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  • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• • A—HUMAN NECESSITIES
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/14011—Baculoviridae
  • C12N2710/14111—Nucleopolyhedrovirus, e.g. autographa californica nucleopolyhedrovirus
  • C12N2710/14141—Use of virus, viral particle or viral elements as a vector
  • C12N2710/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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  • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
  • C12N2760/00011—Details
  • C12N2760/20011—Rhabdoviridae
  • C12N2760/20111—Lyssavirus, e.g. rabies virus
  • C12N2760/20123—Virus like particles [VLP]

Definitions

• This invention relates to a delivery system which can be untilised to deliver and express a foreign gene in eukaryotic cells.
• the delivery system can be used to deliver an RNA gene which will direct synthesis of an encoded anti-sense RNA, catalytic RNA, peptide or polypeptide within specific target cells which can be of a selected type.
• the delivery system can be used in vitro to target eukaryotic cells in culture or can be used in vivo to deliver a prophylactic or therapeutic agent to specific cells in an animal or human that is diseased or infected or at risk of disease, infection or infestation.
• conventional systems for delivery of therapeutic agents included pharmaceutical dosage forms such as capsules which are made principally of gelatin blends and which contain small amounts of other components such as dyes, plasticisers, preservatives and opaquing agents. These capsules function as a soluble external shell or envelope for delivery of drugs to a required location. Soft capsules are used for liquids while hard capsules are used for delivery of free flowing powders. Microencapsulation techniques are also well known.
• Other types of pharmaceutical dosage forms have included compressed tablets prepared by compaction of a formulation containing the drug and certain excipients selected to aid the processing and to improve the properties of the drug. These excipients can include binders, disintegrants, fillers, or diluents and lubricants.
• Film coated tablets are compressed tablets with a film coat applied. An example is an enteric coated tablet which allows the drug to be delivered to the intestines because the coating is insoluble in the stomach. Also known are sustained release tablets which allow release of the drug over a period of time.
• the drugs normally reach the gastro-intestinal tract (GI tract) and diffuse across the gastro-intestinal membrane into the bloodstream.
• the drug contained in the tablet dosage form will disintegrate in the GI tract prior to entry into the bloodstream due to the presence of the disintegrant and the capsule dosage form will dissolve prior to the drug entering the bloodstream.
• permeability barrier imposed by the plasma membrane.
• Pharmaceutical dosage forms comprising tablets or capsules are unable to penetrate the permeability barrier, especially in relation to macromolecules which can comprise polypeptides such as toxins, enzymes or antibodies, or polynucleotides such as DNA or RNA.
• Various methods have been used for delivery of macromolecules into cells. These include physical treatments such as microinjection, permeabilisation by lytic agents or high voltage electric fields and induced uptake of calcium phosphate or polyethylene glycol co-precipitates.
• Cell entry by fusion of a delivery vehicle with the cell plasma membrane has been achieved by use of liposomes and reconstituted viral envelopes (RVEs) .
• RVEs viral envelopes
• Live virus vectors and other engineered viral delivery vehicles have also been used. While many of these methods have been useful for in vitro delivery of macromolecules to cells in culture, few have been successfully applied to delivery of macromolecules in vivo.
• Liposomes comprise artificial lipid envelopes which can be generated in vitro by condensation of phospholipid into a bilayer membrane which can enclose a soluble macromolecule. Trapping efficiency into liposomes can be as high as 20-30% but the efficiency of delivery of macromolecules is poor, especially in vivo where rapid clearance from the bloodstream and high uptake by the liver and spleen present difficulties. In general, liposomes do not allow specific cell targeting but covalent attachment of virus-specific antibodies to the liposome surface has been used to achieve delivery of macromolecules to virus infected cells in vitro .
• RVEs comprise viral envelopes which have been formed by solubilising intact virus in detergent and reassembling the viral envelope on removal of the detergent.
• RVEs can be formed in the presence of therapeutic agents including macromolecules which become encapsulated and can be used for drug delivery in vitro and in vivo. Encapsulation efficiency for macromolecules is lower than for liposomes (3-5%) but delivery efficiency and cell targeting are enhanced by the presence of viral spike glycoproteins in the RVE membrane.
• the spike glycoproteins recognise receptors in the plasma membrane of the target cell. Cells which lack the specific receptor are not recognised by the RVEs and so are not targeted for delivery.
• the spike glycoproteins also contain a fusion domain which enhances fusion of the RVE with the cell membrane.
• Live virus vectors have been used both in vitro and in vivo to deliver genes encoding prophylactic and therapeutic agents such as vaccine antigens and interleukins and to effect synthesis of the products in the target cells.
• a gene encoding the therapeutic agent is engineered into the viral genome and the product is expressed upon infection of target cells.
• the virus In live DNA virus vectors, the virus is engineered to contain a foreign gene or genes at a site in the genome which does not inhibit the infectivity of the virus.
• the virus can also be engineered to have reduced virulence for the target host. After infection of host cells, the virus expresses viral products as well as the foreign product.
• DNA viruses which have been engineered as live, replicating delivery vehicles include poxviruses herpesviruses, adenoviruses, papovaviruses, parvoviruses and baculoviruses of insects.
• live replicating virus vectors can be effective and efficient delivery vehicles, they are not usually acceptable for general human or veterinary use because of the risk of causing disease and because of potential environmental risks due to infection of non-target species.
• DNA viruses can also incorporate integration elements which can modify the host genetic structure and present the risk of inducing tumours and related disorders.
