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Definitions

• the present invention relates to phagemid vectors and associated phagemid particles, and in particular to hybrid and recombinant phagemid vectors, particles and expression systems.
• the invention extends to the use of such phagemid particles and expression systems as a research tool, and for the delivery of transgenes in a variety of gene therapy applications, DNA and/or peptide vaccine delivery and imaging techniques.
• the invention extends to in vitro, in vivo or in situ methods for producing viral vectors, such as recombinant adeno-associated viruses (rAAV) or lentivirus vectors (rLV), and to genetic constructs used in such methods.
• rAAV recombinant adeno-associated viruses
• rLV lentivirus vectors
• Adeno-associated virus (AAV)-mediated gene therapy is a case in point, as vector production has been a bottleneck for clinical translation.
• Recombinant AAV (rAAV) is an attractive vector in gene therapy.
• efficient vector production at commercial scales is not yet possible.
• a variety of expression systems have been developed for rAAV production.
• transfection-based protocols have been the gold standard for high-purity laboratory-scale production, they cannot be efficiently translated to commercial-scale protocols.
• Current methods for commercial-scale production rely on the use of eukaryotic viruses to rescue AAV vectors from a producer cell line.
• infectious eukaryotic viruses is a major point of concern, not only when purifying viral particles, but also regarding safeness for in vivo use.
• AAV are non-enveloped viruses with a 4.7Kb wild type genome that is flanked by inverted terminal repeats (ITRs).
• the genome contains two open reading frames, rep and cap, which provide proteins necessary for replication and encapsidation of the viral genome.
• wild type AAV is found in the presence of adenovirus (Ad) as they provide adeno-helper proteins that are essential for packaging of the AAV genome in to icosahedral virions. Therefore, AAV production relies on three key elements: the ITR- flanked genome, rep and cap, and adeno-helper genes.
• laboratory scale production of rAAV uses DNA transfection to introduce all three genetic elements in to human embryonic kidney HEK293 cells, which is a suitable mammalian producer cell as they inherently express an adeno-helper protein from immortalization.
• laboratory scale production provides rAAV of high purity, transfection methods are not suitable for large-scale production and face major limitations, including inefficiency, which leads to low rAAV yields and high costs.
• live viruses such as adenovirus or herpes simplex virus, are used to efficiently supply the helper functions, which present significant health and safety concerns for in vivo use.
• AAVP Adeno-associated Virus/Phage
• rAAV can be rescued from HEK293 cells transduced with AAVP following transfection with rep- and cap-expressing plasmid, and subsequent infection with wild- type adenovirus type 5.
• the AAVP vector possesses favourable characteristics of mammalian and prokaryotic viruses, and does not suffer from the disadvantages that those individual vectors normally carry.
• the AAVP still has certain inherent limitations of bacteriophage and thus leaves room for significant improvement of AAVP or phage vectors in general, and so there is a need for designing novel superior phage-based vectors.
• AAVP are a hybrid between two virus species (i.e. bacteriophage and AAV), AAVP vectors contain the genome of both the eukaryotic and prokaryotic viruses. Despite being essential for viral reproduction, the prokaryotic genome is functionally or
• phage viral genome thus deleteriously affects vector efficiency and the production method, and ultimately leads to AAVP's relatively low gene transduction efficacy when compared to mammalian viruses.
• hybrid phagemid viral vector system with the new phagemid particle being referred to as Phagemid/Adeno- associated Virion (i.e. PAAV).
• PAAV Phagemid/Adeno- associated Virion
• Another name used by the inventors for the novel vectors they have created is "phasmid”.
• PAAV genome of the invention does not contain any structural bacteriophage genes, and so a prokaryotic helper virus is required to facilitate vector assembly in the host.
• a recombinant phagemid particle for expressing a transgene in a target cell transduced with the particle comprising at least one transgene expression cassette which encodes an agent which exerts a biological effect on the target cell, characterised in that the phagemid particle comprises a genome which lacks at least 50% of its bacteriophage genome.
• re-engineering hybrid viral vectors e.g. AAV or lentivirus
• substantially lacking the phage genome from which the particle is derived dramatically enhances the functional properties of the resultant vector (i.e. the phagemid particle).
• Altering the viral expression system to a phagemid-based system according to the invention expands the possibility of applying phagemid viral vectors in a much broader context. By eliminating at least the particle's genome, the resultant particle size of the phagemid particle is
• phagemid particle can refer to a hybrid phagemid genome encapsulated by phage-derived coat proteins.
• the hybrid phagemid genome is a "phagemid genome” (i.e. a genetic construct containing two origins of replication - one from bacteriophage (e.g. Fi), and one from bacteria (e.g. pUCi)).
• the phagemid genome may contain an incorporated "recombinant transgene cassette from AAV" (rAAV), and is therefore a hybrid and not a conventional phagemid genome with a normal (i.e. generic, non-viral) recombinant transgene expression cassette.
• the phagemid particle can refer to the hybrid phagemid genome (i.e.
• phage proteins derived from a trans-acting agent such as a helper phage
• a trans-acting agent such as a helper phage
• these smaller phagemid particles also display added advantages in enhanced gene transfer, production yield, biodistribution and evasion from eukaryotic cellular barriers.
• Another significant advantage of using the phagemid particle of the invention is that they have the ability to accommodate extremely large and numerous transgene cassettes or gene inserts, such as genes of the three plasmids used for recombinant virus (e.g. rAAV or lentivirus) production by transfection, as described hereinafter.
• the phagemid particle comprises a virion.
• the genome of the recombinant phagemid particle comprises a packaging signal for enabling replication of the phagemid genome into single-stranded DNA, which can subsequently be packaged into the phagemid particle inside a prokaryotic host.
• the packaging signal may preferably comprise an origin of replication.
• the origin of replication preferably comprises an Fi ori, more preferably from an Fi bacteriophage.
• the DNA sequence of one embodiment of the Fi ori is represented herein as SEQ ID No: l, as follows:
• the genome of the recombinant phagemid particle comprises an origin of replication for enabling replication of double-stranded vector inside a prokaryotic host.
• the origin of replication enables high copy number replication of the vector inside the host.
• the origin of replication comprises a pUC ori.
• the DNA sequence of one embodiment of the pUC ori is represented herein as SEQ ID No: 2, as follows:
• the phagemid particle may be designed such that it integrates into the genome of a host cell.
• nucleic acid sequences which favour targeted integration (e.g. by homologous recombination) of the particle's genome are envisaged.
• the genome of the recombinant phagemid particle may comprise one or more DNA sequence, which favours targeted integration into a host genome.
• the phagemid particle may be used as an experimental research tool, and used ex vivo or in vitro.
• the phagemid particle may be used as a
• the at least one transgene expression cassette comprises a viral transgene expression cassette, more preferably a mammalian viral transgene expression cassette.
• the at least one transgene expression cassette may, in one preferred embodiment, comprise a lentivirus transgene expression cassette.
• the at least one transgene expression cassette is preferably an adeno-associated virus (AAV) transgene expression cassette.
• the transgene expression cassette may comprise any nucleic acid encoding an agent, which may have therapeutic or industrial utility in the target cell or tissue.
• the nucleic acid may be DNA, which may be genomic DNA or cDNA. Non-naturally occurring cDNA maybe preferred in some embodiments.
• the nucleic acid may be RNA, such as antisense RNA or shRNA.
• the transgene expression cassette may comprise shRNA configured to target mTOR expression in a tumour cell.
