set as per manufacturer's instructions.

For TRAIL ELISA, we coated the plates with capture antibodies. Next, the plates were washed twice with wash buffer (PBS containing 0.05% tween 20) and blocked at room temperature for 1 hour with PBS containing 1% BSA. The plate was washed twice with wash buffer, the sample was added to the plate and incubated for 2 hours at room temperature. Next, the detection antibody was incubated at room temperature for 1 hour. Then, avidin-horseradish peroxidase D (avidin-HRP D) was added to each well, followed by the substrate solution.

Results

Example 1 construction of DNA backbone for hybridization of two self-complementary transgene expression cassettes

Referring to FIGS. 1 and 2, there are shown schematic diagrams of a known adeno-associated virus/phage ("AAVP") vector (left side) and a known phagemid adeno-associated virus ("PAAV") vector (middle), both derived from a single-stranded M13 filamentous phage. AAVP comprises a complete phage genome and a single mammalian transgene expression cassette flanked by ITR sequences derived from AAV2 virus, while PAAV is based on a phagemid design comprising a single transgene expression cassette flanked by ITRs and requires helper phage to provide structural genes during production. AAVP and PAAV have a problem in that, in the treatment of mammalian cells, both vectors deliver single-stranded DNA of the transgene expression cassette, which must be converted to double-stranded DNA for gene expression and transduction to occur. This process is dependent on mammalian cytokines, inefficient, results in delayed onset of gene expression, and over time, slow and inefficient increases in gene delivery.

The inventors have previously found that transduction of cells with two phage vectors carrying sequences complementary to mammalian transgene expression cassettes did not enhance gene delivery. Thus, the inventors tried to provide the complementary sequences of the transgene expression cassette in a single phage vector (fig. 1), in other words, to design a phage vector carrying both the transgene expression cassette and its complementary sequences to induce hybridization during (i) at the time of cell transduction, or (ii) during production and preparation in a bacterial host (fig. 2).

FIGS. 1 and 2 (right side) show the single-chain (SS) autophagy backbone ("autophagy particle or scPP") for phage production of the invention. In effect, the self-complementing phagemid scaffold enables two single-stranded self-complementing transgene expression cassettes to hybridize and form a double-stranded transgene expression cassette. AAVP and PAAV comprise only one copy of the expression cassette, whereas the scPP of the invention carries an additional transgene expression cassette compared to AAVP and PAAV. The two cassettes are identical and are separated by an Inverted Terminal Repeat (ITR) linker, but their sequence reads are opposite, 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 FIG. 1. As shown in FIG. 2, the phagemid of the invention allowed hybridization between two self-complementing transgene expression cassettes flanking the ITR linker, thereby forming double stranded DNA resembling a hairpin loop structure. Another AAV ITR is included to flank one of the transgene expression cassettes.

To avoid the potential impact of the circular phage genome on the double-stranded DNA formation process, the inventors used phagemids rather than phages to remove the phage genome, leaving only the f1 origin of replication, enabling the transgene expression cassette to replicate in bacteria and be packaged (fig. 1 and 2). Since phage genomes are not present, helper phage are used to infect bacteria to provide structural genes encoding the capsid proteins required for packaging (FIGS. 1 and 2). Phages are non-mesophilic to mammalian cells, and therefore, to enable the vector to enter cells, the inventors have displayed a bicyclic RGD4C ligand on helper phages (fig. 1 and 2), which has been extensively characterized and used for phage-mediated gene delivery. This ligand allows phage entry into mammalian cells by binding to αvβ3 integrin heterodimer receptors expressed primarily on the surface of cancer cells. The inventors used RGD4C/αvβ3 as a ligand-receptor system to demonstrate the conceptual feasibility of this new platform technology.

In this novel design, two complementary mammalian transgene expression cassettes are linked by an ITR derived from AAV2 (fig. 1 and 2). The inventors also added a second ITR, flanking the parental transgene expression cassette, to protect it during cell transduction and to increase its persistence over time (figures 1 and 2).

Example 2-scPP showed a significant increase in gene transfer compared to PAAV

In a first set of experiments, the inventors tried to study the gene delivery profile of a newly designed phage vector (scPP) to verify whether the scPP vector performs better in mammalian cells than the corresponding single-stranded phage vector control (PAAV). Thus, the inventors have performed parallel comparisons of scPP with PAAV gene expression. To transduce cells, the inventors constructed tumor-targeting phage particles that displayed a double-loop RGD4C in the pIII gene of the filamentous M13KO7 helper phage. RGD4C ligand binds to the αvβ3 integrin heterodimeric receptor, which is highly expressed on tumor cells and tumor blood vessels, but barely detectable on healthy tissues. This ligand has been widely used to introduce M13 phage vectors into mammalian cells. Non-targeting vectors without RGD4C were also included in the experiment and added to the cells as negative controls.

First, the inventors used a vector expressing a Green Fluorescent Protein (GFP) reporter gene (fig. 3) and treated murine melanoma B16-F1 cells, as the cells expressed the αvβ3 receptor for the RGD4C ligand. Microscopic analysis of GFP expression on day 4 post transduction showed that GFP was produced in large amounts in rgd4c.scpp-GFP transduced B16-F1 tumor cells, significantly higher than rgd4c.paav-GFP treated cells (fig. 4A). Furthermore, analysis of GFP expression by flow cytometry showed a dose-dependent RGD4 C.scPP-GFP-induced GFP expression with a GFP positive cell proportion of more than 35% at 10 ⁶ TU/cell, in contrast to a GFP positive cell proportion of less than 5% at 10 ⁶ TU/cell for RGD4C.PAAV-GFP (FIG. 4B). Importantly, GFP expression was not detected in cells treated with non-targeted phage particles (NTs) that did not contain RGD4C ligand, demonstrating that gene delivery of the targeted particles was selective for integrin-expressing cells and mediated by RGD4C ligand (fig. 4B).