• RNA viruses used for delivery of foreign genes to animal cells include retroviruses, alphaviruses, Semliki forest virus, Sindbis virus and influenza virus.
• Retrovirus vectors are usually constructed by transfection of helper cells with a DNA molecule which contains the terminal domains (LTRs) and assembly elements (psi region) of a retrovirus and includes the coding region of a foreign gene.
• the helper cells express retrovirus structural proteins.
• the transfected DNA molecule is integrated into the DNA of the helper cells and an RNA molecule corresponding to a modified retrovirus genome is expressed.
• the modified genome including the foreign gene can be assembled into retrovirus-like particles by using the structural proteins expressed in the helper cells.
• Retrovirus vectors can be used for delivery of foreign genes into cells in the form of RNA which is transcribed into DNA and can be integrated into the host chromosomes and subsequently expressed by the host cell.
• retroviral vectors are a useful laboratory tool and have been used in particular cases for gene therapy, more general use is restricted by concerns that the host genetic structure can be modified resulting in tumours and related disorders.
• Sindbis virus causes an acute febrile illness in humans and is transmitted by biting insects. Unlike retroviruses, the virus does not synthesise DNA or induce tumours in infected animals. Sindbis virus vectors have been constructed by deleting genes encoding the capsid structural proteins from the genome and substituting a foreign gene. However, as a (+) sense RNA virus, Sindbis does not carry a viral RNA transcriptase as a structural component of the particle. Efficient expression of the foreign gene in target cells requires expression of the viral replicase and transcriptase components which are encoded in the 5' two-thirds of the genome.
• Sindbis virus expression vectors for delivery of therapeutic agents in vivo has three disadvantages: (i) the gene encoding the therapeutic agent cannot be delivered and expressed without prior expression of some viral proteins (replicase and transcriptase proteins); (ii) a limited amount of cloning capacity, approximately 3475 nucleotides, remains for insertion of foreign genes in the absence of infectious helper virus; and, (iii) the vector may become contaminated with wild type infectious virus due to recombination between the vector and helper virus during vector preparation.
• influenza virus has been described in which the influenza A virus NS gene was replaced by a foreign indicator gene.
• influenza A virus NS gene was replaced by a foreign indicator gene.
• purified influenza virus polymerase complex and helper virus recombinant virus particles were formed.
• the foregoing recombinant influenza virus vector has the disadvantages that the vector remains capable of expressing influenza proteins in the target cell and can revert to virulence by recombination with live virus.
• Pattnaik and Wertz have described infectious defective interfering (DI) vesicular stomatitis virus particles produced by infecting cells with DI particles where the cells harboured vectors for the expression of all five vesicular stomatitis virus proteins.
• DI infectious defective interfering
• a retrovirus vector In vivo delivery of therapeutic proteins to keratinocytes using a retrovirus vector is known as is a drug delivery virion in a retrovirus envelope which contains a protein drug sequence useful as an anti-leukaemia and anti-tumour agent.
• Poxvirus expression systems for delivery of vaccine antigens and a system for delivery of genetic material into brain cells using a virus vector have also be described.
• VLPs virus-like particles
• the prior art includes descriptions of particles, referred to as virus-like particles (VLPs), which can be constructed by expressing viral structural genes in cultured eukaryotic cells.
• the procedure has been used to construct synthetic VLPs of several animal and human viruses.
• the insertion of the complete polycistronic mRNA of poliovirus in the baculovirus polyhedrin gene has been reported.
• VLPs empty poliovirus-like particles
• CLPs core-like particles
• VLPs VLPs of several other viruses including bluetongue, hepatitis B virus and bovine immunodeficiency virus.
• RNA or DNA nucleic acid
• the technology has not yet been applied to the generation of VLPs of (-) sense RNA viruses which appear to require an RNA genome or genome fragment for initiation of the particle assembly process.
• infectious viral particles can be recovered from cDNA clones representing the entire genome of some viruses.
• cDNA is inserted into plasmid vectors containing promoters operative in eukaryotic cells. Transfection of eukaryotic cells with such vectors results in the production of infectious virus.
• This general approach has been used in relation to a number of viruses of humans, animals and plants including poliovirus, Sindbis virus and brome mosaic virus.
• the method has only been applied to some DNA viruses and (+) sense RNA viruses with a genome that can function directly as an mRNA.
• the object of this invention to provide an effective and completely non-infectious system for delivery of foreign genes to animal or human cells.
• the foreign gene will be in the form of a (-) sense RNA.
• a vector for delivering a foreign gene to a target cell for expression of said foreign gene comprising a (-) sense RNA genome contained within a ribonucleoprotein complex within a virus-like particle constituted from structural proteins of a (-) sense RNA virus, wherein said (-) sense RNA genome includes one or more foreign genes but does not include genes for replication of said (-) sense RNA virus.
• a method of preparing a vector for delivering a foreign gene to a target cell for expression of said foreign gene comprising a (-) sense RNA genome contained within a ribonucleoprotein complex within a virus-like particle constituted from structural proteins of a (-) sense virus, wherein said (-) sense RNA genome includes one or more foreign genes but does not include genes for replication of said (-) sense RNA virus, which method comprises the following steps:
• step (i) introducing the expression vector prepared in step (i) into a eukaryotic host cell together with DNA for the expression of proteins for the formation of virus-like particles;
• step (iv) harvesting said virus-like particles from the eukaryotic cell culture of step (iii).
