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  • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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  • C12N2740/10011—Retroviridae
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  • C12N2740/15051—Methods of production or purification of viral material
  • C12N2740/15052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

• the present invention relates to a composition
• the present invention relates to a novel system for producing retroviral particles
• the present invention relates to a composition that is capable of expressing a retroviral particle that is capable of delivering a nucleotide sequence of interest (hereinafter abbreviated to 'NOI”) - or even a plurality of NOIs - to a site of interest
• 'NOI nucleotide sequence of interest
• the present invention relates to a composition useful in gene therapy
• Gene therapy mcludes any one or more of the addition, the replacement, the deletion, the supplementation, the manipulation etc of one or more nucleotide sequences in, for example, one or more targeted sites - such as targeted cells If the targeted sites are targeted cells, then the cells may be part of a tissue or an organ General teachings on gene therapy may be found in Molecular Biology (Ed Robert Meyers, Pub VCH, such as pages 556-558)
• Bv way of further example gene therapy also provides a means by which anv one or more of a nucleotide sequence, such as a gene, can be applied to replace or supplement a defective gene, a pathogenic gene or gene product can be eliminated, a new gene can be added in order, for example, to create a more favourable phenotype, cells can be mampulated at the molecular level to treat cancer (Schmidt- Wolf and Schmidt-Wolf, 1994 Annals of Hematology 69.273-279) or other conditions - such as immune, cardiovascular, neurological, inflammatory or infectious disorders, antigens can be mampulated and/or introduced to elicit an immune response - such as genetic vaccination
• retroviruses have been proposed for use in gene therapy Essentially, retroviruses are RNA viruses with a life cycle different to that of lytic viruses In this regard, when a retrovirus infects a cell, its genome is converted to a DNA form In otherwords, a retrovirus is an infectious entity that replicates through a DNA intermediate More details on retroviral infection etc. are presented later on.
• retroviruses There are many retroviruses and examples include: murine leukemia virus (MLV), human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), and Avian erythroblastosis virus (AEV).
• MMV murine leukemia virus
• HCV human immunodeficiency virus
• EIAV equine infectious anaemia virus
• MMTV mouse mammary tumour virus
• RSV Rous sarcoma virus
• FuSV Fujinami sarcoma
• retroviruses contain three major coding domains, gag, pol, env, which code for essential virion proteins. Nevertheless, retroviruses may be broadly divided into two categories: namely, "simple” and "complex". These categories are distinguishable by the organisation of their genomes. Simple retroviruses usually carry only this elementary information. In contrast, complex retroviruses also code for additional regulatory proteins derived from multiple spliced messages.
• Retroviruses may even be further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses. A review of these retroviruses is presented in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: M Coffin, SM Hughes, HE Varmus pp 1- 25).
• HTLV-BLV human T-cell leukemia virus-bovine leukemia virus group
• RSV Rous sarcoma virus
• MMTV mouse mammary tumour virus
• MLV murine leukemia virus
• HTLV human T-cell leukemia virus
• the lentivirus group can be split even further into “primate” and "non-primate” .
• primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human auto-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
• the non-primate lentiviral group includes the prototype "slow virus” visna/maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiencey virus (FIV) and bovine immunodeficiencey virus (BIV).
• lentivirus family and other types of retroviruses are that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis et al 1992 EMBO. I 11; 3053-3058, Lewis and Emerman 1994 I. Virol. 68: 510-516).
• retroviruses - such as MLV - are unable to infect non-dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.
• a retrovirus initially attaches to a specific cell surface receptor.
• the retroviral RNA genome is then copied to DNA by the virally encoded reverse transcriptase which is carried inside the parent virus.
• This DNA is transported to the host cell nucleus where it subsequently integrates into the host genome.
• the provirus is typically referred to as the provirus.
• the pro virus is stable in the host chromosome during cell division and is transcribed like other cellular proteins.
• the provirus encodes the proteins and packaging machinery required to make more virus, which can leave the cell by a process sometimes called "budding" .
• each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs).