Next, to validate these data, the inventors performed a comprehensive quantitative analysis of gene delivery using particles carrying a reporter gene encoding secreted gaussian luciferase (Lucia) (8, 16) (fig. 5). Gene expression is quantified by assaying luciferase activity in the growth medium. The inventors tested different doses of particles and assessed gene expression over a period of several days. Furthermore, the inventors have collected a set of tumor cell lines from different species and different histological sources to rule out the possibility that the observed gene delivery efficiency of rgd4c.scpp is species-specific or histological-specific. The transduction experiments used murine melanoma B16-F1 cells, B16-F10 cells and RMS metastatic melanoma cells. The inventors also incorporated human MCF7 breast cancer cells, a549 lung cancer cells, and human osteosarcoma cells. In addition, the inventors tested these vectors on human embryonic kidney HEK293 cells, as the cells have been widely used for routine gene delivery, viral and non-viral transduction, and DNA transfection experiments, and have previously also been used as standard in vitro models for phage-mediated gene delivery. The data show that at all doses tested, gene expression of rgd4c.scpp particles was detectable at the earliest 1 to 2 days post treatment and increased gradually over time (fig. 6-14). In contrast, the onset of gene expression of rgd4c.paav was delayed and its expression level was always significantly lower than rgd4c.scpp at all time points, doses and cell lines tested (fig. 6-14). The non-targeted particles as controls did not show any gene expression (fig. 6-14). These findings suggest that the observed enhanced gene expression may be due to earlier induction, more successfully transduced cells, or may be the result of the co-action of these two non-mutually exclusive events.

Finally, to demonstrate that this advantage of scPP in gene delivery is not limited to reporter genes, i.e., GFP or Lucia, but can also be applied to therapeutic genes, the inventors constructed vectors carrying both tumor necrosis factor α (tnfα) and interleukin 15 (IL 15), which are used in cancer immunotherapy (8). After phage transduction, protein expression levels of both cytokines in the cell culture medium were detected by ELISA. Similarly, the data show that the newly designed scPP particles expressed significantly higher levels of both tnfα and IL15 in cell culture medium compared to PAAV (fig. 15-17). The inventors also constructed a vector carrying another cytokine, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and again demonstrated that scPP produced significantly higher levels of TRAIL in human osteosarcoma cell supernatants compared to PAAV-treated human osteosarcoma cells (fig. 18).

Example 3 comparison of in vivo Gene delivery to mouse solid tumors after systemic administration

To translate these findings into in vivo studies, the inventors compared the gene delivery of scPP and PAAV after intravenous administration to tumor-bearing mice. The inventors used a vehicle for delivery of TRAIL and injected the vehicle into immunodeficient mice with established human subcutaneous xenografts (osteosarcoma). To this end, 5×10 ¹⁰ TU/mouse was administered to tumor-bearing mice at the dose previously used for phage vectors, followed by identification of TRAIL MRNA transcript expression in tumors by reverse transcription real-time quantitative PCR (RT-qPCR) (fig. 19). Parallel biodistribution studies of scPP and PAAV were also performed in tumor-bearing mice to analyze gene delivery in tumors and major internal organs. These biodistribution experiments were performed to ensure that after intravenous administration of rgd4c.scpp particles, gene expression was selective for established tumors in mice, whereas no expression was in healthy tissue (expression in healthy tissue may lead to off-target effects). The detected TRAIL MRNA transcript expression was significantly higher in the rgd4c.scpp injected mice tumors compared to rgd4c.pssaav injected mice (fig. 19). Furthermore, TRAIL expression in healthy tissue is negligible, similar to the control group, suggesting that rgd4c.scpp can target tumors efficiently and systemically without affecting other major internal organs. Non-targeted particles did not show significant expression in either the tumor or any organ studied.

EXAMPLE 4 study of transgene expression mechanism

To understand the molecular mechanism of scPP-mediated transgene expression and its reasons for its superiority over PAAV, the inventors studied the extracellular and intracellular targeting of particles after treatment of mammalian cells and compared scPP in parallel with PAAV at various steps of gene delivery.

Diffusion through the extracellular matrix (ECM)

The diffusion efficiency was assessed using FITC-labeled particles and their ability to migrate in matrigel supports (fig. 20). No significant differences were detected between the two constructs, indicating that their diffusion characteristics through the extracellular matrix (ECM) are similar. This is consistent with what was reported in previous studies, i.e., the diffusion of the M13 phage vector is determined by the particle size (8). Indeed, extracellular matrix analysis of scPP and PAAV showed that there was no difference in size between the two particles.

Internalization

Next, the inventors tried to investigate the entry of phage into transduced cells. To this end, B16-F1 cells were transduced and treated with two different methods 6 hours after transduction. The first method is to stain the particles with anti-fd phage antibodies and quantitate them by flow cytometry (fig. 21A). The second method was to extract DNA and quantitate it by qPCR with the ampicillin gene present in the phagemid as target (FIG. 21B). Again no difference was detected between the two particles.

Example 5-improvement of the efficiency of scPP Gene delivery is not due to the presence of two transgene expression cassettes

Since the two vectors do not show any differences in extracellular matrix diffusion and cell entry, to gain further insight into the mechanism behind the difference between scPP and PAAV, the inventors investigated whether the gene delivery advantage of scPP arises from the fact that it carries an additional transgene expression cassette. Thus, the inventors constructed a control vector containing two copies of the transgene expression cassette, but in the same direction, clockwise (cw), to avoid any sequence complementarity and hybridization. This vector was designated clockwise phage particle or cwPP (FIG. 22A). To take into account any potential effects that may be generated by the specific orientation of the two transgenic expression cassettes, both of which are in the counterclockwise (aw) orientation, a second control vector was constructed, designated awPP (FIG. 22A). Subsequently, the scPP vector was transduced in parallel with the control vector, and the results showed that the efficiency of the scPP vector was better than both controls cwPP and awPP (fig. 22B). Furthermore, to investigate whether intermolecular hybridization between complementary transgene sequences provided in different vectors can achieve efficiency comparable to scPP, the inventors performed synchronous transduction with cwPP and awPP vectors (fig. 23A). Although the efficiency of this synchronous transduction was higher than cwPP and awPP used alone, it was still lower than scPP (fig. 23B). Overall, these data indicate that the observed improvement in the efficiency of the scPP vector correlates with its ability to achieve successful intramolecular hybridization.