• a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, adjuvant and/or excipient together with a vector for delivering a foreign gene to a target cell for expression of said foreign gene, said vector comprising a (-) sense RNA genome contained within a ribonucleoprotein complex within a virus-like particle constituted from structural proteins of a (-) sense virus, wherein said (-) sense RNA genome includes one or more foreign genes but does not include genes for replication of said (-) sense RNA virus, and wherein said ribonucleoprotein complex includes a polymerase for synthesis of (+) sense RNA from said (-) sense RNA.
• a method of delivering the expression product of a foreign gene to a target cell comprising contacting said target cell with a vector according to the first embodiment and co-transforming or co-transfecting said cell with a vector which provides an RNA-dependent RNA polymerase activity.
• a method of delivering the expression product of a foreign gene to a target cell comprising contacting said target cell with a vector according to the first embodiment which further comprises within said ribonucleoprotein complex a polymerase for synthesis of (+) sense RNA from said (-) sense RNA genome.
• a method of delivering the expression product of a foreign gene to cells of a tissue of a mammalian subject comprising administering to said subject a vector according to the first embodiment which further comprises within said ribonucleoprotein complex a polymerase for synthesis of (+) sense RNA from said (-) sense RNA genome, or a pharmaceutical composition according to the third embodiment.
• the (-) sense RNA genome of the vector of the first embodiment incorporates terminal fragments of the genome of a (-) sense RNA virus to facilitate packaging of the genome into the virus-like particles (VLPs).
• the VLPs contain the necessary viral proteins to target and enter specific cells and preferably contains a protein to synthesize (+) sense RNA (ie. mRNA) transcripts of the foreign gene.
• the expression product of the foreign gene can be a peptide or polypeptide.
• the peptide expression product can be a biologically active molecule, specific therapeutic agent or immunogen.
• the polypeptide expression product can be a biologically active protein, specific therapeutic agent or immunogen.
• the VLP vector also permits delivery of anti-sense RNA or catalytic RNA to a targetcell.
• Figure 1 is a schematic representation of the method of preparing the VLP vectors of the invention and the use of the VLP vectors for delivering a gene product to a target cell.
• Figure 2 is a representation of a DNA construct comprising 5' and 3' domains. ribozyme domains R1 and R2 and a filler domain into which a foreign gene can be inserted at a preferably unique restriction endonuclease site such as the NcoI site shown.
• Figure 3 depicts a process for preparing a genome construct comprising 5' and 3' domains, ribozyme domains and a filler domain.
• Figure 4 depicts a process for preparing VLP particles from the genome construct resulting from the process depicted in Figure 3.
• the following abbreviations are used for restriction endonuclease sites: B, BamHI; E, EcoRI; P, PstI; and S, SmaI.
• Figures 5a to 5d depict typical steps in the construction and cloning of a chimeric G protein gene.
• Figure 5a depicts the construction of an anchor" gene fragment
• Figure 5b the construction of a "donor” gene fragment
• Figure 5c the construction of a chimeric G protein gene
• Figure 5d depicts the cloning of the chimeric G protein gene.
• Figure 6 is a schematic representation of the construction of baculovirus transfer vectors harbouring TB2-CAT genome constructs. The position of thepolyhedron genepromoter therefrom in pAcYM1 and and the direction of transcription
• Figure 7 presents nucleotide sequences of the CAT and CAT3 PCR products with indication of the positions of the terminal and internal Wcol restriction enzyme sites.
• the CAT3 sequence is indicated in full with nucleotide differences in the CAT sequence shown above the sequence.
• the CAT sequence (1) has a nucleotide modification at position 357 (T substituted for A) resulting in an amino acid change from lie to Leu; and, (2) does not contain the rabies virus transcription termination/ polyadenylation sequence CATG[A] 7 immediately following the CAT3 gene translation stop codon (TAA).
• TD a solution of 0.8 mM tris-HCl (pH 7.4), 150 mM NaCl, 5 mM KCl and 0.7 mM Na 2 HPO 4 which is adjusted to pH 7.5 with HC1 and autoclaved
• X-gal 5-bromo-4-chloro-3-indoyl- ⁇ -D-galactoside The term "foreign gene” is used in the following description and claims to denote a gene that is not normally present in the specific cells targeted by the VLP vector or if normally present in the specific cells, is not expressed at the level attainable after delivery of the foreign gene by the VLP vector of the invention.
• the M1, P and NS genes and respective expression products are equivalent as are the M2 and M genes and respective expression products.
• VLP vectors can be prepared and utilised to deliver the expression product of a foreign gene to a target cell, inlcudes the following steps:
• step (ii) inserting the DNA molecule prepared in step (i) into an expression vector suitable for transfection of eukaryotic cells;
• step (iii) transfecting a eukaryotic cell with the recombinant expression vector prepared in step (ii) and simultaneously transfecting the same eukaryotic cell with vectors which express structural proteins of the (-) sense RNA virus and optionally with a vector for the expression of a protein with RNA-dependent RNA polymerase activity;
• VLPs virus-like particles
• a target cell with the VLPs produced in step (iv) to deliver the foreign gene expression product to the cells or preparing a composition for delivering the VLPs to target cells of tissue of an animal to deliver the foreign gene expression product to those cells.
• the structural proteins and protein having RNA-dependent RNA polymerase activity referred to in step (iii) include those with similar functions to the L protein, G protein, N protein, M1 protein and M2 protein of rabies virus.
• the G protein can be a chimeric G protein incorporating a modified external domain.