• LTRs are responsible for pro viral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral gene. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
• the LTRs themselves are indentical sequences that can be divided into three elements, which are called U3, R and U5.
• U3 is derived from the sequence unique to the 3' end of the RNA.
• R is derived from a sequence repeated at both ends of the RNA and
• U5 is derived from the sequence unique to the 5'end of the RNA.
• the sizes of the three elements can vary considerably among different retroviruses.
• the site of transcription initiation is at the boundary between U3 and R in the left hand side LTR (as shown above) and the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR (as shown above).
• U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins.
• Some retroviruses have any one or more of the following genes that code for proteins that are involved in the regulation of gene expression: tat, rev, tax and rex.
• the basic molecular organisation of a retroviral RNA genome is (5') R - U5 - gag, pol, env - U3-R (3')- In a retroviral vector genome gag, pol and env are absent or not functional.
• the R regions at both ends of the RNA are repeated sequences.
• U5 and U3 represent sequences unique, respectively, to the 5' and 3' ends of the RNA genome.
• LTRs long terminal repeats
• the LTRs in a wild type retrovirus consist of (5')U3 - R - U5 (3'), and thus U3 and U5 both contain sequences which are important for proviral integration
• Other essential sequences required in the genome for proper functioning include a primer binding site for first strand reverse transcription, a primer binding site for second strand reverse transcription and a packaging signal
• gag encodes the internal structural protein of the virus Gag is proteolytically processed into the mature proteins MA (matrix), CA (capsid), NC (nucleocapsid)
• the gene pol encodes the reverse transcriptase (RT), which contains both DNA polymerase, and associated RNase H activities and mtegrase (IN), which mediates replication of the genome
• RT reverse transcriptase
• I mtegrase
• the gene env encodes the surface (SU) glycoprotein and the transmembrane (TM) protein ol the vi ⁇ on which form a complex that interacts specifically with cellular receptor proteins This interaction leads ultimately to fusion of the viral membrane with the cell membrane
• the envelope glycoprotein complex of retroviruses includes two polypeptides an external, glycosylated hydrophihc polypeptide (SU) and a membrane-spanning protein (TM) Together, these form an oligome ⁇ c "knob” or “knobbed spike” on the surface of a vi ⁇ on
• Both polypeptides are encoded by the env gene and are synthesised in the form of a polyprotein precursor that is proteolytically cleaved during its transport to the cell surface
• uncleaved Env proteins are able to bind to the receptor, the cleavage event itself is necessary to activate the fusion potential of the protein, which is necessary for entry of the virus into the host cell
• both SU and TM proteins are glycosylated at multiple sites
• TM is not glycosylated
• the SU and TM proteins are not always required for the assembly of enveloped vi ⁇ on particles as such, they do play an essential role in the entry process
• the SU domain binds to a receptor molecule - often a specific receptor molecule - on the target cell.
• this binding event activates the membrane fusion- inducing potential of the TM protein after which the viral and cell membranes fuse
• viruses notably MLV
• a cleavage event - resulting in the removal of a short portion of the cytoplasmic tail of TM - is thought to play a key role in uncovering the full fusion activity of the protein (Brody et al 1994 I Virol 68 4620-4627, Rein et al 1994 I Virol 68 1773-1781).
• This cytoplasmic "tail”, distal to the membrane-spanning segment of TM remains on the internal side of the viral membrane and it varies considerably in length in different retroviruses.
• the specificity of the SU/receptor interaction can define the host range and tissue tropism of a retrovirus. In some cases, this specificity may restrict the transduction potential of a recombinant retroviral vector. For this reason, many gene therapy experiments have used MLV.
• a particular MLV that has an envelope protein called 4070A is known as an amphotropic virus, and this can also infect human cells because its envelope protein "docks" with a phosphate transport protein that is conserved between man and mouse. This transporter is ubiquitous and so these viruses are capable of infecting many cell types. In some cases however, it may be beneficial, especially from a safety point of view, to specifically target restricted cells.
• mice ecotropic retrovirus which unlike its amphotropic relative normally only infects mouse cells, to specifically infect particular human cells.