• shRNA configured to target mTOR expression in a tumour cell.
• down-regulation of mTOR expression in tumour cells e.g. medulloblastoma cells
• RGD4C-phagemid carrying the mTOR/ shRNA
• the agent encoded by the nucleic acid may be a polypeptide or protein.
• the transgene may encode the Herpes simplex virus thymidine kinase gene, which may subsequently exert a therapeutic effect on the target tumour cell.
• the transgene expression cassette may encode TNFa for expression in a tumour cell.
• RGD4C- phagemid can successfully deliver TNFa to DIPG in a selective manner, resulting in apoptosis induction. Therefore, RGD4C-phagemid-TNFa has therapeutic potential for use in targeted therapy against DIPG.
• recombinant phagemid particle depends on the type of cell-targeting ligand expressed on the surface of the particle.
• the transgene expression cassette may comprise one or more functional elements required for expression of the nucleic acid in the target cell.
• the transgene expression cassette comprises a promoter, such as the CMV promoter.
• the DNA sequence of one embodiment of the CMV promoter is represented herein as SEQ ID No: 3, as follows:
• the transgene expression cassette comprises a grp78 promoter.
• the nucleic acid sequence of one embodiment of the grp78 promoter is represented herein as SEQ ID No: 8, as follows:
• the transgene expression cassette comprises nucleic acid for encoding a polyA tail attachable to the expressed agent.
• the DNA sequence of one embodiment of the nucleic acid for encoding a polyA tail is represented herein as SEQ ID No: 4, as follows:
• the transgene expression cassette comprises left and/or right Inverted Terminal Repeat sequences (ITRs).
• ITR can be specific to an AAV or lentivirus serotype, and can be any sequence, so long as it forms a hairpin loop in its secondary structure.
• the AAV serotype may be AAV1-9, but is preferably AAVi, AAV2, AAV5, AAV6 or AAV8.
• the DNA sequence of one embodiment (left ITR from a commercially available AAV plasmid) of the ITR is represented herein as SEQ ID No: 5, as follows:
• the DNA sequence of another embodiment (right ITR from a commercially available AAV plasmid) of the ITR is represented herein as SEQ ID No: 6, as follows:
• the genome of the recombinant phagemid particle comprises a selection marker, which will depend on the host cell in which the vector is harboured, for example for conferring ampicillin resistance in a host cell, preferably a bacterium.
• the marker provides selection pressure during production of the phagemid particle in the host cell.
• the recombinant phagemid particle comprises one or more capsid minor coat protein.
• the recombinant phagemid particle may comprise a pill capsid minor coat protein that is configured to display a cell-targeting ligand for enabling delivery of the particle to the target cell.
• the recombinant phagemid particle comprises one or more capsid major coat protein.
• the recombinant phagemid particle may comprise at least one pVIII capsid major coat protein that is configured to display a foreign peptide thereon.
• the recombinant phagemid particle may comprise a modification of the capsid structure, for example by treatment, or chemical or biochemical conjugation. Examples of suitable modifications may include cross-linking peptide residues on to the phagemid particle.
• the recombinant phagemid particle may comprise one or functional peptide attached to the capsid thereof.
• a functional peptide may comprise a nuclear translocation signal.
• the phagemid particle may therefore be multifunctional, and use features disclosed in WO 2014/184528.
• the recombinant phagemid particle may be combined with a cationic polymer to form a complex having a net positive charge, as described in WO 2014/ 184529.
• the cationic polymer may be selected from a group consisting of:
• the cationic lipid is selected from the group consisting of fugene®, lipofectamine ®, and DOTAP (N-[i-(2,3-Dioleoyloxy)propyl]-N,N,N- trimethylammonium methyl-sulfate).
• the cationic polymer comprises DEAE, more preferably DEAE.DEX.
• the phagemid particle comprises a genome which substantially lacks the phage genome from which the particle is derived.
• the genome of the recombinant phagemid particle lacks at least 60%, more preferably at least 70%, and even more preferably at least 80% of the bacteriophage genome from which it is derived. More preferably, the genome of the recombinant phagemid particle lacks at least 90%, more preferably at least 95%, and even more preferably at least 99% of the bacteriophage genome from which it is derived.
• the genome of the recombinant phagemid particle lacks all of the bacteriophage genome from which it is derived.
• the genome of the phagemid viral particle may, in some embodiments, comprise the bacteriophage origin of replication for enabling replication of the particle into single-stranded DNA, i.e. Fi bacteriophage ori.
• the phagemid particle lacks bacteriophage structural genes in its genome required for the formation, packaging or extrusion of the particle from a prokaryotic host. Such structural genes encode the capsid proteins etc.
• the phagemid particle comprises a genome which lacks a gene encoding a minor or major coat protein from which the particle is derived.
• the phagemid particle comprises a genome which lacks a pill capsid minor coat protein,or which lacks a pVIII capsid major coat protein.
• the phagemid particle comprises a genome which lacks both a pill capsid minor coat protein, and a pVIII capsid major coat protein.
• the recombinant phagemid particle preferably comprises a replication-deficient, virus-like-particle or virion constructed from, and displaying, the structural
• components including but not limited to proteins and other conjugated compounds, derived from a bacteriophage, despite the genome of the particle not containing the structural genes of a bacteriophage from which it is derived.
• the genome of the recombinant phagemid particle of the first aspect lacks the derivative phage genome, including the structural genes
• an alternative system is required in order to provide the necessary structural (i.e. capsid) genes that are required to package the recombinant phagemid genome in a bacteriophage capsid to produce the particle of the invention.
• the inventors have devised a novel system for producing the particles of the first aspect, involving the use of a separate so- called "helper virus" vector.
• the particle of the first aspect is a hybrid phagemid vector, which includes components of a phagemid and a eukaryotic virus.
• a system for producing a recombinant phagemid particle from a prokaryotic host comprising:-
• a first vector configured to persist inside a prokaryotic host, and comprising at least one transgene expression cassette, and a packaging signal for enabling replication of the vector into single-stranded DNA;
• a second vector comprising nucleic acid encoding structural proteins required for packaging the single-stranded DNA, resulting in the formation and extrusion of a recombinant phagemid particle from the prokaryotic host.
• the novelty of the system of the second aspect is its ability to package the genome of eukaryotic viruses (such as AAV or lentivirus), which is provided by the first vector, into a prokaryotic virus capsid (i.e. bacteriophage), which is provided by the second vector.
• eukaryotic viruses such as AAV or lentivirus
• bacteriophage i.e. bacteriophage
• PAAV PAAV
• the system of the second aspect is used to produce the recombinant phagemid particle according to the first aspect.
• the first vector therefore comprises the genome of the recombinant phagemid particle.
• the packaging signal of the first vector may preferably comprise an origin or replication.
• the origin of replication in the first vector comprises an Fi ori, more preferably from an Fi bacteriophage.
• the first vector comprises a second origin of replication for enabling replication of double-stranded vector inside a prokaryotic host.
• the origin of replication enables high copy number replication of the vector inside the host.
• the origin of replication comprises a pUC ori.
• the first vector may comprise one or more DNA sequence, which favours targeted integration into a host genome, thus removing the requirement for any origin of replication.
• the transgene expression cassette comprises a viral transgene expression cassette, more preferably a mammalian viral transgene expression cassette.
• the at least one transgene expression cassette may comprise a lentivirus transgene expression cassette or a AAV transgene expression cassette.
• An AAV transgene expression cassette is preferred.