• the VLP formed in step (iv) will thus consist of the modified genome or genome fragment transcribed from the DNA molecule constructed in step (i), complexed with the L protein and M1 protein and surrounded by a sheath of N protein in a ribonucleoprotein complex which is surrounded by an internal matrix comprising the M2 protein and enclosed within a lipid envelope including the G protein (or chimeric G protein incorporating a modified external domain).
• the process is not limited to rabies virus however and the structural proteins, protein having RNA-dependent RNA polymerase activity and subgenomic (-) sense RNA fragments can be obtained from any (-) sense RNA virus having either a segmented or non-segmented genome.
• Such viruses include, but are not limited to, viruses from the following families: Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Bunyaviridae, Arenaviridae and Filoviridae.
• the preferred viruses are viruses from the rhabdovirus and paramyxovirus genera.
• the DNA molecule referred to in step (i) above typically comprises domains containing DNA sequences corresponding to 5' terminal and 3 ' terminal non-coding regions of the particular (-) sense RNA viral genome in addition to the sequences corresponding to the coding regions of the one or more foreign genes.
• the 5' and 3' domains are derived from the sequences of the 5 ' and 3 ' non-coding regions of the genome of a rhabdovirus or paramyxovirus.
• the DNA molecule includes domains encoding ribozymes.
• the ribozyme domains can be constructed from any of the known ribozyme structures, some of which have been described by Haseloff and Gerlach (Nature 334 , 585-591 (1988)).
• the ribozyme domains will be active during step (iii) of the above process and will ensure that the (-) RNA transcript expressed in eukaryotic cells will have a structure suitable for assembly of VLPs.
• the foreign gene contained within the DNA construct can be the complete coding region of a selected foreign polypeptide including initiation and termination codons or can be a fragment of a gene corresponding to a functional domain or domains of a polypeptide.
• the polypeptide encoded by the foreign gene can be an immunogen, a therapeutically or biologically active peptide or polypeptide, or an engineered protein such as an antibody-like molecule.
• the foreign gene can encode anti-sense RNA or catalytic RNA directed against an intracellular RNA molecule.
• Multiple foreign genes can be inserted in tandem.
• a restriction enzyme site, ZVcol for example, is advantageously included in a generic DNA construct to facilitate insertion of the selected foreign gene or genes to generate the DNA molecule.
• the DNA molecule can also include a filler domain comprising sequences of viral or other origin to give the construct sufficient length to be efficiently packaged in VLPs.
• the filler domain can constitute any nucleotide sequence that has characteristics which will allow the formation of VLPs.
• the filler domain will constitute a fragment derived from a portion of the L protein coding region of a rhabdovirus or paramyxovirus which is adjacent to the 5' terminal non-coding region of the (-) RNA genome.
• the filler domain will ensure that the genome to be expressed in step (iii) will be of sufficient size to allow formation of VLPs. That size is preferably greater than about 1000 nucleotides.
• the DNA molecule incorporates cohesive ends suitable for insertion of the molecule at selected restriction enzyme sites of plasmid vectors.
• a DNA construct suitable for carrying a foreign gene is described in international application No. PCT/AU92/00363 (WIPO publication No. WO 93/01833), the entire disclosure of which is incorporated herein by cross-reference. That construct, TB-2, after incorporation into a eukaryotic expression vector as described in step (ii) above and used as described in steps (iii) to (v) above, allows the formation of rabies VLPs. Inclusion of a foreign gene or gene(s) at the Ncol site of the TB-2 construct permits construction by steps (i) to (v) above of a rabies VLP which can be used as a vector for delivery of the foreign gene into a eukaryotic cell for expression of the gene in that cell.
• the 5' and 3' domains are derived from the known nucleotide sequence of the 5' and 3' terminal regions of the genome of rabies virus ( PV and CVS strains).
• the R1 domain is designed to target a site within the (-) RNA transcript of the TB-2 DNA construct.
• the R1 ribozyme in the transcript will cleave the RNA to ensure that extraneous parts of the transcript are removed so that the 5' terminus of the transcript corresponds to, or approximates, that of the 5' terminus of the rabies virus genome.
• the R2 ribozyme domain is designed to target a site within the (-) RNA transcript of the TB-2 DNA construct.
• the R2 ribozyme will cleave the RNA to ensure that extraneous parts of the 3' region of the transcript (including the R2 domain) are removed so that the 3' terminus of the transcript approximates that of the 3' terminus of the rabies virus genome.
• the filler domain in the TB-2 construct is derived from the known nucleotide sequence of a 1167 nucleotide region at the 5' end of the rabies virus (PV strain) L protein gene.
• the TB-2 construct also includes an NcoI site at which any selected foreign gene or genes can be inserted.
• TB-2 DNA is constructed from 3 fragments (Fragment A, Fragment B, and Fragment C in Figure 3).
• Fragment A incorporates the 5' domain and R1 domain of TB-2 and can be prepared from overlapping complementary oligonucleotides.
• Suitable oligonucleotides are PJW.5R1A and PJW.5R1B, the sequences of which, together with other oligonucleotides suitable for use in other steps of the procedure, follow:
• Oligonucleotides PJW.5R1A and PJW.5R1B are annealed and end-filled using T4 DNA polymerase to produce a blunt-end double-stranded DNA molecule of the required nucleotide sequence which can then be cloned into, for example, the Smal site of a suitable plasmid vector such as pBluescript IIKS+.
• the DNA can then be excised from the vector by using suitable restriction enzymes, BamHI and EcoRI for example, to generate the required fragment with cohesive ends in the required orientation (Fragment A, Figure 3).