• Replacement of a fragment of an envelope protein with an erythropoietin segement produced a recombinant retrovirus which then bound specifically to human cells that expressed the erythropoietin receptor on their surface, such as red blood cell precursors (Maulik and Patel 1997 "Molecular Biotechnology: Therapeutic Applications and Strategies” 1997. Wiley-Liss Inc. pp 45.).
• the complex retroviruses also contain "accessory" genes which code for accessory or auxiliary proteins.
• Accessory or auxiliary proteins are defined as those proteins encoded by the accessory genes in addition to those encoded by the usual replicative or structural genes, gag, pol and env. These accessory proteins are distinct from those involved in the regulation of gene expression, like those encoded by tat, rev, tax and rex. Examples of accessory genes include one or more of vif, vpr, vpx, vpu and nef. These accessory genes can be found in, for example, HIV (see, for example pages 802 and 803 of "Retroviruses" Ed. Coffin et al Pub. CSHL 1997).
• Non-essential accessory proteins may function in specialised cell types, providing functions that are at least in part duplicative of a function provided by a cellular protein.
• the accessory genes are located between pol and env, just downstream from env including the U3 region of the LTR or overlapping portions of the env and each other.
• the complex retroviruses have evolved regulatory mechanisms that employ virally encoded transcriptional activators as well as cellular transcriptional factors. These tra ⁇ s-acting viral proteins serve as activators of RNA transcription directed by the LTRs.
• the transcriptional trans-activators of the lentiviruses are encoded by the viral tat genes. Tat binds to a stable, stem-loop, RNA secondary structure, referred to as TAR, one function of which is to apparently optimally position Tat to trans-activate transcription.
• retroviruses have been proposed as a delivery system (other wise expressed as a delivery vehicle or delivery vector) for inter alia the transfer of a NOI, or a plurality of NOIs, to one or more sites of interest.
• the transfer can occur in vitro, ex vivo, in vivo, or combinations thereof.
• the retroviruses are typically called retroviral vectors or recombinant retroviral vectors.
• Retroviral vectors have even been exploited to study various aspects of the retrovirus life cycle, including receptor usage, reverse transcription and RNA packaging (reviewed by Miller, 1992 Curr Top Microbiol Immunol 158:1-24).
• At least part of one or more of the gag, pol and env protein coding regions may be removed from the virus. This makes the retroviral vector replication-defective. The removed portions may even be replaced by a NOI in order to generate a virus capable of integrating its genome into a host genome but wherein the modified viral genome is unable to propagate itself due to a lack of structural proteins. When integrated in the host genome, expression of the NOI occurs - resulting in, for example, a therapeutic effect.
• the transfer of a NOI into a site of interest is typically achieved by: integrating the NOI into the recombinant viral vector; packaging the modified viral vector into a virion coat; and allowing transduction of a site of interest - such as a targeted cell or a targeted cell population.
• retroviral vectors e.g. to prepare suitable titres of the retroviral vector
• propagation and isolation may entail isolation of the retroviral gag, pol and env genes and their separate introduction into a host cell to produce a "packaging cell line" .
• the packaging cell line produces the proteins required for packaging retroviral DNA but it cannot bring about encapsidation due to the lack of a psi region.
• the helper proteins can package the pApositive recombinant vector to produce the recombinant virus stock. This can be used to infect cells to introduce the NOI into the genome of the cells.
• the recombinant virus whose genome lacks all genes required to make viral proteins can infect only once and cannot propagate. Hence, the NOI is introduced into the host cell genome without the generation of potentially harmful retrovirus.
• a summary of the available packaging lines is presented in "Retroviruses" (1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 449).
• packaging cells have been developed in which the gag, pol and env viral coding regions are carried on separate expression plasmids that are independently transfected into a packaging cell line so that three recombinant events are required for wild type viral production.
• This strategy is sometimes referred to as the three plasmid transfection method (Soneoka et al 1995 Nucl. Acids Res. 23: 628-633).
• Transient transfection can also be used to measure vector production when vectors are being developed.
• transient transfection avoids the longer time required to generate stable vector-producing cell lines and is used if the vector or retroviral packaging components are toxic to cells.