EXAMPLE 6 particle size

The similarity of cPP to PAAV particles in terms of diffusion and internalization suggests that there is no difference in the size of these two particles, consistent with recent reports (8). Thus, the inventors speculate that scPP should be able to package the compressed genome, thereby forming particles similar to PAAV particle sizes. As a preliminary study, the inventors analyzed scPP and PAAV particles by Transmission Electron Microscopy (TEM) and quantified the length of individual phage particles (fig. 24). Helper phages were also analyzed to help identify helper phage populations in the scPP and PAAV formulations. Importantly, TEM imaging showed that the scPP and PAAV particles were very close in size (fig. 24). To further confirm these findings, the inventors also analyzed cwPP particle sizes by electron microscopy, and found an increase in cwPP carrier size compared to scPP (fig. 24). The above results were confirmed in parallel by agarose gel electrophoresis of whole phage particles (FIG. 25).

Taken together, these findings indicate that scPP is a more efficient vector than PAAV. Since no differences were detected in terms of diffusion and cell entry, such differences are likely to be related to their different genome designs. This is also supported by the similar particle size of the two vectors, probably because the self-complementing phage particles (scPP) pack a more compact genome, which is likely the result of self-hybridization between the two complementing transgene expression cassettes. Indeed, the fact that control particles encoding copies of two co-rotating transgene expression cassettes cannot reach the transduction efficiency of scPP suggests that the presence of dual transgene expression cassette loading does not directly lead to the cause of the efficiency improvement (whether alone or in combination), but rather supports self-hybridization to form a double stranded DNA transgene expression cassette during phage production, a mechanism behind the advantages of scPP.

EXAMPLE 7 scPP capsid packaging double-stranded transgenic DNA expression cassette

To confirm that the scPP genome is capable of forming a double-stranded DNA structure and is packaged as a double-stranded DNA by the phage capsid, the inventors extracted the scPP genome from the phage capsid/particle and then digested with BamHI, a double-stranded DNA digestive enzyme whose target sequence is located within the transgene expression cassette. In other words, successful digestion of BamHI can only occur in the presence of double stranded DNA (FIG. 26A). As expected, a 1898bp band was detected in the digested sample, whereas it was not detected in the undigested control (FIG. 26B). To further confirm that this band was indeed produced by self-hybridization of a single-stranded DNA molecule, the inventors speculate that under denaturing conditions leading to DNA dehybridization, the molecule will unfold and migrate as a 3796kb band (fig. 26A).

Under denaturing conditions, the 5000bp DNA ladder band produced two distinct bands (due to separation of their complementary strands) (FIG. 26C), indicating that the 4000bp reference band corresponds to the sixth band in the denaturing ladder. As assumed, the extracted band in denatured form was expected to be 3796bp in length with a migration rate approximately the same as that of the 4000bp band in the denaturation step (FIG. 26D).

These findings provide strong evidence that phage capsids package double stranded DNA transgene expression cassettes during production in bacteria, due to self-hybridization of their two complementary transgene expression cassettes during the preparation of scPP in the host bacteria.

Example 8 delivery of double-stranded transgenic expression cassettes by scPP particles upon transduction of mammalian cells

Next, the inventors sought to investigate whether scPP vectors could deliver a double stranded transgene expression cassette, and the expression cassette did not require host cells to synthesize the complementary strand of the transgene expression cassette during transduction and gene expression. Indeed, the inventors speculate that if these vectors are capable of delivering double-stranded transgene expression cassettes upon entry into mammalian cells, then host cell DNA synthesis may be omitted from playing a role in transduction.

The inventors compared the expression of the scPP-Lucia vector with PAAV-Lucia in B16-F1 cells, which were pretreated with Hydroxyurea (HU) 24 hours prior to transduction to inhibit host cell DNA synthesis. The hydroxyurea treatment was continued at the same concentration without interruption after transduction and continued until the expression of Lucia was detected. Importantly, unlike the traditional single-stranded phage vector (PAAV), the DNA replication inhibitor hydroxyurea did not affect transduction of the scPP vector (fig. 27). In contrast, hydroxyurea inhibited PAAV gene expression (fig. 27). These data indicate that transduction of scPP is independent of DNA synthesis and thus transformation of the transgene expression cassette from single-stranded to double-stranded.

Example 8 comparison of Lucia reporter gene delivery

Referring to fig. 28, it is shown that in human metastatic osteosarcoma 143B cells PAAV, compared to the luca reporter gene delivery of scPP, the vehicle dose used was gradually increased over the time frame from day 1 to day 3 post-treatment. No expression of the Lucia gene was observed in the control group (untreated and non-targeted groups) on days 1 and 2 post transduction. However, on day 1 post-transduction, at 500000 and 1000000 TU/cell doses, expression of the Lucia gene was observed in the RGD4 C.scPP-treated group, whereas no expression was observed in the PAAV-treated group. This demonstrates the effectiveness of the self-complementing scPP vector in the immediate expression of the transgene. At days 2 and 3 post transduction, the expression level of Lucia was higher in the rgd4c.scpp treated group than in the rgd4c.paav treated group at doses of 100000 to 1000000TU per cell.