• Fragment C incorporates the 3' domain, R2 domain and the foreign gene insertion site (NcoI site) of TB-2 and can be constructed from overlapping complementary oligonucleotide primers PJW.3R2A and PJW.3R2B.
• the oligonucleotides are annealed and end-filled using T4 DNA polymerase to produce a blunt-end double-stranded DNA molecule of the required nucleotide sequence which can be similarly cloned into, for example, the SmaI site of a vector such as pBluescript IISK+.
• the DNA can then be excised from the vector by using suitable restriction enzymes such as BamHI and PstI, to generate the required fragment with cohesive ends in the required orientation (Fragment C, Figure 3).
• Fragment B incorporates the filler domain and can be constructed, for example, from the rabies virus (PV strain) genome using primer PJW.L2R (above) and reverse transcriptase to prepare a single-stranded cDNA copy of the required portion of the rabies L protein gene and then by using primers PJW.L2R and PJW.L4R (above) and the polymerase chain reaction (PCR) to amplify a double-stranded DNA molecule of the required nucleotide sequence.
• the DNA molecule can then be cloned into, for example, the Smal site of a suitable plasmid vector such as pUC8.
• the DNA can then be excised from the vector using suitable restriction enzymes, EcoRI and PstI for example, to generate the required fragment with cohesive ends in the required orientation (Fragment B, Figure 3).
• the TB-2 DNA construct can then be assembled by ligation of Fragment A, Fragment B and Fragment C with T4 DNA ligase to join the cohesive ends in the required orientation ( Figure 3).
• the TB-2 DNA construct is inserted into a vector for synthesis of (-) sense RNA.
• the (-) sense RNA is synthesised in an insect cell using a baculovirus expression vector.
• the TB-2 construct is inserted into a baculovirus transfer vector such as pAcUW31 at the BamHI site to form pAcUW31.TB2.
• Recombinant baculovirus capable of expressing TB-2 (-) sense RNA is formed by recombination in insect cells between pAcUW31.TB2 and a baculovirus such as AcNPV to form AcNPV.TB2.
• the transfer vector may be any transfer vector containing baculovirus promotors, such as Pol and p10.
• rabies virus VLPs containing a (-) sense RNA modified genome or genome fragment are produced by co-infection of an insect cell with the recombinant baculovirus AcNPV.
• TB2 and other recombinant baculoviruses which express rabies virus L protein, G protein, N protein, M1 protein and M2 protein as shown in Figure 4.
• one or more foreign genes are included in the DNA molecule.
• the construct can be modified to incorporate any selected foreign gene or genes by insertion of the selected gene or genes at the Ncol site.
• the selected gene or genes can be positioned within the construct so that the initiation codon will substitute for the initiation codon of the nucleoprotein (N) gene of the virus from which the terminal domains are derived.
• the foreign gene or genes can be inserted at any suitable site within the filler domain, if present, or proximal the DNA sequences corresponding to the 5' or 3' domains of the (-) sense RNA genome.
• DNA comprising the foreign gene includes the initiation codon, termination codon and coding region of the selected foreign gene, all or a part of the 3' noncoding region including the polyadenylation site of the rabies virus N protein mRNA, or equivalent sequence, and cohesive ends suitable for insertion of the DNA into the DNA molecule.
• the DNA will have Ncol restriction termini for insertion of the DNA at the NcoI site of the TB-2 construct. It will be understood by one of skill in the art that the DNA comprising the foreign gene is inserted into the DNA molecule so that (+) sense RNA formed in a target cell contains the sense strand of the foreign gene.
• the foreign gene can be obtained by established procedures of molecular cloning well known in the art. Addition of the 3' noncoding region, polyadenylation site and cohesive ends can be conducted, for example, by using PCR and suitable oligonucleotide primers which contain the desired sequences. Other methods of modification that are known in the art can also be used, such as ligation of oligonucleotide linkers to DNA comprising the gene.
• rabies VLPs can be applied to any other (-) sense unsegmented RNA virus, particularly rhabdoviruses and paramyxoviruses.
• the required VLP will contain a suitably modified genome or genome fragment containing a foreign gene or genes including essential assembly and transcription signals provided by the 5' and 3' domains of the DNA construct. Ribozyme domains R1 and R2 can be provided to ensure that the RNA transcript has suitable terminal sequences.
• the selected foreign gene can be inserted at any suitable site within the DNA construct.
• the RNA transcript of the DNA construct when co-expressed in eukaryotic cells with the structural proteins of the homologous (-) sense RNA virus is incorporated in a VLP.
• a VLP vector comprising a (-) sense genome which includes a foreign gene can be used to deliver the foreign gene to a eukaryotic cell and to express the polypeptide product or RNA of the foreign gene in the target cell.
• RNA-dependent RNA-polymerase such as the L protein of rabies virus.
• RNA-dependent RNA-polymerase in the ribonucleoprotein complex is not essential however and the activity can be provided by co-transfection of the target cell with a vector from which an RNA-dependent RNA-polymerase activity is expressed.
• the vector may be a plasmid or a virus.
• the vector is an homologous (-) sense RNA virus.
• VLP vectors of the present invention can therefore be changed by modifying the structure of the envelope glycoprotein. This can be achieved by constructing chimeric envelope protein genes which can be substituted for the envelope glycoprotein gene(s) during step (iii) of the process described above. Methods for the construction and expression of chimeric viral glycoproteins are known and are described, for example, by Puddington et al. (Proc. Natl . Acad. Sci USA 84, 2756-2760 (1987)), Schubert et al. (J. Virol. 66, 1579-1589 (1992)) and Owens and Rose (J. Virol. 67, 360-365 (1993)).