• Components typically used to generate retroviral vectors include a plasmid encoding the Gag/Pol proteins, a plasmid encoding the Env protein and a plasmid containing a NOI.
• Vector production involves transient transfection of one or more of these components into cells containing the other required components.
• the vector encodes toxic genes or genes that interfere with the replication of the host cell, such as inhibitors of the cell cycle or genes that induce apotosis, it may be difficult to generate stable vector-producing cell lines, but transient transfection can be used to produce the vector before the cells die. Also, cell lines have been developed using transient infection that produce vector titre levels that are comparable to the levels obtained from stable vector-producing cell lines (Pear et al 1993, PNAS 90:8392-8396).
• HIV vectors are usually made by transient transfection of vector and helper virus.
• Some workers have even replaced the HIV Env protein with that of vesicular stomatis virus (VSV). Insertion of the Env protein of VSV facilitates vector concentration as HIV/VSV-G vectors with titres of 5 x 10 5 (10 8 after concentration) were generated by transient transfection (Naldini et al 1996 Science 272: 263-267).
• transient transfection of HIV vectors may provide a useful strategy for the generation of high titre vectors (Yee et al 1994 PNAS. 91: 9564-9568).
• a drawback, however, with this approach is that the VSV-G protein is quite toxic to cells.
• retroviral vectors are used extensively in biomedical research and for gene therapy.
• Current methods for the production of retroviral vectors make use of the fact that the two roles of the wild-type retrovirus genome, that is protein encoding and as a template for new genome copies, can be de-coupled (e.g. Soneoka et al 1995 Nucl. Acids Res. 23, 628 and references therein).
• Protein that is required for the assembly of new virus particles and for enzyme and regulatory functions can be produced by non-genome sequences in, for example, a mammalian packaging cell line (e.g. Miller 1990 Hum. Gene Therapy 1, 5).
• a genome sequence lacking the protein encoding functions is provided, so that the resulting retroviral vector particles are capable of infecting but not of replicating in a target cell.
• the genome sequence can also be designed for delivery and integration of a therapeutic gene (Vile and Russel 1995 Brit. Med. Bull 51, 12).
• Standard methods for producing murine leukaemia virus (MLV)-based vectors include use of cell lines expressing the gag-pol and env genes (the packaging components) of MLV. These will package a compatible retroviral vector genome introduced by transduction or by transfection with an appropriate plasmid.
• An alternative method involves simultaneous transfection of gag-pol, env, and vector genome plasmids into suitable cells.
• Retroviral vector particles are generally harvested by removing supernatant from a culture of particle-producing cells. The resulting suspension may be concentrated with respect to the vector particles, using physical methods, but only to a limited degree as problems such as aggregation and damage tend to arise. Thus, it may only be possible to concentrate a suspension of vector particles by up to 100-fold.
• the present invention seeks to provide an improved system for preparing viral particles that may be of subsequent use in medicine.
• the present invention seeks to provide an improved system for preparing a high titre of viral particles that may be of subsequent use in medicine.
• composition comprising at least one baculoviral component and at least one retroviral component, wherein the retroviral component is capable of being packaged into a retroviral particle.
• composition wherein the composition is a baculovirus expression system comprising at least one retroviral component, wherein the retroviral component is capable of being packaged into a retroviral particle.
• a retroviral particle obtainable from expression of the composition according to the present invention.
• a process for preparing a retroviral particle comprising expressing the composition according to the present invention.
• an insect cell comprising the composition according to the present invention.
• a retroviral vector particle production system comprising the composition according to the present invention in an insect cell.
• a retroviral vector particle produced by the retroviral vector particle production system according to the present invention.
• an expression vector comprising a polynucleotide sequence which encodes a retroviral vector genome having a 5' and a 3' end, which retroviral vector genome is capable of being expressed and packaged into a retroviral vector particle in a baculovirus expression system.
• the retroviral component corresponds to a retroviral genome.