The method comprises the following steps:

143B cells were seeded in 96-well plates to achieve 60-70% confluency 48 hours after seeding. The average cell number per well or per plate was calculated on the day of transduction, and based thereon the amount of tumor targeted RGD4C-PAAV or scpP particles carrying the secreted luciferase (Lucia) gene that had to be added to the culture system was calculated. Non-targeted (NT) phage carrying the same gene and untreated cells were used as controls. Subsequently, an appropriate amount of the pellet stock solution was diluted in DMEM medium containing 10% serum and thoroughly mixed to prepare a transduction mixture. The concentration of the transduction mixture ranges from 100000 to 1000000 Transduction Units (TUs) per cell. The recommended volume of transduction mixture used per well is the minimum amount (50 ul) that can completely cover the cell monolayer. After 24 hours of transduction, the medium was supplemented with DMEM medium containing 10% serum to 150uL. The transduced cells were continued to be cultured until analysis (from day 1 to day 3).

For evaluation and quantification of gene expression, for phage (PAAV or scPP) particles carrying secreted luciferase reporter gene, 10 μl of culture medium was taken for each day after transduction to detect luciferase activity by mixing the sample with 25 μ l QUANTI-Luc ^TM reagent (InvivoGen Co., U.S.) for 5min, followed byNavigator microplate luminometer (Promega Co., U.S.A.) with integration time of 0.1 seconds.

Example 9-comparison of delivery of secreted cytokine sTRAIL to human metastatic osteosarcoma 143B cells

Referring to FIG. 29, the expression of sTRAIL gene in the medium of human metastatic osteosarcoma 143B cells transfected with PAAV or scPP DNA constructs is shown. Untreated cells and cells treated with transfection reagents were used as controls. The level of sTRAIL protein (pg/ml) was detected by TRAIL ELISA kit. The experiment was performed in triplicate. Statistical analysis used independent t-test, one-way anova and Tukey's HSD post hoc test. All results are expressed as mean ± Standard Error (SEM). * P <0.01, P <0.001. The data show that cells transfected with the scPP-STRAIL DNA construct expressed higher levels of sTRAIL in the medium than cells transfected with the PAAV-STRAIL DNA construct.

The method comprises the following steps:

143B cells were seeded into 6-well format plates to reach 80% confluency at 24 hours of culture. The medium was replaced with low serum medium (Opti-MEM, company Thermofisher, uk) for 2 hours prior to transfection. The transfection mixture was prepared by mixing 2. Mu. g PAAV-sTRAIL or scPP-STRAIL DNA construct with 6. Mu.l in low serum medium HD (Promega company, uk) were mixed. The mixture was incubated at room temperature for 20-25 minutes. Next, the mixture was added drop-wise to a culture plate containing low serum medium and cells. Thereafter, the cells were returned to the incubator and cultured for 48 hours. Finally, the medium was collected and TRAIL levels were quantified by ELISA. * sfil = secretory TRAIL. Secreted sTRAIL levels in the supernatant were measured using the human TRAIL/TNFSF10 DuoSet ELISA kit (R & D systems, UK). The detection operation is performed according to the manufacturer's instructions.

EXAMPLE 10 Induction of osteosarcoma cell death in vitro following treatment with scPP-sTRAIL encoding secreted sTRAIL

Referring to fig. 30, it is shown that the rgd4c.scpp-sTRAIL particle treated group had a dose-dependent lower cell viability. Data are expressed as percent cell viability relative to untreated cells. The experiment was performed in triplicate. Statistical analysis used one-way analysis of variance and Tukey's HSD post hoc testing. All results are expressed as mean ± Standard Error (SEM). * P <0.05.

The method comprises the following steps:

143B cells were seeded into 6-well format plates to achieve 60-70% confluency 48 hours after seeding. The average cell number per well was calculated on the day of transduction and based thereon the amount of tumor targeted rgd4c.scpp particles carrying the secreted TRAIL(s) gene to be added to the culture was calculated. Non-targeted (NT) phage carrying the same gene and untreated cells were used as controls. Subsequently, an appropriate amount of the pellet stock solution was diluted in DMEM medium containing 10% serum and thoroughly mixed to prepare a transduction mixture. The concentration of the transduction mixture ranged from 500000 to 1000000 Transduction Units (TUs) per cell (shown as 0.5 and 1.0, respectively, in the graph). The recommended volume of transduction mixture used per well is the minimum amount (1 ml) that can completely cover the cell monolayer. After 24 hours of transduction, the medium was supplemented with DMEM medium containing 10% serum to 2mL. The transduced cells were continued to be cultured for 3 days. Cell death was assessed by cell viability assay.

CellTiter-Glo luminous cell viability assay an equal volume of CellTiter-Glo reagent (Promega, UK) was added to the medium in the culture wells containing the transduced cells, followed by mixing for two minutes on an orbital shaker to induce cell lysis. Next, the mixture was incubated at room temperature for 10 minutes to stabilize the luminescence signal, followed by transfer to a read plate luminometer. Signals were detected using GloMax Navigator microplate luminescence detector (Promega, uk).

Example 11-toxicity assessment. Lactate Dehydrogenase (LDH), a biomarker of toxicity in mice, was not elevated

Referring to FIG. 31, there is shown that none of the serum LDH levels were elevated in the mice treated with the scPP-sTRAIL and PAAV-sTRAIL particles compared to untreated mice. This data indicates that PAAV and scPP are safe for in vivo treatment. LDH data are expressed as relative values to untreated groups. The experiment was performed in triplicate. Statistical analysis used one-way analysis of variance and Tukey's HSD post hoc testing. There were no statistically significant differences in this experiment.