• One preferred embodiment of the second vector is illustrated in Figure 7, with preferred components being shown in Figure 8.
• the second vector or "helper phage" is preferably a bacteriophage engineered specifically for rescuing the phagemid particles carrying the first vector (i.e. the phagemid particle's genome) from prokaryotic hosts, an embodiment of which is shown in Figure 3.
• the second vector i.e.
• the helper phage is therefore provided to lend its proteins and polypeptides to the first vector (i.e. the phagemid particle's genome), or any other DNA entity that contains a functional packaging signal and/or a single stranded origin or replication.
• the second vector is most preferably replication-defective.
• the second vector comprises a disrupted packaging signal, which significantly deters its ability to package itself into phage particles.
• the second vector comprises a disrupted origin of replication.
• the disrupted origin of replication is a medium copy number origin, such as pi5a.
• the disrupted origin of replication is a low copy number origin, such as a pMBi.
• the first vector i.e. the phagemid particle's genome
• the second vector i.e. the helper phage
• the genome of the second vector may be engineered to give the resultant recombinant phagemid particle targeting properties (or multifunctional properties as described in WO 2014/ 184528). Hence, it provides the structural capsid proteins for phagemid particle assembly.
• the second vector comprises nucleic acid encoding one or more capsid minor coat proteins, or one or more capsid major coat proteins. All capsid proteins may either be wild type or recombinant, present in single or multiple copies, and modified to display chimeric or synthetic peptides. This includes the display of antigens of other viruses for peptide vaccine delivery or as an adjuvant in the case that a DNA vaccine (delivered by the phagemid particle of the first aspect) is desired.
• the second vector may comprise a first nucleic acid sequence encoding a pill capsid minor coat protein that is configured to display a cell- targeting ligand for enabling delivery of the recombinant phagemid particle to a target cell (e.g. a tumour). Therefore, it may be desired to induce a 9-amino acid mutation in the pill minor coat protein of the recombinant phagemid particle in order to confer its specificity to tumour cells and angiogenic tumour-associated endothelial cells that express ⁇ ⁇ ⁇ 3 and ⁇ ⁇ ⁇ 5 integrins.
• the genome of the second vector may comprise the RGD4C targeting peptide (CDCRGDCFC - SEQ ID No: 7) ⁇
• the second vector may comprise a second nucleic acid sequence encoding at least one pVIII capsid major coat protein that is configured to display a foreign peptide thereon.
• it may be desired to induce a mutation in the wild pVIII major coat protein of the recombinant phagemid particle in order to display a short peptide, for example less than 10 amino acids long.
• the short peptide maybe a targeting moiety or have inherent biological/chemical functionality in vivo or in vitro. For example, immune stimulation in vivo via antigen display, or binding to
• nanoparticles e.g. gold
• nanoparticles in vitro via displaying a gold-binding peptide.
• the first vector may be a member of the Retroviridae family, or of the Orthoretrovirinae Sub-family.
• the first vector may be a member of the Lentivirus genus.
• the first vector is a member of the Parvoviridae family or sub-family.
• the first vector is a member of the Dependoparvovirus, or adeno-associated virus species.
• the packaging signal e.g. the origin of replication
• the second vector i.e. the helper phage structural proteins
• the first vector comprises a nucleic acid sequence substantially as set out in SEQ ID No: 9, or a fragment or variant thereof, wherein SEQ ID No: 9 is represented as follows:
• the second vector (helper phage with RGD sequence) comprises a nucleic acid sequence substantially as set out in SEQ ID No: 10, or a fragment or variant thereof, wherein SEQ ID No: 10 is represented as follows:
• the second vector (helper phage without RGD sequence) comprises a nucleic acid sequence substantially as set out in SEQ ID No: n, or a fragment or variant thereof, wherein SEQ ID No: n is represented as follows:
• Example 1 the inventors have devised two alternative approaches (see Figures 9 and 10) for producing the recombinant phagemid particle of the invention in a prokaryotic host.
• a method for producing a recombinant phagemid particle from a prokaryotic host comprising: -
• helper phage comprising nucleic acid encoding bacteriophage structural proteins
• this embodiment results in very high yields of particles.
• the first vector i.e. the phagemid particle's genome
• the host cell may then be transformed with the helper phage, which results in the production of the recombinant phagemid particle.
• the method comprises a purification step following the culturing step. Purification may comprise centrifugation and/or filtration.
• a method for producing a recombinant phagemid particle from a prokaryotic host comprising: -
• the prokaryotic host persist inside the prokaryotic host, and comprising at least one transgene expression cassette, and a packaging signal for enabling replication of the vector into single-stranded DNA, and (b) a second vector comprising nucleic acid encoding structural proteins required for packaging the single-stranded DNA; and (ii) culturing the host under conditions which result in the single-stranded DNA being packaged by the structural proteins to form and extrude a recombinant phagemid particle from the prokaryotic host.
• the second vector i.e. the helper phage
• the second vector maybe introduced into the host cell, for example by infection.
• the host cell may then be transformed with the first vector (i.e. the phagemid particle's genome), which results in the production of the recombinant phagemid particle.
• the method comprises a purification step following the culturing step. Purification may comprise centrifugation and/or filtration.
• helper phage comprising nucleic acid encoding viral vector structural proteins to produce the recombinant phagemid particle according to the first aspect from a prokaryotic host.
• a host cell comprising the first and/or second vector as defined in the second aspect.
• the host cell is preferably prokaryotic, more preferably a bacterial cell.
• suitable host cells include:
• the particle or system can be used ex vivo or in vitro.
• the particle is used therapeutically or in diagnostic methods, preferably in vivo.
• the recombinant phagemid particle according to the first aspect, or the system according to the second aspect for use in therapy or diagnosis.
• the invention may be used for the treatment of a wide variety of diseases due to the target-specific nature and the improved transduction efficiency of the recombinant phagemid particle of the invention. Consequently, the therapeutic opportunities of recombinant bacteriophages used in gene therapy may be significantly increased by the invention due to its ability to carry one or more transgene expression cassettes.
• the invention may be used prophylactically to prevent disease, or after the development of a disease, to ameliorate and/or treat it.
• recombinant phagemid particle according to the first aspect, or the system according to the second aspect for use in a gene therapy technique.
• a ninth aspect there is provided a method of treating, preventing or ameliorating a disease in a subject using a gene therapy technique, the method comprising
• the invention may be used to create a variety of different recombinant phagemid particles that can be used for the treatment and/ or diagnosis of a variety of diseases depending on the nature of the particles and the displayed foreign proteins.
• the recombinant phagemid particle comprises a tumor-targeting ligand and/or which comprises a transgene expressing an anti-tumor gene (e.g. the HSVtk gene)
• the target cell in the gene therapy technique is preferably eukaryotic, and preferably mammalian.
• the gene therapy technique therefore is preferably used to treat, prevent or ameliorate cancer.
• Tumours maybe in the brain, e.g. medulloblastoma, or diffuse intrinsic pontine glioma (DIPG).
• the recombinant phagemid particle may be used in combination with conventional treatments, such as chemotherapeutic drugs (i.e. doxorubicin, temozolomide, lomustine), radiation therapy, or other drugs/xenobiotic compound, including but not limited to inhibitors of histone deacetylases (HDAC inhibitors), proteasome inhibiting drugs and anticancer products from natural and dietary sources (i.e. genistein).