• the composition comprises an RNA transcription start site for the retroviral vector genome, and wherein the nucleotide sequence encoding the retroviral component is operably linked to a promoter comprising an upstream promoter component located upstream of the RNA transcription start site and a downstream promoter component located downstream of the RNA transcription start site.
• downstream promoter component is upstream of the polynucleotide sequence encoding the retroviral vector genome.
• the promoter is a baculovirus promoter.
• the promoter is the polyhedrin promoter, the plO promoter and/or the polh promoter. In one embodiment, preferably the promoter is a non-baculovirus promoter.
• the promoter is the T7 promoter or the sp6 Salmonella phage promoter.
• composition comprises at least one RNA cleavage component.
• RNA cleavage component(s) Preferably manipultaion of at least one of the RNA cleavage component(s) would yield a retroviral genome free of any baculoviral components.
• At least one of the RNA cleavage component(s) is located between the promoter and the sequence encoding the retroviral component.
• RNA cleavage component(s) is located immediately adjacent the sequence encoding die retroviral vector component for subsequent cleavage at the 5' end of the vector component.
• At least one of the RNA cleavage component(s) is located downstream of the sequence encoding the retroviral component.
• RNA cleavage component(s) has a cleavage site immediately adjacent the sequence encoding the retroviral vector component for subsequent cleavage at the 3 ' end of the vector component.
• RNA cleavage component(s) is a ribozyme cleavage site for subsequent cleavage thereof.
• each RNA cleavage component is a ribozyme cleavage site for subsequent cleavage thereof.
• each ribozyme cleavage site may be cleaved by a ribozyme which is independently derived from the composition.
• a ribozyme which is independently derived from the composition.
• any one or more of the ribozyme cleavage sites is cleaved by a ribozyme derived from the second viral component.
• Ribozymes are RNA molecules that can function to catalyse specific chemical reactions within cells without the obligatory participation of proteins.
• group I ribozymes take the form of introns which can mediate their own excision from self-splicing precursor RNA.
• Other ribozymes are derived from self-cleaving RNA structures which are essential for the replication of viral RNA molecules.
• ribozymes can fold into secondary and tertiary structures that provide specific binding sites for substrates as well as cofactors, such as metal ions. Examples of such structures include hammerhead, hairpin or stem-loop, pseudoknot and hepatitis delta antigenomic ribozymes have been described.
• Each individual ribozyme has a motif which recognises and binds to a recognition site in a target RNA.
• This motif takes the form of one or more "binding arms” but generally two binding arms.
• the binding arms in hammerhead ribozymes are the flanking sequences Helix I and Helix III which flank Helix II. These can be of variable length, usually between 6 to 10 nucleotides each, but can be shorter or longer. The length of the flanking sequences can affect the rate of cleavage.
• reducing the total number of nucleotides in the flanking sequences from 20 to 12 can increase the turnover rate of the ribozyme cleaving a HIV sequence, by 10-fold (Goodchild, JVK, 1991 Arch Biochem Biophys 284: 386-391).
• a catalytic motif in the ribozyme Helix II in hammerhead ribozymes cleaves the target RNA at a site which is referred to as the cleavage site. Whether or not a ribozyme will cleave any given RNA is determined by the presence or absence of a recognition site for the ribozyme containing an appropriate cleavage site.
• Each type of ribozyme recognizes its own cleavage site.
• the hammerhead ribozyme cleavage site has the nucleotide base triplet GUX directly upstream where G is guanine, U is uracil and X is any nucleotide base.
• Hairpin ribozymes have a cleavage site of BCUGNYR, where B is any nucleotide base other than adenine, N is any nucleotide, Y is cytosine or thymine and R is guanine or adenine. Cleavage by hairpin ribozymes takes places between the G and the N in the cleavage site.
• a broad aspect of this embodiment of the present invention relates to a composition
• a composition comprising at least a first viral component obtainable from a first virus and a second viral component component obtainable from a second virus; wherein the first virus is different from the second virus; wherein the second viral component is flanked by at least two cleavage sites (which may be the same or different); wherein at least a part of the second viral component is capable of being packaged into a viral particle; which viral particle is substantially free of any first viral component.
• At least one of the cleavage sites is a ribozyme cleavage site.