The method comprises the following steps:

Athymic mice (BALB/c nu/nu,8-10 weeks old) were purchased from Charles river laboratories, UK. 143B cells were inoculated subcutaneously into thymus-free mice in an amount of 2X 10 ⁶ cells per mouse to establish human OS cells (human osteosarcoma cells). Tumor-bearing mice were injected intravenously with either targeted (RGD 4C) or non-targeted (NT) phage (PAAV or scPP) particles carrying the sTRAIL gene at doses of 5 x 10 ¹⁰ TU per mouse on days 3, 5 and 9 of the experiment. At the end of the experiment (day 10), mice were sacrificed by cardiac perfusion. Subsequently, whole blood was collected from the heart, and serum samples were prepared by centrifugation at 1600g for 15 minutes. LDH levels in serum were tested to assess phage-treated toxicity. The experiment uses Cytotox Non-radioactive cytotoxicity assay kit (Promega, uk), the assay procedure was performed according to the manufacturer's protocol.

Example 12-biodistribution of sTRAIL delivery in tumor-bearing mice with established osteosarcoma models

Referring to FIG. 32, the data shows the relative expression levels of human TRAIL gene (relative to untreated groups) in different organs of mice after treatment with PAAV or scPP particles carrying the sTRAIL gene. The rgd4c.scpp-sTRAIL particles are most effective in targeting and delivering genes to tumors, followed by rgd4c.paav-sTRAIL. Non-targeted particles do not show significant expression in tumors or any other organ. The experiment was performed in triplicate. Statistical analysis was performed using a two-way ANOVA (two-way ANOVA) and a multiple comparison t-test. All results are expressed as mean ± Standard Error (SEM). * P <0.01.

The method comprises the following steps:

Athymic mice (BALB/c nu/nu,8-10 weeks old) were purchased from Charles river laboratories, UK. 143B cells were inoculated subcutaneously into athymic mice in an amount of 2X 10 ⁶ cells per mouse to establish human OS cells. Tumor-bearing mice were injected intravenously with either targeted (RGD 4C) or non-targeted (NT) phage (PAAV or scPP) particles carrying the sTRAIL gene at doses of 5 x 10 ¹⁰ TU per mouse on days 3,5 and 9 of the experiment. At the end of the experiment (day 10), mice were sacrificed by cardiac perfusion. Tumors and normal organs including lung, liver, spleen, heart, kidney, pancreas and brain were collected. Total RNA was extracted from these organs and the expression of human TRAIL was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR).

Example 13 immunofluorescent staining of tumors displaying sTRAIL protein expression after RGD4C.PAAV and RGD4C.scPP treatment with encoded sTRAIL

Referring to fig. 33, confocal microscopy analysis showed that TRAIL expression was only detected in tumors of rgd4c.paav.strail and rgd4c.scpp.strail treated groups (green). Higher TRAIL expression was observed in scPP treated tumors. These findings indicate that rgd4c.scpp.strail is able to target tumors effectively and comprehensively. Non-targeted (NT) phage particles did not show significant TRAIL expression in tumors.

The method comprises the following steps:

Athymic mice (BALB/c nu/nu,8-10 weeks old) were purchased from Charles river laboratories, UK. 143B cells were inoculated subcutaneously into athymic mice in an amount of 2X 10 ⁶ cells per mouse to establish human OS cells. Tumor-bearing mice were injected intravenously with either targeted (RGD 4C) or non-targeted (NT) phage (PAAV or scPP) particles carrying the sTRAIL gene at doses of 5 x10 ¹⁰ TU per mouse on days 3,5 and 9 of the experiment. At the end of the experiment (day 10), mice were sacrificed by cardiac perfusion. Tumors were collected and frozen for sectioning. Human TRAIL expression in tumor tissues was detected by immunofluorescent staining. The expression of TRAIL was assessed using anti-human TRAIL antibodies on optimal cutting temperature compound (OCT) frozen sections (6 μm) of tissue. Sections were fixed in 4% paraformaldehyde (damschtatt Merck, germany) for 15 minutes at room temperature. The sections were then incubated for 1 hour in 5% normal goat serum in TBS (Tris-buffered saline) containing 0.3% Triton-X, followed by incubation with primary antibodies (rabbit anti-human TRAIL polyclonal antibodies, thermo FISHER SCIENTIFIC, UK). Subsequently, tissue sections were incubated with Alexa in TBS containing 1% filtered bovine serum albumin and 0.3% Triton-X 488 Conjugated goat anti-rabbit IgG was incubated for 30 min. After three washes with PBS, the slides were blocked with Prolong Gold anti-fluorescence quenching blocks (company Life Technoligies, uk). Cell sections were imaged using a DMi8 advanced confocal fluorescence microscope (Wei Cila mol Leica Microsystems, germany).

EXAMPLE 14 hematoxylin-eosin staining of tumors showing extensive lesions after systemic treatment

Referring to fig. 34, hematoxylin-eosin staining results of tumors are shown, with extensive lesions in tumors after systemic treatment with rgd4c.scpp-sTRAIL compared to untreated mice or mice treated with non-targeted (NT) vehicle.

The method comprises the following steps:

Athymic mice (BALB/c nu/nu,8-10 weeks old) were purchased from Charles river laboratories, UK. 143B cells were inoculated subcutaneously into athymic mice in an amount of 2X 10 ⁶ cells per mouse to establish human OS cells. Tumor-bearing mice were injected intravenously with either targeted (RGD 4C) or non-targeted (NT) phage particles (PAAV or scPP) carrying the sTRAIL gene at doses of 5 x 10 ¹⁰ TU per mouse on days 3, 5 and 9 of the experiment. At the end of the experiment (day 10), mice were sacrificed by cardiac perfusion. Tumors were collected and processed for frozen sections. An optimal cutting temperature complex (OCT) frozen section (6 μm) of tissue was prepared and hematoxylin-eosin stained.