• HDAC inhibitors histone deacetylases
• proteasome inhibiting drugs i.e. genistein
• a vaccine comprising the recombinant phagemid viral particle according to the first aspect or the system according to the second aspect.
• the vaccine is a peptide vaccine.
• the vaccine is preferably a DNA vaccine.
• the vaccine preferably comprises a suitable adjuvant.
• the recombinant phagemid particle may be used to carry a transgene or DNA cassette encoding an antigen to stimulate the body's immune system.
• the recombinant phagemid particle may also be used to directly display and express the antigen of interest on the major pVIII coat proteins, thus providing an efficient platform for the simultaneous delivery, by a single phage particle, of numerous antigens as vaccine DNA vaccines, or proteins, or adjuvants readily expressed on the phage surface.
• the subject may be mammalian, and is preferably human.
• the recombinant phagemid particle according to the first aspect, or the system according to the second aspect for use in delivering and targeting a foreign antigen to a tumour in a vaccinated subject.
• Animals will first be vaccinated against foreign antigens, or already vaccinated against the antigen used, then the tumour-targeted phagemid will be administered to the vaccinated animals to deliver the foreign antigens to tumours, in order to induce an immune attack against these tumours.
• the recombinant phagemid particle of the invention can also be used in a variety of different genetic-molecular imaging techniques, such as positron emission tomography (PET), Ultrasound (US), SPECT imaging, functional magnetic resonance imaging, or bioluminescence imaging.
• PET positron emission tomography
• US Ultrasound
• SPECT positron emission tomography
• SPECT functional magnetic resonance imaging
• bioluminescence imaging positron emission tomography
• a thirteenth aspect there is provided use of the recombinant phagemid particle according to the first aspect, or the system according to the second aspect, in a genetic-molecular imaging technique.
• the transgene harboured by the phagemid particle may encode HSVtk and/ or the sodium/iodide symporter (NIS), and the particle is preferably used in combination with a radiolabeled substrate.
• NIS sodium/iodide symporter
• the human sodium/iodide symporter ⁇ NIS) imaging gene is preferably used in combination with I 124 for clinically applicable positron emission tomography (PET) imaging, or with pertechnetate for
• the HSVtk gene is preferably used in combination with radiolabeled nucleoside analogues such as the 20-[18F]-fluoro-20-deoxy-i-b-D-arabino-furanosyl- 5-ethyluracil ([18F]FEAU).
• radiolabeled nucleoside analogues such as the 20-[18F]-fluoro-20-deoxy-i-b-D-arabino-furanosyl- 5-ethyluracil ([18F]FEAU).
• the recombinant phagemid particles and systems according to the invention may be used in a medicament which maybe used in a monotherapy, or as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing disease, such as cancer.
• agents i.e. referred to hereinafter as "agents”
• a combined therapeutic approach using the phagemid particles and systems of the invention with existing chemotherapeutics, such as Temozolamide, Doxorubicin or Genistein is preferred.
• therapy may comprise the combination of the recombinant phagemid particle and system of the invention with an extracellular matrix degrading agent, such as enzyme or losartan.
• extracellular matrix degrading agents such as enzyme or losartan. The inventors believe that extracellular matrix degrading agents should enhance phagemid diffusion in the subject being treated, and especially within a solid tumour.
• the agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
• the composition may be in the form of a powder, tablet, capsule, liquid etc. or any other suitable form that may be administered to a person or animal in need of treatment.
• the vehicle of medicaments according to the invention should be one which is well -tolerated by the subject to whom it is given.
• Medicaments comprising the agents according to the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents of the invention may be administered by inhalation (e.g. intranasally).
• compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin.
• Agents according to the invention may also be incorporated within a slow- or delayed-release device.
• Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
• the device maybe located at least adjacent the treatment site.
• Such devices may be
• administration e.g. at least daily injection.
• agents and compositions according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment.
• Injections maybe intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion) or enhanced by convention (convection enhanced delivery - relevant to local injections at disease site).
• the amount of the agent that is required is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent (i.e. recombinant phagemid viral particle or the system), and whether it is being used as a
• Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the disease.
• a daily dose of between o.o ⁇ g/kg of body weight and 500mg/kg of body weight of the agent according to the invention may be used. More preferably, the daily dose is between o.oimg/kg of body weight and 400mg/kg of body weight, and more preferably between o.img/kg and 200mg/kg body weight.
• the agent may be administered before, during the or after the onset of disease.
• the agent may be administered immediately after a subject has developed a disease.
• Daily doses may be given systemically as a single administration (e.g. a single daily injection).
• the agent may require administration twice or more times during a day.
• the agent may be administered as two (or more depending upon the severity of the disease being treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight of 70 kg).
• a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
• a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses.
• a pharmaceutical composition comprising the recombinant phagemid viral particle according to the first aspect, or the system according to the second aspect, and a pharmaceutically acceptable vehicle.
• composition can be used in the therapeutic amelioration, prevention or treatment of any disease in a subject that is treatable with gene therapy, such as cancer.
• the invention also provides, in a fifteenth aspect, a process for making the
• composition according to the twelfth aspect, the process comprising contacting a therapeutically effective amount of the recombinant phagemid particle according to the first aspect, or the system according to the second aspect, and a pharmaceutically acceptable vehicle.
• a "subject” maybe a vertebrate, mammal, or domestic animal.
• agents, compositions and medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or maybe used in other veterinary applications. Most preferably, however, the subject is a human being.
• a “therapeutically effective amount” of agent is any amount which, when administered to a subject, is the amount of drug that is needed to treat the target disease, or produce the desired effect, e.g.
• the therapeutically effective amount of agent used maybe from about o.oi mg to about 8oo mg, and preferably from about o.oi mg to about 500 mg.
• a "pharmaceutically acceptable vehicle” as referred to herein, is any known
• the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
• a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet- disintegrating agents.
• the vehicle may also be an encapsulating material.
• the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
• the active agent e.g.
• the particle or system of the invention may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
• the powders and tablets preferably contain up to 99% of the active agents.
• Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
• the pharmaceutical vehicle maybe a gel and the composition may be in the form of a cream or the like.
• the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
• Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
• the particles or system according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
• the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
• suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g.
• cellulose derivatives preferably sodium carboxymethyl cellulose solution
• alcohols including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
• the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
• Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
• the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
• Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
• the particles or system i.e.
• hybrid vector may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
• the recombinant phagemid particle, system and pharmaceutical compositions of the invention maybe administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 8o (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
• compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
• forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
• adeno-associated virus is often the vector of choice for gene therapy.
• lentiviral vectors also have key several advantages over other systems. Firstly, they have a large packaging capacity of at least 8 Kb of DNA, which is an important feature when packaging sizeable expression cassettes of tissue-specific promoters and transgenes. Secondly, they differ from simpler retroviruses not only in the genome organisation, but also in that they are able to transduce non-dividing cells, which is a very useful quality when considering application as a gene therapy vector to non-proliferating tissues such as muscle, neurons and haematopoietic stem cells. In addition, lentivectors have reduced immunogenicity compared to adenoviral vectors, making it possible to consider systemic delivery routes. However, barrier of using AAV or lentivirus for laboratory and clinical research include their extremely high production cost and low yields.
• the recombinant phagemid particle of the invention can also be used to produce recombinant viral vectors, such as AAV or lentivirus, in vitro or in vivo (including in situ).
• Phage-guided AAV production utilizes the ability of the phagemid particles to package large amounts of single-stranded ssDNA.