• each of the cleavage sites is a ribozyme cleavage site.
• the ribozyme cleavage site may be cleaved by a ribozyme which is independently derived from the composition.
• a ribozyme which is independently derived from the composition.
• any one or more of the ribozyme cleavage sites is cleaved by a ribozyme derived from the second viral component.
• the first virus is a baculovirus.
• the second virus is a retrovirus.
• downstream promoter component is located within the sequence encoding the retroviral component.
• downstream promoter component is located within the sequence encoding the retroviral vector.
• the retroviral component comprises a retroviral R region at either end of a sequence encoding a retroviral vector genome, wherein the downstream promoter component is located in the 5' R region and has a counterpart sequence in the 3' R region.
• the composition comprises in a downstream direction: an upstream baculovirus promoter component, a downstream baculovirus promoter component, a ribozyme sequence, a retroviral 5' R region, a retroviral U5 region, a retroviral vector region for insertion of one or more genes to be delivered by the vector, a retroviral U3 region, a retroviral 3' R region, and optionally a second ribozyme sequence.
• the composition comprises in a downstream direction, an upstream baculovirus promoter component, a retroviral 5' R region comprising a downstream promoter component, a retroviral U5 region, a retroviral vector region for insertion of one or more genes to be delivered by the retroviral vector, a retroviral U3 region, a retroviral 3' R region, and optionally a ribozyme sequence.
• composition comprises one or more nucleotide sequences encoding one or more packaging components for producing retroviral vector particles which particles comprise the retroviral component
• composition further comprises at least one nucleotide sequence of interest (NOI)
• NOI nucleotide sequence of interest
• the NOI is useful in medicine.
• the NOI is part of the retroviral component.
• compositions may include one entity (such as a composition of matter) or two or more entities.
• the composition of the first or second aspect may be one entity - such as a modified baculoviral genome wherein at least a part of the baculoviral genome has been replaced with the retroviral component of the present invention.
• modified includes actual modification of a wild type genome or ab initw construction of an entity that could have been modified from a wild type genome (such as by ligating two or more fragments so as to form an entity corresponding to the modified genome).
• the packaging components of the present invention may be contained in an entity different to that just described.
• the composition of the present invention can be a retroviral particle producer and/or vector.
• the composition may even be a kit comprising a first part which includes the baculoviral component and a second part which includes the retroviral component.
• the kit may include one or more other parts, such as one or more suitable restriction enzymes etc.
• retroviral vector genome includes a retroviral nucleic acid which is capable of infection, but which is not capable, by itself, of replication. Thus it is replication defective.
• a retroviral vector genome carries or is capable of carrying polynucleotide sequences of non-retroviral origin, for delivery to target cells.
• a retroviral vector genome may comprise further non-retroviral sequences, such as non-retroviral control sequences in the U3 region which influence expression of the genome once it is integrated as a provirus into a target cell.
• the retroviral vector genome need not contain elements from only a single retrovirus.
• WO 96/37623 describes retroviral vectors having hybrid LTRs derived from two different retroviruses.
• retroviral vector particle refers to the packaged retroviral vector genome, that is preferably capable of binding to and entering target cells.
• the components of the particle may be modified with respect to the wild type retrovirus.
• the envelope proteins in the proteinaceous coat of the particle may be genetically modified in order to alter their targeting specificity or achieve some other desired function.
• the retroviral component includes an env nucleotide sequence
• all or part of that sequence can be optionally replaced with all or part of another env nucleotide sequence.
• Replacement of the env gene with a heterologous env gene is an example of a technique or strategy called pseudotyping. Pseudotyping is not a new phenomenon and examples may be found in WO-A-98/05759, WO-A-98/05754, WO- A-97/ 17457, WO-A-96/09400, WO-A- 91/00047 and Mebatsion et al 1997 Cell 90, 841-847.
• Pseudotyping can confer one or more advantages.
• the env gene product of the HIV based vectors would restrict these vectors to infecting only cells that express a protein called CD4.
• CD4 a protein that express a protein called CD4.