Conclusion(s)

The inventors constructed a novel phage vector comprising complementary single stranded sequences of a transgene expression cassette to induce hybridization upon cell transduction or during production and manufacturing in a bacterial host. The inventors have conducted in vitro experiments using various cell lines and transgenes and observed a surprising increase (3-to 15-fold) in transduction efficiency of the self-complementing phage particles (scPP) of the invention compared to traditional pure single-stranded DNA phage vectors. In fact, the self-complementing phage vectors exhibited rapid transduction onset and higher transgene expression levels in all cell lines tested. More importantly, unlike traditional single-stranded phage vectors, inhibitors of DNA replication do not affect transduction from the complementary phage vector. Furthermore, in vivo studies showed significant enhancement of gene delivery to solid tumors in mice following systemic administration compared to single-stranded DNA phage particles. All these biological properties demonstrate that the construction and characterization of novel filamentous phage vectors comprising self-complementing single-stranded DNA, which deliver the transgene cassette as double-stranded DNA by hybridization, will make a significant contribution to the continued development of phage-based gene delivery systems.

The techniques described herein have a number of unique features, including packaging hybridizable self-complementary single-stranded DNA (i.e., double-stranded DNA) transgene expression cassettes from M13 phage capsids. Furthermore, in contrast to the prior art, the system is able to package large genomes by using two Inverted Terminal Repeats (ITRs) instead of three ITRs. Moreover, this technique allows the M13 phage to rapidly initiate gene expression in mammalian cells as compared to existing phage vectors. This is also the first demonstration of hybridization between the genome of hybridizable self-complementary single-stranded DNA (i.e., double-stranded adeno-associated virus, dsAAV) and the phage capsid. In other words, this is the first hybrid vector consisting of a phage capsid with hybridizable complementary single-stranded DNA (i.e., double-stranded recombinant adeno-associated virus, ds-rAAV). Furthermore, this is the first report of the ability to package and deliver a hybridizable complementary single-stranded DNA transgene cassette (i.e., a double-stranded transgene cassette), and the transgene cassette can be prepared to initiate gene expression in mammalian cells. In addition, since the inventors used a transgene cassette that was flanked by adeno-associated virus inverted terminal repeats (AAV ITRs), this was the first report showing that double-stranded adeno-associated virus DNA (from hybridizable complementary single-stranded DNA) was packaged with a phage capsid and delivered to mammalian cells, as AAV capsids were not present therein.

In addition, the phage vector of the present invention allows phage to initiate gene expression in mammalian cells faster than existing phage vectors. Furthermore, the delivery of double stranded adeno-associated virus (ds AAV) vectors is costly, and the use of phage capsids to deliver double stranded adeno-associated viral DNA is cost-effective as described herein, because such delivery systems are produced in bacteria, utilizing a cost-effective production and purification process for phage vectors in prokaryotic hosts that is compatible with industrial scale reactors and separation systems. This also contributes to an increase in production scale, thereby directly reducing costs.

The inventors supported their findings using soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL), demonstrating that self-complementing phage particles (scPP) perform surprisingly well in gene delivery. For example, when using genes such as TRAIL, the inventors demonstrated cancer cell death, indicating that the scPP vector can be used to deliver therapeutic genes in vivo or in vitro.

The inventors believe that this technology will have an impact on the field of phage gene delivery, adeno-associated virus (AAV) gene therapy and systemic delivery. This delivery platform is applicable to systemic gene therapy of cancer and other human diseases because the phage capsid is not targeted to human tissue, so it can be delivered systemically to the disease tissue targeting site via ligands shown on the phage capsid, allowing therapeutic DNA to enter and be delivered in place.

Reference to the literature

1-Ivanenkov V.,Felici F.,Menon A.G."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 C.M.,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 tomammalian 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.,WilliamsonR.,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 C.E.,Soghomonyan S.,Alauddin M.M.,Marini F.C.,3rd,Restel B.H.,Ozawa M.G.,Moya C.A.,Rangel R.,Sun Y.,Zaoui K.,Schmidt M.,von Kalle C.,Weitzman M.D.,Gelovani J.G.,Pasqualini R.,Arap W."A hybrid vector for ligand-directed tumortargeting and molecular imaging"Cell 2006,125:385-398.

7-Yata,T.,Lee,E.L.Q.,Suwan,K.,Syed,N.,Asavarut,P.and Hajitou A."Modulation ofExtracellular Matrix in Cancer is Associated with Enhanced Tumor Cell Targeting by BacteriophageVectors"Mol.Cancer 2015,14:110.

8-Asavarut A.,Waramit S.,Suwan K.,Marais G.J.K.,Chongchai A.,Benjathummarak S.,Al-Bahrani M.,Vila-Gomez P.,Williams M.,Kongtawelert P.,Yata T.and Hajitou A."Systemicallytargeted Cancer Immunotherapy and Gene Delivery using Transmorphic Particles"EMBO Mol.Med.2022,14:e15418.

9-Stoneham,C.A.,Hollinshead,M.,and Hajitou A."Clathrin-Mediated Endocytosis andSubsequent 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 systemictransgene 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."DualSystemic Tumor Targeting with Ligand-directed Phage and Grp78 Promoter Induces TumorRegression"Mol.Cancer Ther.2012,11:2566-2577.

12-Przystal J.M.,Waramit S.,Pranjol M.Z.I.,Yan W.,Chu G.,Chongchai A.,Samarth G.,Olaciregui N.G.,Tabatabai G.,Carcaboso A.M.,Aboagye E.O.,Suwan K.and Hajitou A."Efficacyof systemic temozolomide-activated phage-targeted gene therapy in human glioblastoma"EMBO.Mol.Med.2019,11:e8492.

13-Tsafa E.,Bentayebi K.,Topanurak S.,Yata T.,Przystal J.,Fongmoon D.,Hajji N.,WaramitS.,Suwan K.and Hajitou A."Doxorubicin Improves Cancer Cell Targeting by Filamentous PhageGene Delivery Vectors"Int.J.Mol.Sci.2020,21:7867.

14-Monaci P.,Urbanelli L.and Fontana L."Phage as gene delivery vectors"Curr.Opin.Mol.Ther.2001,3:159-169.