• a typical AAV production system consists of three major elements: rAAV, rep- cap and adenohelper genes, which function together to produce rAAV particles.
• a sixteenth aspect there is provided use of the phagemid particle according to the first aspect or the system according to the second aspect, to produce a recombinant viral vector comprising or derived from the viral genome within the genome of the phagemid particle.
• a method for producing recombinant viral vector comprising introducing into, a eukaryotic host cell, the recombinant phagemid particle according to the first aspect, or the system according to the second aspect, and allowing the host cell to produce recombinant viral vector.
• the recombinant virus product is a recombinant mammalian virus, such as AAV or lentivirus.
• the viral vector product is rAAV.
• the phagemid viral particle according to the first aspect, or the system according to the second aspect is used in cis and/or trans together with the delivery and/or presence of other genetic elements required for the production of mammalian viruses, as determined by the phagemid particle's genome, inside the eukaryotic host cell.
• the method used to assist or enhance gene transfer to the host cell by the phagemid particle includes those described in WO 2014/ 184528 (i.e. multifunctional) and WO
• the eukaryotic host cell maybe mammalian.
• the host cell may comprise or be derived from Human Embryonic Kidney Cells (HEK293), Spodoptera frugiperda pupal ovarian tissue (Sf9), or Chinese Hamster Ovary (CHO). Insect cells are also envisaged.
• the host cell may be transformed with one or more phagemid particle genome carrying genes selected from the group consisting of: rAAV, lentivirus, capsid, replication, helper protein encoding genes, and any other genes required for the expression and packaging of mammalian viruses.
• the rAAV gene in hybrid phagemid particle-guided rAAV production, may be carried by the recombinant phagemid viral particle according to the first aspect, as shown in Figure 3, and the adenohelper and rep-cap genes may be carried on separate vectors, or be integrated into the eukaryotic host genome.
• Figure 12 shows the adenohelper genes on one vector
• Figure 13 shows the rep-cap on a separate vector.
• Any combinations of the rAAV, rep-cap and adenohelper genes may be carried on one or more vectors, i.e. in cis or trans configurations.
• rep-cap or adenohelper proteins in the context of rAAV production, could also be integrated or introduced into the eukaryotic host as a stably expressed accessory DNA (e.g. a plasmid), whereby the hybrid phagemid particle supplies the recombinant viral genome for packaging into a recombinant virus, as determined by the transgene cassette inside the phagemid particle's genome.
• a stably expressed accessory DNA e.g. a plasmid
• rAAV, rep-cap and adenohelper genes are carried on a single vector, as shown in Figures 14 and 15. The inventors believe that this is the first time that all three sets of genes have been harboured on the same vector.
• a recombinant vector comprising comprising rAAV, rep-cap and adenohelper genes.
• a recombinant phagemid particle comprising the vector of the eighteenth aspect.
• a twentieth aspect there is provided use of the vector according to the eighteenth aspect or the particle of the nineteenth aspect, to produce a recombinant AAV viral vector comprising or derived from the viral genome of the phagemid particle.
• a method for producing recombinant AAV viral vector comprising introducing into, a eukaryotic host cell, the vector according to the eighteenth aspect or the particle of the nineteenth aspect, and allowing the host cell to produce recombinant viral vector.
• the rep-cap and adenohelper genes on the vector behave as trans-acting or cis-acting or a combination of both elements that facilitate packaging of the rAAV genome in the AAV virus capsid, in the context of rAAV production.
• This production process is comparable to transient co-transfection of multiple plasmids, and usually involving three plasmids.
• the plasmids are replaced with the recombinant phagemid particles of the invention, which are targeted to eukaryotic cells (preferably mammalian cells), which also carry the same elements.
• the method may be carried out in vivo, in vitro, ex vivo, or in situ.
• the recombinant phagemid particles preferably comprise a targeting moiety for the target eukaryotic cell that is the designated eukaryotic host.
• the designated eukaryotic host cell type is a diseased cell.
• the diseased cell is a malignant or benign tumour.
• the eukaryotic host is a derivative of any of the eukaryotic hosts listed above.
• recombinant phagemid particles and genetic elements required for the production of recombinant virus could be in any fashion as indicated earlier, either in cis-acting or trans-acting combinations, inside the eukaryotic host cell. It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comprises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof.
• substantially the amino acid/polynucleotide/polypeptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/polynucleotide/polypeptide sequences of any one of the sequences referred to herein, for example 40% identity with the nucleic acids identified herein.
• amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to is also envisaged.
• sequence identity which is greater than 65%, more preferably greater than 70%, even more preferably greater than 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to is also envisaged.
• amino acids referred to amino acids
• acid/ polynucleotide/ polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90% identity, even more preferably at least 92% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 98% identity and, most preferably at least 99% identity with any of the sequences referred to herein.
• the skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences.
• an alignment of the two sequences must first be prepared, followed by calculation of the sequence identity value.
• the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM250, Gonnet etc.), and gap-penalty, e.g. functional form and constants.
• percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs.
• percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance. Hence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process.
• ClustalW The popular multiple alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the invention.
• a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to a nucleic acid sequence described herein, or their complements under stringent conditions.
• stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in o.2x SSC/ 0.1% SDS at approximately 20-65°C.
• a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown herein.
• nucleic acid sequence could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
• Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
• Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
• small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
• Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
• the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
• the positively charged (basic) amino acids include lysine, arginine and histidine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
• Figure l is a table showing features of the phagemid-AAV (PAAV) virus particle according to the invention compared to prior art AAVP virus particles;
• FIG 2 shows schematic illustrations of embodiments of a Helper Phage and a Phagemid genome (PAAV) according to the invention, and a phagemid-AAV (PAAV) particle that is created by the Helper and phagemid.
• Structural genes are integral to packaging of DNA in to virus particles, and are supplied by the replication-defective Helper phage.
• the phagemid genome is extremely parasitic to the Helper phage.
• FIG. 3 is a schematic representation of one embodiment of a phagemid genome (PAAV);
• Figure 4 shows the respective locations of fi ori and pUC ori on the phagemid genome shown in Figure 3;
• Figure 5 shows the location of a selection marker gene (AmpR) on a recombinant adeno-associated virus (rAAV) transgene cassette on the phagemid genome shown in Figure 3;
• AmpR selection marker gene
• rAAV recombinant adeno-associated virus
• Figure 6 shows the rAAV transgene cassette on the phagemid genome shown in Figure 3, which contains a gene of interest (e.g. GFP), the expression of which is driven by a CMV promoter and/or enhancer sequences, and tailed with a polyA signal.
• the entire transgene cassette is flanked by Inverted Terminal Repeat sequences (ITRs) from AAV;
• Figure 7 shows an embodiment of the Helper phage which is a bacteriophage engineered for rescuing phagemid particles from prokaryotic hosts carrying a phagemid genome, such as that shown in Figure 3;
• Figure 8 shows a section of the genome of the helper phage shown in Figure 5 comprising the RGD4C targeting peptide in the pill minor coat protein;
• Figure 9 shows a first embodiment of a method for producing phagemid-AAV (PAAV) particles
• Figure 10 shows a second embodiment of a method for producing phagemid-AAV (PAAV) particles
• Figure 11 shows one embodiment of a phage-based approach for in vitro AAV production showing the three vectors, (i) phagemid-AAV (PAAV), (ii) Rep-Cap phagemid, and (iii) adenohelper phagemid;
• Figure 12 shows the genome map of an embodiment of the adenohelper phagemid vector shown in Figure 11;
• Figure 13 shows the genome map of an embodiment of a Rep-Cap phagemid vector shown in Figure 11;
• Figure 14 shows an embodiment of a unified adenohelper-Rep-cap phagemid-AAV (PAAV) vector
• Figure 15 shows the genome map of an embodiment of the unified adenohelper- Rep- Cap phagemid vector shown in Figure 11;
• Figure 16 shows an embodiment of in situ AAV production using either the three phagemid vectors shown in Figures 11-13, or the unified adenohelper-Rep-Cap-AAV phagemid vector shown in Figures 14 and 15;
• Figure 17 shows Transmission Electron Microscopy (TEM) of known AAVP vectors and PAAV vectors according to the invention.