• FIG. 5a to 5d A suitable method for the construction of a chimeric glycoprotein gene is illustrated in Figures 5a to 5d.
• the nucleotide sequence of a chimeric glycoprotein is constructed, for example, from the envelope glycoprotein genes of rabies virus and the rhabdovirus, vesicular stomatitis virus (VSV).
• VSV vesicular stomatitis virus
• the chimeric gene illustrated retains internal and transmembrane domains of the rabies glycoprotein but includes the external domain of VSV.
• the chimeric gene components are advantageously synthesised by PCR amplification of template DNA using oligonucleotide primers. Such primers are shown as "OLIGO 21" to "OLIGO 24" in Figures 5a and 5b.
• the chimeric glycoprotein gene can be substituted for the rabies G protein gene in an expression vector, a recombinant baculovirus for example, and used for the construction of rabies VLP vectors.
• VLPs formed using such a chimeric structure will adsorb to and enter cells recognised by the VSV glycoprotein.
• Such a process can be used to construct chimeric envelope proteins which incorporate any selected external domain which can be included in the surface structure of the VLPs .
• the chimeric structure can be selected so that the VLPs can adsorb to, enter and express the foreign gene in specific cells which carry a receptor for the modified external domain.
• External domains that can be used to alter the target cell specificity of the VLP vectors of this invention include, but are not limited to, the external domains of influenza virus hemagglutinin, human immunodeficiency virus (HIV) gpl60, and paramyxovirus hemagglutinin-neuraminidase.
• the chimeric envelope protein can comprise an external domain from a virus fused to the trans-membrane and internal domains of the virus on which the VLP is based, wherein the first mentionedvirus is different to the second mentioned virus.
• Suitable vector-host cell system can be used to express the modified genome or genome fragment and viral proteins for VLP formation.
• Suitable host cells include higher eukaroytic cells such as vertebrate cells using poxvirus, papillomavirus or retrovirus vectors, or lower eukaryotic cells such as yeast cells.
• the preferred expression system is, however, an insect host cell such as Spodoptera frugiperda harbouring a recombinant baculovirus vector.
• the VLP vectors of the invention may be administered as follows: by topical treatment of mucous membranes; by intramuscular, subcutaneous, intraperitoneal or intravenous injection into tissue; or, by delivery to the intestinal mucosa either naked or in acid- and pepsin-resistant capsules.
• topical treatment of mucous membranes are oral, nasal, occular, respiratory, anal, vaginal or urethral routes.
• the VLP vectors may be administered to cells or tissue in vitro by directly contacting the cells or tissue with the VLPs.
• VLP vectors of the invention are prepared by combining the VLPs with pharmaceutically acceptable carriers, diluents, adjuvants or excipients or combinations thereof.
• the number of VLPs administered to a target cell or target cell of a tissue will depend on the expression product of the foreign gene. In some instances a single VLP per cell will be sufficient whereas in other instances a large number of VLPs will be required per cell, such as, where the foreign gene expression product is an anti-sense RNA.
• One of skill in the art would be able to determine the number of VLPs to be administered from a consideration of the expression product of the foreign gene.
• synthetic (-) sense RNA virus VLPs can be produced without helper virus, defective-interfering particles or synthetic transcription complexes.
• the VLPs synthesised by this process are modified to contain a foreign gene which can be delivered to and expressed in eukaryotic cells.
• the VLP vectors of the invention can be modified to include an external domain whih allows adsorption to and entry into cells of a selected type.
• the VLPs do not contain complete genes from the homologous (-) sense RNA virus so the synthetic particles are non-infectious.
• Plasmids used for constructing the recombinant plasmids were obtained from the following sources: pSVL-CAT (Cameron and Jennings (1989) Proc. Natl . Acad. Sci . USA 86, 9139) was obtained from CSIRO Division of Biomolecular Engineering, North Ryde, NSW, Australia; pBluescript KSOO+ was obtained from Promega Corporation, Madison, WI, USA; and, pAcYM1 (Matsuura et al . (1987) J. Gen. Virol . 68, 1233) was obtained from the Institute of Virology and Environmental Microbiology, Oxford, UK.
• Plasmid pTB2 is described in international application No. PCT/AU92/00363.
• the BamHI TB-2 insert of pTB2 is also contained within the pAcTB2 vector described in PCT/AU92/00363.
• pAcTB2 has been deposited with the Australian Government Analytical Laboratories, 1 Suakin Street, Pymble, NSW 2073, Australia, under accession No. 92/32588.
• Oligonucleotide primers were synthesised for PCR amplification of required DNA fragments (CPRl, CPR2 and CPR3 ) and DNA sequencing (Bac1 and Bac2).
• CPRl, CPR2 and CPR3 required DNA fragments
• Bac1 and Bac2 DNA sequencing
• a full-length copy of the CAT gene was obtained by PCR using primers CPR1 and CPR2 and plasmid pSVL-CAT DNA as a template for amplification.
• the reaction was performed using Taq Buffer, 3.5 mM MgCl 2 , 0.25 mM of each dNTP, 5 units of Taq DNA polymerase ( Promega Corp.), 1 ⁇ g of each primer and 7.5 ng of pSVL-CAT plasmid DNA.