15-Burg M.A.,Jensen-Pergakes K.,Gonzalez A.M.,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 X.O.,TannousB.A."A secreted luciferase for ex vivo monitoring of in vivo processes"Nat Methods 2008,5:171-173.

Claims

1. A phage vector, characterized in that it comprises:

At least two single-stranded self-complementary transgenic expression cassettes separated by a linker hybridize to form a double-stranded transgenic expression cassette.

2. The phage vector according to claim 1, characterized in that:

The phage vector contains a packaging signal that enables the replication of the at least two single-stranded self-complementary transgenic expression cassettes, which hybridize in bacteria and are subsequently packaged as double-stranded transgenic expression cassettes into the phage vector within a prokaryotic host.

3. The phage vector according to claim 2, characterized in that:

The packaging signal includes a phage replication origin, which may optionally be an F1 origin.

4. The phage vector according to any one of the preceding claims, characterized in that:

The phage vector contains a bacterial replication origin, optionally a pUC origin.

5. The phage vector according to any one of the preceding claims, characterized in that:

The phage vector contains one or more DNA sequences that enable it to target and integrate into the host genome.

6. The phage vector according to any one of the preceding claims, characterized in that:

The at least two self-complementary transgene expression cassettes include viral transgene expression cassettes, preferably mammalian viral transgene expression cassettes.

7. The phage vector according to any one of the preceding claims, characterized in that:

The at least two self-complementary transgene expression cassettes comprise either a lentiviral transgene expression cassette or an adeno-associated virus (AAV) transgene expression cassette.

8. The phage vector according to any one of the preceding claims, characterized in that:

The at least two self-complementary transgenic expression cassettes contain any nucleic acid encoding a factor that may have therapeutic or industrial use in target cells or tissues. Optionally, the nucleic acid is DNA, cDNA, RNA, antisense RNA, or shRNA.

9. The phage vector according to claim 8, characterized in that:

The factor encoded by the nucleic acid is a polypeptide or a protein.

10. The phage vector according to any one of the preceding claims, characterized in that:

Each of the at least two transgene expression cassettes contains a promoter, optionally wherein the promoter is a cytomegalovirus promoter, a glucose regulatory protein 78 promoter, a tumor-specific promoter, or a tissue-specific promoter.

11. The phage vector according to any one of the preceding claims, characterized in that:

Each of the at least two transgenic expression cassettes contains nucleic acids for polyA tails.

12. The phage vector according to any one of the preceding claims, characterized in that:

The phage vector contains four single-stranded self-complementary transgenic expression cassettes separated by linkers, which hybridize to form two double-stranded transgenic expression cassettes.

13. The phage vector according to any one of the preceding claims, characterized in that:

The two single-stranded self-complementary transgenic expression cassettes are positioned in opposite directions within the phage vector. Preferably, the first transgenic expression cassette extends in the 5' to 3' direction, while the corresponding second transgenic expression cassette extends in the 3' to 5' direction.

14. The phage vector according to any one of the preceding claims, characterized in that:

Wherein, the percentage of sequence identity between the first transgenic expression cassette and the second transgenic expression cassette is at least 65%, 70%, or 75%, or wherein the percentage of sequence identity between the first transgenic expression cassette and the second transgenic expression cassette is at least 80%, 85%, 90%, or 95%.

15. The phage vector according to any one of the preceding claims, characterized in that:

The linker separating the at least two self-complementary transgenic expression cassettes is a terminal inverted repeat (ITR).

16. The phage vector according to claim 15, characterized in that:

The phage vector contains a second ITR, wherein the second ITR is located on the flank of one of the at least two self-complementary transgene expression cassettes.

17. The phage vector according to claim 15 or 16, characterized in that:

Wherein, the first ITR and/or the second ITR are AAV ITRs.

18. The phage vector according to any one of claims 15-17, characterized in that:

The phage vector contains only two ITRs, preferably fewer than three ITRs.

19. The phage vector according to any one of claims 1-14, characterized in that:

The linker separating the at least two self-complementary transgenic expression cassettes is an unrelated DNA fragment, wherein the percentage of sequence identity between the linker and the first and second transgenic expression cassettes is less than 50%, 45%, or 40%, preferably less than 35%, 30%, or 25%. Optionally, the length of the unrelated DNA fragment is between 60 bp and 300 bp, between 80 bp and 280 bp, between 100 bp and 260 bp, between 120 bp and 240 bp, between 140 bp and 220 bp, or between 160 bp and 200 bp.

20. The phage vector according to any one of the preceding claims, characterized in that:

The phage vector contains a selection marker, optionally wherein the selection marker is an ampicillin resistance gene.

21. The phage vector according to any one of the preceding claims, characterized in that:

The phage vector comprises one or more capsid minor coat proteins; optionally, the phage vector comprises a pIII capsid minor coat protein configured to display a cell-targeting ligand to enable delivery of the vector to target cells; and/or

The phage vector contains one or more capsid major shell proteins, and optionally, the phage vector contains at least one pVIII capsid major shell protein configured to display an exogenous peptide thereon.

22. The phage vector according to any one of the preceding claims, characterized in that:

The phage vector contains a genome that is substantially missing from the phage genome from which the vector originates. Optionally, the genome of the phage vector is missing at least 60%, more preferably at least 70%, or even more preferably at least 80% of the phage genome from which it originates.

23. The phage vector according to any one of the preceding claims, characterized in that:

The phage vector lacks in its genome the phage structural genes required for the formation, packaging, or release of particles from a prokaryotic host.

24. A system for generating a bacteriophage vector from a prokaryotic host, characterized in that it comprises:

(i) a first vector, configured to persist within a prokaryotic host, and comprising at least two single-stranded self-complementary transgenic expression cassettes separated by linkers and hybridizing to form a double-stranded transgenic expression cassette, and a packaging signal for enabling the replication of the at least two single-stranded self-complementary transgenic expression cassettes; and

(ii) A second vector containing nucleic acid for encoding structural proteins required for packaging the double-stranded transgenic expression cassette, thereby enabling the formation and release of the phage vector within the prokaryotic host.