• RGD.AAVP.GFP filament pink
• RGD. PAAV.GFP filament blue
• helper phage present in virus sample green
• Figure 19 shows quantification of GFP-positive cells 9 days post-transduction in (A) 293AAV, (B) 293AAV with the addition of DEAE.DEXTRAN, (C) U87 and (D) U87 with the addition of DEAE.DEXTRAN.
• Figure 21 shows immunofluorescence staining of UW228 and DAOY human medulloblastoma cells to demonstrate expression of civ, ⁇ 3 and ⁇ 5 integrin subunits, receptor for RGD4C-phagemid.
• Tumour cells were stained using primary rabbit anti- Ov, ⁇ 3 or ⁇ 5 antibodies (diluted 1:50 in PBS-i%BSA), then with goat anti-rabbit
• AlexaFluor-488 secondary antibody shown in green
• 0.05 ⁇ g/ml DAPI in blue
• Figure 22 shows targeted gene delivery to paediatric medulloblastoma cells by
• RGD4C-phagemid Medulloblastoma cells (UW228) were grown on 96 well-plates, then transduced with RGD4C-phagemid vector carrying the Luciferase gene (RGD).
• Untreated cells or cells treated with the non-targeted vector (M13) were used as negative controls. Luciferase expression was monitored over a time course from day 2 to 4 after transduction;
• Figure 23 shows Western blot analyses showing down regulation of mTOR expression in paediatric UW228 and DAOY medulloblastoma cells following treatment with RGD4C-phagemid carrying the mTOR/ shRNA (RGD4C-mTOR/ shRNA)).
• Cell lysates were collected at day 4 post vector treatment, and total proteins were measured by BCA assay.
• Western blot was probed with a monoclonal antibody to human mTOR (Cell Signalling). Untreated cells (CTR) and cells treated with RGD4C-phagemid, lacking mTOR/shRNA, (RGD4C) were used as negative controls;
• Figure 24 shows combination treatment of temozolomide (TMZ) and RGD4C- phagemid carrying shRNA for mTOR in medulloblastoma.
• Medulloblastoma cells UW228 and DAOY
• RGD4C RGD4C-phagemid
• RGD4C-mTOR/shRNA RGD4C-mTOR/shRNA
• Figure 25 shows treatment of medulloblastoma cells with TNFa vectors.
• UW228 cells were treated with RGD4C-phagemid-TNFa (RGD4C/TNFa) and non-targeted (ctr).
• RGD4C/TNFa RGD4C-phagemid-TNFa
• ctr non-targeted
• Figure 26 shows immunofluorescence staining of DIPG cells to demonstrate expression of ⁇ , ⁇ 3 and ⁇ 5 integrin subunits, receptor for RGD4C-phagemid.
• Cells were stained using primary rabbit antibodies then with goat anti-rabbit AlexaFluor-488 secondary antibody. Control cells received secondary antibody alone. Images were taken using a confocal microscope;
• Figure 27 shows selective and dose dependent delivery of gene expression to DIPG cells by RGD4C-phagemid/AAV.
• Increasing vector dose 1 x 10 6 or 2 x 10 6 TU/cell of RGD4C-phagemid-Luc (RGD4C) carrying the reporter Luc (luciferase) gene was used to treat DIPG cells.
• Luc expression was measured daily.
• Non-targeted vector lacking RGD4C (ctr) was used as negative control for targeting. Error bars: mean ⁇ SEM;
• Figure 28 shows Treatment with RGD4C-phagemid-TNFa.
• DIPG cells were transduced with 2 x 10 6 TU/cell RGD4C-phagemid-TNFa (RGD4C) and non-targeted vector as negative control (ctr).
• Apoptotic activity was measured at day 9 post-vector treatment using caspase-Glo assay (caspase 3/7, caspase 8, and caspase9). Error bars: mean ⁇ SEM. * P ⁇ 0.05, ** P ⁇ 0.01, ***P ⁇ 0.001;
• Figure 29 shows luciferase expression after transduction with RGD.PAAV at various concentrations of transducing units
• Figure 30 shows luciferase expression after transduction with NT.PAAV at various concentrations of transducing units
• Figure 31 shows the percentage of PAAV vectors bound to the cell surface of 293 AAV cells.
• RGD.PAAV vectors had 58.2% binding efficiency, whereas M13. PAAV vectors had 7.1% binding efficiency relative to their respective controls.
• this includes the genetic design of the vector, which carries ramifications in its production and therapeutic properties. Ultimately, this leads to AAVP's relatively low gene transduction efficacy when compared to mammalian viruses.
• the research described herein relates to the design of the most advanced version of phage gene delivery vectors and their superiority to the known and existing phage vector, AAVP, by using a so-called "phagemid system", with the new phagemid vector being referred to as Phagemid/Adeno-associated Virion Phagemid (i.e. PAAV).
• PAAV Phagemid/Adeno-associated Virion Phagemid
• the PAAV genome does not contain any structural phage genes - a prokaryotic helper virus is required to facilitate vector assembly (Mol Ther3, 476-484; Pharmaceutical research 27, 400-420 (2010)).
• PAAV Phagemid - AAV Vector
• PAAV particles according to the invention i.e. virions
• the PAAV particles (6kb) of the invention are much smaller than the known AAVP particles (i4kb), i.e. 42% less DNA, and 50% shorter viral particles, and the PAAV particles are produced at yields that far surpass prior art systems (100X) the yield of AAVP).
• PAAV particles of the invention can carry larger payloads, which is very useful for delivering multiple transgenes in gene therapy approaches.
• PAAV modified bacteriophage expression system
• PAAV can be used as a highly viral vector for gene therapy, or for large-scale production of viral vectors.
• FIG 2 there is shown an embodiment of a Helper Phage genome and a Phagemid genome (PAAV DNA) according to the invention, which are used together upon expression in a prokaryote to produce the phagemid-AAV (PAAV) particle, also shown in Figure 1.
• Structural genes are integral to packaging of DNA in to virus particles, and are supplied by the replication-defective Helper phage, which is discussed in detail below.
• the phagemid genome is extremely parasitic to the Helper phage, meaning it outcompetes the replication-defective helper phage in both replication and packaging.
• phagemid genome which is a plasmid containing two origins of replication and two other genetic elements.
• Phagemid genomes require two origins of replication to facilitate both its replication inside the prokaryotic (e.g. bacterial) host and packaging into phagemid particles when rescued by a helper virus.
• the first origin of replication is a high-copy number origin of replication (pUC ori) that enables replication of the double-stranded phagemid
• the second origin of replication is a phage origin of replication (fi ori) that enables replication of the plasmid into single-stranded DNA, which can subsequently be packaged into a phagemid vector particle (PAAV).
• fi ori phage origin of replication
• the phagemid genome includes a selection marker gene.