• the env gene in these vectors has been substituted with env sequences from other RNA viruses, then they may have a broader infectious spectrum (Verma and Somia 1997 Nature 389:239-242).
• - workers have pseudotyped an HIV based vector with the glycoprotein from VSV (Verma and Somia 1997 ibid).
• retroviral vector particles can be produced in a baculovirus expression system and those particles can successfully deliver one or more NOI(s) to target cells.
• Another important advantage of the present invention is that higher titres of vector particles may be produced than in the mammalian systems. Again, this is an unexpected finding.
• baculovirus expression systems may be free from endogenous mammalian genetic material, so reducing the risk of mammalian contaminants being present in the resulting retroviral vector preparation.
• insect contaminants are very unlikely to be biologically active in a mammalian system and are therefore expected to present less of a problem than mammalian contaminants .
• composition of the present invention comprises a baculoviral component.
• baculoviruses are a diverse group of viruses found mainly in insects with no supposedly known arthropod hosts.
• They can accommodate relatively large insertions of heterologous DNA - such as an NOI according to the present invention - (O'Reilly et al. in Baculovirus Expression Vectors, A Laboratory Manual 1994, Oxford University Press).
• They have the potential for very strong expression of heterologous genes DNA - such as an NOI according to the present invention - for example expression levels of 25 to 50% of the total cellular protein have been reported.
• baculoviruses have large double-stranded, circular DNA genomes within a rod-shaped capsid. Within the capsid, the DNA is condensed into a nucleoprotein stucture known as the core. The capsid plus the core are collectively referred to as the nucleocapsid.
• the length of a baculoviral DNA is between 80 and 120 kilobasepairs (kbp).
• AcMNPV Autographa California multiple nuclear polyhedrosis virus
• BmNPV Bombyx mori nuclear polyhedrosis virus
• Nucleocapsids are made in the nucleus of the infected cells and are subsequently enveloped (that is, they acquire a membrane) by one of two processes. Nuclecapsids can bud through the plasma membrane of the infected cell or they can acquire an envelope within the nucleus where they are produced. Membrane-enveloped nucleocapsids are referred to as virus particles or virions. The plasma-membrane budded form of the virus is referred to as the budded virus (BV).
• BV budded virus
• the viruses produce nuclear occlusion bodies which comprise enveloped nucleocapsid(s) embedded in a crystalline protein matrix, the major component of which is a virus-encoded protein called polyhedrin.
• Polyhedrin is the product of a single gene in the baculovirus genome and is produced, late in infection, accounting for 50% or more of all proteins being made by infected insect cells. Transcription of the polyhedrin (polh) gene is driven by an extremely active promoter, which is therefore ideally suited as a promoter for driving expression of foreign genes. Similiar levels of protein production can occur if the polyhedrin gene is replaced by a foreign gene.
• the insect baculovirus expression system provides a eukaryotic environment that is generally conducive to the proper folding, disulfide bond formation, oligomerisation and/or other posttranslational modifications required for the biological activity of some eukaryotic proteins.
• Post-translational modifications that have been reported to occur in baculovirus- infected insect cells include signal cleavage, proteolytic cleavage, N-glycosylation, 0- glycosylation, acylation, amidation, phosphorylation, prenylation, and carboxylation.
• the sites of such modifications are usually at identical positions on the proteins produced in insect and mammalian cells (O'Reilly et al ibid).
• baculovirus expression vectors An advantageous feature of baculovirus expression vectors is the potential for very strong expression of a heterologous gene DNA - such as an NOI according to the present invention.
• the highest expression levels reported using baculovirus expression vectors is 25% -50% of the total cellular protein. Such levels are equivalent to polh gene (Polyhedrin) expression and correspond to approximately 1 gram of protein product per 10 cells (eg a litre culture).
• heterologous proteins are not produced at the same level as polyhedrin and levels approaching 25 % of the total cellular protein have been achieved in a few cases. Most of these cases involved expression of structural genes of other virus families, the products of which are quite stable. Most heterologous proteins are produced at levels ranging from 10 mg to 100 mg per 10 cells. Nevertheless, in the cases where different eukaryotic expression systems have been compared, the baculovirus system has usually outperformed other expression systems in overall protein production.