• reaction mixes were heated at 85°C, 3 min before the addition of Taq DNA polymerase and subjected to 40 cycles at 95°C for 90 s, 51°C for 90 s, 72°C for 90 s followed by incubation at 72°C for 5 min before maintainance at 25oC until DNA products were processed.
• the CAT DNA product was applied to a 0.8% LMT agarose gel and a discrete DNA band of approximately 0.7 kb was excised.
• An equal volume of TE buffer pH 7.6 was added and the mixture was incubated at 68°C for 5-8 min with occasional vortexing.
• the DNA was extracted once with phenol, twice with phenol:chloroform:isoamyl alcohol (25:24:1) and was precipitated by the addition of 0.3 M sodium acetate pH 5.2, 20 mg glycogen (Boehringer Mannheim) as a carrier and 2.5 vol of ethanol. After incubation at -20°C for 30 min, DNA was collected by microcentrifugation, washed with 70% ethanol and dried under vacuum.
• 3'-Terminal adenosine overhangs resulting from Taq DNA polymerase extension were removed using the 3'->5' exonuclease activity associated with the Klenow fragment of DNA polymerase 1.
• Purified DNA products were reacted with 2.5 units of Klenow fragment (Promega Corp.) in restriction enzyme buffer H (Boehringer Mannheim) at 22°C for 15 min and extracted with phenol: chloroform: isoamyl alcohol and precipitated as above.
• the CAT gene DNA was blunt-end ligated into the dephosphorylated EcoRV site of pBluescript KSII+ (Stratagene) followed by transformation of XLl-Blue E. coli host cells (Stratagene) and the selection of ampicillin-resistant white colonies on agar plates prepared with TYM medium and containing ampicillin, X-gal and IPTG. Plasmids containing the CAT gene were identified and the sequences of the inserts in pBlue-CAT were determined using T3 and T7 sequencing primers (Promega Corp.) and SequenaseTM (United States Biochemicals) sequencing reagents.
• Plasmid pBlue-CAT was subjected to partial digestion with NcoI and the resulting DNA fragments were resolved in a 1.2% LMT agarose gel.
• the full-length CAT gene fragment of approximately 0.7 kb was isolated, purified and subcloned into the dephosphorylated NcoI site of pTB2 as described above with the exception that no blue/white selection system was available.
• Plasmids containing inserts were identified and sequenced using the T7 sequencing primer to determine the orientation of inserts in the NcoI site of the TB2 genome construct. Two clones containing the CAT gene in forward (pTB2-CAT) and reverse (pTB2-CAT-R) orientation were selected as shown in Figure 6.
• Plasmids pTB2-CAT and pTB2-CAT-R were digested with BamHI to obtain inserts containing the TB2 DNA construct incorporaiting the CAT gene in both orientations.
• the inserts of approximately 2.1 kb were isolated and purified as above and subcloned into the dephosphorylated BamHI site of the baculovirus transfer vector pAcYM1.
• Recombinant plasmids were identified and the orientation of the inserts was determined by sequencing as described above with primers Bac1 and Bac2 which allowed strand extension across the two reformed BamHI sites of the recombinant pAcYM1 vector. The sequences of the Bacl and Bac2 primers are shown above. Clones possessing inserts in the required orientation, pAcTB2-CAT and pAcTB2-CAT-R, were selected and plasmid DNA purified by CsCl gradient centrifugation.
• Plasmids pAcTB2-CAT3 and pAcTB2-CAT3-R were prepared exactly as described above for plasmids pAcTB2-CAT and pAcTB2-CAT-R except that the CAT gene was obtained from plasmid pSVL-CAT DNA by PCR amplification using primers CPR1 and CPR3. This resulted in the inclusion of the rabies virus polyadenylation sequence (CATG[A] 7 ) immediately after the CAT gene termination codon in addition to the polyadenylation sequence present in TB-2.
• baculovirus AcPAK6 was grown in Spodoptera frugiperda (Sf9) cells and purified by sucrose gradient centrifugation. DNA was isolated and purified by CsCl gradient centrifigation and digested to completion with Bsu36l.
• Sf9 cells were co-transfected with 100 ng Bsu36l-linearized AcPAK6 DNA and 1 ⁇ g of each of the four plasmid constructs (pAcTB2-CAT, pAcTB2-CAT-R, pAcTB2-CAT3 and pAcTB2-CAT3-R) using LipofectinTM (Gibco/BRL) transfection reagent.
• Cells were incubated at 28°C for 4 days and recombinant baculoviruses were identified by plaque selection.
• Cells were treated with X-gal to differentiate wild type (blue) from recombinant (white) baculovirus clones and the plaques were visualized by staining with neutral red.
• Clearly defined white plaques were selected and grown in duplicate 96-well cultures of Sf9 cells at 28 °C for 3-6 days.
• DIG-labelled probe was prepared by PCR using 12 ng gel-purified CAT3 PCR product as a template, 0.5 ⁇ g CPRl and CPR3 primers, reaction mixes containing 4mM MgCl 2 , 0.5 mM dATP, dCTP and dGTP, 0.32 mM dTTP, 8 nmol DIG-11-dUTP (Boehringer Mannheim), 2.5 units Taq DNA polymerase (Promega Corp.) and the cycling temperatures described above. Dot hybridizations with the DIG-labelled CAT probe identified recombinant baculoviruses containing the TB2-CAT, TB2-CAT-R, TB2-CAT3 and TB2-CAT3-R genome constructs.