• a selection marker e.g. ampicillin resistance
• an antibiotic resistance gene with its own promoter. This ensures expression (and replication) of the phagemid genome when the prokaryotic host is cultured in the presence of the antibiotic that the selection marker confers resistance to.
• the phagemid genome further includes a recombinant (adeno- associated virus, AAV) transgene cassette which contains a transgene of interest.
• AAV adeno- associated virus
• This can include, but is not limited to, polypeptides/proteins, short hairpin/small interfering/short guiding RNAs, or a combination of both.
• the transgene shown in Figure 6 encodes GFP and human Beta-globin.
• Expression of the transgene is driven by a viral promoter (e.g. CMV) and/or enhancer sequences, and tailed with a polyA signal to prevent degradation.
• the promoter can also be a mammalian and tumour specific promoter in cancer gene therapy applications (i.e.
• the entire transgene cassette is flanked by Inverted Terminal Repeat sequences (ITRs) from AAV, which form a protective hairpin structure allowing the transgene cassette to be stably maintained as concatameric episomal (extra-chromosomal) DNA in the mammalian cell nucleus transduced by the phagemid particle.
• ITRs enable AAV transgene cassettes to be stably expressed over a long period of time.
• the phagemid despite having a small genome, is unable to package itself into particles as it lacks structural phage genes. As a result, it requires "rescuing" by a helper virus, as shown in Figure 7, which provides structural (i.e. capsid) proteins required for formation and extrusion of particles from the prokaryotic host. Conventionally speaking, genetic elements in the vector are generic and used widely in genetic engineering.
• Helper phage provides structural (i.e. capsid) proteins required for formation and extrusion of particles from the prokaryotic host.
• genetic elements in the vector are generic and used widely in genetic engineering.
• helper phage (referred to herein as M13KO7) is a
• the helper phage contains a disrupted origin of replication (pi5a, medium copy number) and packaging signal, which significantly deters its ability to package itself into phage particles. Consequently, the phagemid genome will outcompete the helper phage in both replication and packaging.
• the genome of the helper phage must be engineered to do so, as it provides the structural capsid proteins for phagemid particle assembly.
• the helper genome may encode a pill capsid minor coat protein that is configured to display a cell-targeting ligand for enabling delivery of the resultant PAAVP particle to a desired target cell (e.g. tumour). It can also encode at least one pVIII capsid major coat protein that is configured to display a foreign peptide on the resultant PAAV particle.
• the genome of the helper phage comprises the RGD4C targeting peptide (CDCRGDCFC - SEQ ID No: 7).
• PAAVP phagemid genome and the Helper phage are used together to produce, in a prokaryotic host, the Phagemid - AAV Vector (PAAV) particle, as discussed below.
• PAAV Phagemid - AAV Vector
• - TGi a strain of E. coli that carries the fertility factor (F pilus).
• - 2xYT liquid broth used to culture TGi E. coli.
• - TYE top agar solid media used to culture TGi E. coli, adapted from 2x TY by the addition of 1.25% bacteriological agar.
• helper phage M13KO7
• PAAV Purify phagemid
• Method 1 The benefits of Method 1 are its very high yields.
• Phagemid/AAV Vector (PAAV) Production Method 2 Stable producer cell-line
• Kanamycin supplemented with 1% glucose.
• Examples 1 and 2 describe the components of the invention (i.e. phagemid genome shown in Figure 3 and helper phage shown in Figure 7) required to produce the Phagemid - AAV Vector (PAAV) particle and two methods of production. Once produced and purified, the PAAV particles can have a range of uses, such as in gene therapy.
• PAAV Phagemid - AAV Vector
• the PAAVP particles described herein carry the GFP transgene, as it is readily detectable in known assays to show successful delivery to a target cell.
• any transgene may be selected and engineered into the phagemid genome shown in Figure 3, to be carried in the resultant PAAV particles.
• the transgene may be any gene encoding a protein, which may have therapeutic or industrial utility.
• the transgene may encode dystrophin, a blood coagulation factor, insulin or a cytokine receptor sub-unit.
• the transgene may also encode a short hairpin/small interfering/short guiding RNA molecule using in RNAi therapy.
• the transgene may encode multiple polypeptides, nucleic acids, or a combination of both, fused together using an internal ribosomal entry site (IRES) or a viral fusion peptide (T2A peptides for in-frame fusion).
• IRS internal ribosomal entry site
• T2A peptides viral fusion peptides for in-frame fusion.
• PAAVP Phagemid - AAV Vector
• PAAVP particles described herein can be used in novel methods for producing adeno-associated virus (AAV).
• Phage-guided AAV production utilizes the ability of the phagemid particles to package large amounts of dsDNA.
• a typical AAV production system consists of three major elements: rAAV, rep-cap and adenohelper genes, which function together to production recombinant AAV particles. The inventors have devised two different strategies.
• the first strategy employed is to produce three different phagemid vectors that carry the rAAV-producing elements. These are the Phagemid - AAV Vector (PAAV) (see Figure 3), the adenohelper phagemid particle (see Figure 12), and the rep-cap phagemid particle (see Figure 13).
• PAAV Phagemid - AAV Vector
• the basic structures of these particles are similar, as they contain two origins of replication and a selection marker, as described in the phagemid/AAV construction section.
• the key difference, however, is the transgene cassette.
• Phagemid - AAV (PAAV) genome contains an AAV transgene cassette, as shown in Figure 3, the adenohelper and rep-cap particles contain the adenohelper transgene or rep-cap transgene, as shown in Figures 12 and 13, respectively.
• the inventors have genetically engineered a so-called "unified construct" that contains all of the required elements inside a single vector genome, as shown in Figures 14 and 15.
• the rep-cap and adenohelper genes behave as trans-acting elements that facilitate packaging of the rAAV genome in the phagemid/AAV vector.
• This production process is comparable to transient co- transfection of three plasmids.
• the plasmids are replaced with phagemid vectors carrying the very same elements.
• AAV adeno- associated virus
• DMEM Dulbecco's Modified Eagle Medium.
• FBS Foetal Bovine Serum, a growth supplement.
• EDTA Ethyl-diamine tetra-acetic acid, an ion chelator used to dissociate cells by sequestering calcium ions required for tight junction formation.
• GlutaMax a growth supplement, analogue of L-Glutamine. Protocol for phagemid-guided AAV production:
• the inventors have devised a method for the in situ production of AAV particles using the PAAV.
• an optimal dose (or multiple doses) of the three phagemid vectors or the unified vector are introduced in vivo through intravenous/thecal/peritoneal or
• the diseased tissue is a tumour displaying the relevant integrins and so the targeting moiety on the phagemid PAAV particles is the RGD4C sequence.
• the tumour should start to produce rAAV containing the viral transgene encoded in the hybrid phagemid particle and not wild-type AAV. These AAV particles should autoinfect nearby sites, as they naturally have high affinity to mammalian tissue, and eradicate the tumour over a given time.
• Example 6 Engineering Pseudovirions for Large-scale Targeted Gene Transfer and Recombinant Adeno-associated Virus Production
• the inventors imaged PAAV particles to show that vector size is substantially reduced when using the phagemid-based vector system.
• Transmission Electron Microscopy the inventors imaged and measured the length of PAAV of the invention and known AAVP particles on mesh copper TEM grids after negative staining with uranyl acetate (see Figure 17). It was found that the average AAVP particle was 1455.02nm in length (Fig. 17A), while a typical PAAV particle according to the invention is only 729.96nm in length (Fig. 17B) - which equates to approximately 50% reduction in particle size.