25. The system according to claim 24, characterized in that:

The system is used to generate a phage vector according to any one of claims 1-23.

26. The system according to claim 24 or 25, characterized in that:

The first vector contains the genome of the phage vector.

27. The system according to any one of claims 24-26, characterized in that:

The packaging signal of the first vector includes a phage replication origin, preferably an F1 origin.

28. The system according to any one of claims 24-27, characterized in that:

The first carrier includes a second replication origin, preferably a pUC origin.

29. The system according to any one of claims 24-28, characterized in that:

The connector of the first carrier is an ITR, preferably an AAV ITR.

30. The system according to any one of claims 24-29, characterized in that:

The second vector is a bacteriophage specifically designed to rescue the genome of the first vector from the prokaryotic host. Preferably, the second vector is a replication-defective vector.

31. The system according to any one of claims 24-29, characterized in that:

The second vector contains a disrupted packaging signal that significantly inhibits the ability of the second vector to be packaged into phage particles. Preferably, the second vector contains a disrupted origin of replication.

32. The system according to claim 31, characterized in that:

The destroyed replication origin is either a medium replication origin, optionally p15a, or a low replication origin, optionally pMB1.

33. The system according to any one of claims 24-32, characterized in that:

The second vector comprises a first nucleic acid sequence and/or a second nucleic acid sequence, wherein the first nucleic acid sequence encodes a pIII capsid minor outer shell protein configured to display a cell-targeting ligand to enable the phage vector to be delivered to a target cell, and the second nucleic acid sequence encodes at least one pVIII capsid major outer shell protein configured to display an exogenous peptide thereon.

34. A method for generating a bacteriophage vector from a prokaryotic host, characterized in that it comprises:

(i) Introducing a first vector into a prokaryotic host cell, the first vector being configured to persist within the prokaryotic host and comprising at least two single-stranded self-complementary transgenic expression cassettes separated by linkers and hybridizing to form a double-stranded transgenic expression cassette, and a packaging signal for enabling the at least two single-stranded self-complementary transgenic expression cassettes to replicate.

(ii) Introducing a helper phage into a host, said helper phage containing nucleic acid encoding a phage structural protein; and

(iii) The host is cultured under conditions that produce a double-stranded transgenic expression cassette, which is packaged with structural proteins to enable the formation and release of a phage vector carrying the double-stranded transgenic expression cassette in the prokaryotic host.

35. A method for generating bacteriophage particles from a prokaryotic host, characterized in that it comprises:

(i) Introducing the following into a prokaryotic host cell: (a) a first vector configured to persist within the prokaryotic host and comprising at least two single-stranded self-complementary transgenic expression cassettes separated by linkers and hybridizing to form a double-stranded transgenic expression cassette, and a packaging signal for enabling the replication of the at least two single-stranded self-complementary transgenic expression cassettes; and (b) a second vector comprising structural proteins encoded by nucleic acids required for packaging the double-stranded transgenic expression cassette; and

(ii) The host is cultured under conditions that produce a double-stranded transgenic expression cassette packaged with structural proteins, so that the phage vector is formed in and released from the prokaryotic host.

36. The use of an auxiliary bacteriophage, characterized in that:

The helper phage contains nucleic acid encoding a viral vector structural protein for generating a phage vector from a prokaryotic host as described in any one of claims 1-23.

37. A host cell, characterized in that it comprises:

The first carrier and/or the second carrier as described in any one of claims 24-33.

38. The phage vector according to any one of claims 1-23, or the system according to any one of claims 24-33, characterized in that it is used as an experimental research tool, optionally, the tool is used in vitro or ex vivo.

39. The phage vector according to any one of claims 1-23, or the system according to any one of claims 24-33, characterized in that it is used for treatment or diagnosis.

40. The phage vector according to any one of claims 1-23, or the system according to any one of claims 24-33, characterized in that it is used in gene therapy technology.

41. The phage vector or system according to claim 40, wherein the gene therapy technology is used to treat, prevent or manage cancer.

42. A vaccine, characterized in that it comprises:

The phage vector as described in any one of claims 1-23 or the system as described in any one of claims 24-33.

43. The phage vector according to any one of claims 1-23, or the system according to any one of claims 24-33, characterized in that it is used to deliver a vaccine to a subject.

44. The phage vector according to any one of claims 1-23, or the system according to any one of claims 24-33, characterized in that it is used for targeted delivery of exogenous antigens to the tumor of a vaccine subject.

45. Use of a phage vector as described in any one of claims 1-23 or a system as described in any one of claims 24-33, characterized in that it is used in genetic molecular imaging techniques.

46. A pharmaceutical composition, characterized in that it comprises:

The phage vector as described in any one of claims 1-23, or the system as described in any one of claims 24-33, and a pharmaceutically acceptable vector.

47. A method for preparing the pharmaceutical composition of claim 46, characterized in that it comprises:

Contact a therapeutically effective amount of the phage vector as described in any one of claims 1-23 or the system as described in any one of claims 24-33 with a pharmaceutically acceptable carrier.

48. Use of a phage vector as described in any one of claims 1-23 or a system as described in any one of claims 24-33, characterized in that it is used to generate a recombinant viral vector comprising or derived from a viral genome within the genome of the phage vector.

49. A method for generating a recombinant viral vector, characterized in that it comprises:

The phage vector as described in any one of claims 1-23 or the system as described in any one of claims 24-33 is introduced into a eukaryotic host cell, such that the host cell is able to produce a recombinant viral vector.

50. The use according to claim 48 or the method according to claim 49, characterized in that:

The recombinant viral vector is a recombinant mammalian virus, rAAV, recombinant self-complementary AAV vector, or recombinant lentiviral vector.