• Amplification products were resolved in 0.8% agarose gels and fragments of the appropriate size, approximately 2.1 kb, were identified confirming that the recombinant baculoviruses contained full length TB2-CAT, TB2-CAT-R, TB2-CAT3 and TB2-CAT3-R constructs.
• virus seed stocks from the 24-well cultures were subjected to a second round of plaque purification in Sf9 cells.
• Well- separated plaques were selected, isolated and used to produce stocks of cloned recombinant baculoviruses for use in subsequent manipulations.
• Recombinant baculoviruses expressing rabies virus structural proteins (N/M1 and M2/G in dual expression vectors) and each of the four recombinant baculoviruses expressing genome constructs TB2-CAT, TB2-CAT-R, TB2-CAT3 and TB2-CAT3-R were used to infect spinner cultures of Sf9 cells. Cultures were incubated at 28 °C for 3 days, the medium harvested, clarified by centrifugation and VLPs were collected by ultracentrifugation at
• VLPs were resuspended in TD buffer supplemented with 1 mM EDTA and centrifuged through TD-buffered 10% (w/w) sucrose onto a cushion of TD-buffered 40% (w/w) sucrose at 35000 rpm for 30 min at 4°C in a Beckman SW40T1 rotor.
• the band at the interface was harvested, diluted and the semi-purified VLPs collected by centrifugation at 30000 rpm for 90 min at 4°C in a Beckman SW40Ti rotor.
• VLP formation was demonstrated by SDS-PAGE of disrupted pellets and Western blotting using polyclonal rabies virus antiserum.
• Rabies virus structural proteins G, N, M1 and M2 were identified in VLPs produced with all four TB2 genome constructs containing the CAT gene - TB2-CAT, TB2-CAT-R, TB2-CAT3 and TB2-CAT3-R.
• Visual comparison of the intensity of the structural proteins in VLPs produced using the four TB2-CAT genome constructs with that observed for VLPs produced with the TB2 genome suggested that the Sf9 cells shed similar quantities of VLPs irrespective of the nature of the TB2 genome employed.
• Monolayers of 5 ⁇ 10 5 baby hamster kidney cells (BHK-21, BSR clone) were infected with 8 ⁇ 10 6 plaque-forming units of rabies virus (CVS strain).
• CVS strain rabies virus
• the infected monolayers and uninfected BHK-21 cell monolayers were treated with 2 ⁇ 10 9 VLPs containing the following genome constructs: (a) TB2-CAT; (b) TB2-CAT3; (c) TB2-CAT-R; (d) TB2-CAT3-R; or, (e) no VLPs.
• TB2-CAT TB2-CAT3
• TB2-CAT-R TB2-CAT3-R
• no VLPs no VLPs.
• all monolayers were harvested and assayed for CAT gene expression by using the CAT-ELISA (Boehringer Mannheim).
• BHK-21 cell monolayers were treated as described in Experiment 1 except that rabies virus infections and VLP treatments were conducted simultaneously: that is, a 4 hour interval was not allowed between infection and VLP treatment.
• Table I demonstrate that a foreign gene, in this case CAT, can be expressed from sequence information contained within a (-) sense VLP genome. Expression was dependent on correct orientation of the foreign gene with CAT being detectable only in those cells transfected with TB2-CAT and TB2-CAT3 VLP genomes. Expression was also dependent on viral RNA-dependent RNA-polymerase activity as CAT was only detectable in cells infected with rabies virus (left-hand column for each experiment in Table I).
• VLPs of the invention which can express an immunogenic protein to animals or humans who are at risk of disease, infection or infestation will cause immunity in much the same way as existing vaccines incorporating inactivated or attenuated viruses.
• the VLPs of this invention can be used to deliver therapeutic agents to diseased or infected tissue.
• the VLPs can be targeted to specific cells or tissues, can allow synthesis and hence amplification of the therapeutic agent in the target tissue, are completely non-infectious and typically do not carry genes of an infectious agent.
• the VLPs contain no DNA, no integration elements and no enzyme capable of DNA synthesis, there is no risk of modification of the host genome which can result in the induction of tumours or related disorders.
• a vector for delivering a foreign gene to a target cell for expression of said foreign gene comprising a (-) sense RNA genome contained within a ribonucleoprotein complex within a virus-like particle constituted from structural proteins of a (-) sense RNA virus, wherein said (-) sense RNA genome includes one or more foreign genes but does not include genes for replication of said (-) sense RNA virus.
• a vector according to claim 1 which further comprises within said ribonucleoprotein complex a polymerase for synthesis of (+) sense RNA from said (-) sense RNA genome.
• said (-) sense RNA genome comprises a 5' domain from the genome of rabies virus, a filler domain comprising rabies virus genomic RNA, said one or more foreign genes and a 3' domain from the genome of rabies virus;
• said ribonucleoprotein complex comprises said (-) sense RNA genome together with rabies M1 and L proteins surrounded by a sheath of rabies N protein;
• said ribonucleoprotein complex is surrounded by an internal matrix comprising rabies M2 protein and is enclosed in a lipid envelope including rabies G protein.
• virus-like particle includes modified glycoprotein comprising an external domain which targets said virus-like particle to a selected cell type.
• modified glycoprotein comprises the internal and transmembrane domains of rabies virus G protein fused to an external domain comprising a polypeptide ligand for a receptor on the surface of said selected cell type.
• the expression product of said foreign gene is selected from the group consisting of a peptide, a polypeptide, an anti-sense RNA and a catalytic RNA.

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