• the relative vector size is approximately 38% shorter than the helper virus.
• PAAV may be more efficient as a gene delivery vector than the AAVP, not only in terms of production yield, but also in subsequent infection processes when entering and expressing genes in mammalian cells.
• the inventors probed vector efficiency at various stages of infection, including binding, internalisation, and gene expression in 293AAV (a derivative of Human Embryonic Kidney 293) and U87 glioblastoma cell lines.
• the inventors performed a GFP-expression assay using RGD and NT PAAV.GFP and AAVP.GFP vectors (see Fig 19). In this experiment, they also tested whether addition of the cationic polymer DEAE.DEXTRAN (Dex) could enhance gene transfer by increasing the bioavailability and endosome-escape of PAAV vectors, as described in WO2014/184529.
• DEAE.DEXTRAN cationic polymer
• the targeted RGD.PAAV.GFP vector transduces target cells with higher efficacy (7.7%, p ⁇ o.oi and 1.4%, p ⁇ 0.05 GFP +ve cells in 293AAV and U87 cells, respectively) - compared to AAVP, this translates to a 2.44 and 1.56 fold increase respectively in 293AAV and U87 cells (Fig 19A, C).
• the inventors transduced 293AAV cells with three targeted vectors, which are normally plasmids that require transfection for gene transfer. They were able to harvest rAAV particles from the cell lysate and quantify the rAAV gene copy number (GC) per mL over three time-points after phagemid-guided transduction (Fig. 20A).
• GC gene copy number
• phagemid-guided rAAV production provides over 1.9-fold increase at 168 hours (7.69eii GC/mL, Fig 21A) in rAAV yield. Because phagemid-guided gene transfer requires extensive intracellular processing (unlike transfection), it requires a longer time for viral genes to be expressed and packaged in to functional particles. When yields are compared at the same 72-hour time-point however, transfection produced i.76eii GC/mL higher than phage-guided rAAV production. The rAAV yield per mL culture supernatant from transfection or phagemid-guided production dishes at all time points were approx. 8-9eio GC/mL with no observable trends (data not shown).
• the tripeptide, RGD is found in proteins of the extracellular matrix, including fibronectin.
• the integrins act as receptors for fibronectin by binding to the RGD motif located in fibronectin in the site of cell attachment to ⁇ ⁇ ⁇ 3 integrin, and so the inventors induced a 9-amino acid mutation in the pill minor coat protein of the recombinant phagemid particle in order to confer its specificity to tumour cells and angiogenic tumour-associated endothelial cells that express ⁇ ⁇ ⁇ 3 and ⁇ ⁇ ⁇ 5 integrins.
• the genome of the second vector comprises the RGD4C targeting peptide (CDCRGDCFC - SEQ ID No: 7).
• FIG. 21 there is shown immunofluorescence staining of UW228 and DAOY human medulloblastoma cells, which demonstrates the expression of civ, ⁇ 3 and ⁇ 5 integrin subunits, receptor for RGD4C-phagemid.
• Figure 23 shows Western blot analyses showing down-regulation of the mammalian target of rapamycin (mTOR) expression in paediatric UW228 and DAOY
• RGD4C-phagemid carrying the mTOR/ shRNA (RGD4C-1T1TOR/ shRNA)).
• Figure 24 shows combination treatment of temozolomide (TMZ) and RGD4C- phagemid carrying shRNA for mTOR in medulloblastoma cells, known for their resistance to temozolomide.
• TMZ temozolomide
• RGD4C- phagemid carrying shRNA for mTOR in medulloblastoma cells, known for their resistance to temozolomide.
• the data demonstrate that targeted the RGD4C- mTOR/ shRNA can re-sensitize medulloblastoma cells to TMZ and achieve complete tumor cell eradication. Therefore, targeted knockdown of mTOR expression by the RGD4C-phagemid is an efficient strategy to use in combination with temozolomide against chemoresistant tumor cells, such as medulloblastoma.
• FIG 25 shows treatment of medulloblastoma cells with TNFa vectors. Therefore, RGD4C/TNFa has therapeutic potential for use in targeted tumor killing such as medulloblastoma.
• Figure 26 shows immunofluorescence staining of DIPG cells to demonstrate expression of civ, ⁇ 3 and ⁇ 5 integrin subunits, receptor for RGD4C- phagemid. These data demonstrate that the phagemid vector containing the RGD4C targeting peptide can be used for targeted gene delivery and gene therapy in the paediatric brain tumors, DIPG.
• Figure 27 shows selective and dose dependent delivery of gene expression to DIPG cells by RGD4C-phagemid/AAV.
• HEK cells were plated in a 48-well plate in complete media (DMEM, 10% FCS, 1% glutamine, 1% penicillin/streptomycin) and incubated for at least 48 hours until 70- 80% confluence was reached. Cells were then washed with PBS and transduced with hybrid phage/phagemid vectors suspended in serum-free media (DMEM) for 12 hours before the media was supplemented with complete media. Luciferase expression was measured by adding IOUL of culture media to 50UL of prepared Quanti-luc (InvivoGen, USA) reagent. The emission of photos was measured using a plate reader equipped with a luminometer (promega, USA).
• Figure 29 shows luciferase expression after transduction with RGD.PAAV at various concentrations of transducing units
• Figure 30 shows luciferase expression after transduction with NT.PAAV at various concentrations of transducing units.
• the graphs demonstrate a dose-dependent exponential relationship between time and expression of luciferase after incubation with hybrid phage/phagemid vectors at various concentrations.
• the figures demonstrate that quantifiable gene expression can be achieved by phagemid vectors via an assay for secreted luciferase.
• 293AAV cells were seeded on 24-well plates in complete media (DMEM +io%FCS, i%Glutamine, 1% Penicillin/Streptomycin), and were left to reach 70-90% confluence for a minimum of 48 hours.
• the cells were washed twice with 50OUL PBS and placed on ice before being transduced with 200000 TU/cell (transducing units/cell) of PAAV vectors suspended in 200uL of serum-free DMEM. After 1 hour of incubation on ice, the media was recovered from the wells and the amount of phagemid particles were titrated on TGi E.coli and quantified by colony-counting.
• internalisation assay utilises staining of intracellular phage capsid for signal detection, the smaller overall size (and available capsid protein per particle) of the PAAV means that the proportional number of particles internalised cannot be compared directly to that of AAVP, which we have shown using TEM is twice in length compared to PAAV particles. Accordingly, methods of the invention involve a purification step (e.g. FPLC) to remove the helper phage.
• a purification step e.g. FPLC
• phagemid-guided rAAV production may benefit greatly from decreased competitive inhibition by helper phage contamination and yield multiple fold higher rAAV particles compared to conventional transfection protocols.
• rAAV recombinant adeno-associated virus
• Hybrid phagemid vectors that are highly efficient at gene transfer to mammalian cells are described.
• a rAAV transgene cassette to the phage capsid, it is possible to create a vector system that is easily produced at commercial scales.
• These phagemid/AAV (PAAV) vectors have very large cloning capacities and are targeted to mammalian cells, meaning transfection reagents are not required.
• PAAV phagemid/AAV
• a novel large-scale rAAV production system using PAAV and bacteriophage vectors has been developed, in both adherent cells and in cell-suspensions. This platform technology will enable commercial virus production for clinical translation at GMP standards and pave the way for commercial production of other biosynthetics.

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