• the present invention provides a production facility (e.g. a vat, or even an organism or cell thereof) for producing a quantity of an NOI or the expression product thereof; wherein the facility contains a medium comprising a baculovirus composition which comprises the NOI.
• the baculoviral composition is a composition according to the first aspect of the present invention.
• the rod-shaped nucleocapsids of baculoviruses can extend to accomodate larger viral DNA genomes and it is likely that a baculovirus vector can accomodate an additional 100 kbp of DNA or more
• the number of genes that might be expressed simultaneously using baculovirus vector systems is also likely to be high
• it may be necessary to construct a series of transfer plasmids that allow the building of the insert in successive increments This limitation has more to do with the fragility of large DNAs in vitro rather than the vector system per se
• additional transfer plasmids are required
• baculovirus expression system does carry out at least some splicing and its usefulness in identifying and obtaimng specific gene products from multigene families has been noted.
• the present invention provides the use of a baculoviral composition to express an NOI comprising at least one mtron
• the baculoviral composition is a composition according to the first aspect of the present invention.
• Baculovirus vectors are helper-virus independent and therefore relatively simple to use. Constructing a recombinant baculovirus is considerably faster and simpler than constructing a cloned, high-expressing, recombinant eukaryotic cell line.
• AcMNPV- and BmNPV- based vectors have a number of advantages for most common applications.
• the cell lines sypporting AcMNPV replicaton are superior in growth characteristics and expression leves than cell lines supporting BmNPV replication.
• the range of transfer plasmids and parent viruses available for the AcMNPV-based system is much greater than that for the BmNPV -based system.
• the use of a genetically modified AcMNPV-based vector is advantageous because it replicates only in insect cells and is able to carry large (greater than 15kb inserts) (Boyce and Butcherl996 PNAS 93: 2348-2352).
• the cell lines that are most commonly used with AcNPV -based vectors are the S podoptera frugiperda (SF) cell lines because they are well-tested and have excellent growth and handling properties. These cells have been reported to produce some proteins at levels approaching 20% or more of the total cell protein, and it is unlikely that any other generally available cell line will provide more than threefold higher yields of protein than these.
• Sf cell lines such as the SF cell line IPBL-SF-21 AE (Vaughn et al., (1977) In Vitro, 13, 213-217) is preferred.
• the derivative cell line Sf or Sf8 may be preferred.
• Tricoplusia ni 368 Korean and Marmorosch, (1976) Invertebrate Tissue Culture Applications in Medicine, Biology and Agriculture. Academic Press, New York, USA
• pBAC4x-lare commercially available (e.g. from Stratagene, La Jolla, CA, USA and Novagen).
• NOIs are generally introduced into baculovirus genomes by allelic replacement.
• allelic replacement strategies the foreign gene is inserted into a transfer plasmid so that it is downstream of the required viral promoter and flanked on both sides by viral sequences that will target the gene and promoter to a particular region in the viral genome.
• the plasmid and the parental viral DNA are cotransfected into host cells and enzymes in the cells recombine the DNAs. This primarily involves homologous recombination between the regions of the plasmid DNA flanking the foreign gene and their homologous counterparts in the viral genome.
• “Late” and “early” promoters are used in baculovirus expression systems.
• the polh and plO promoters are examples of strong promoters which are expressed late in infection. The regulation of transciption from these promoters is presently not well understood even though a number of viral gene products that may be important for transcription have been identified (Hasnain et al 1997 Gene 190: 113-118). Promoters that drive gene expression earlier in the infection process are also being considered for nontoxic proteins that are slowly processed in baculovirus-infected cells.
• the baculovirus expression system also has an excellent track record for expressing genes as nonfusion proteins (that is, for expressing genes using their natural, translation initiation codon and N-terminal sequence) but some proteins are expressed to higher levels as fusions.
• VLPs retroviral virus-like particles
• Such insect baculovirus systems have been used extensively for the analysis of the Gag assembly reaction and a number of the observations made are relevant to the production of gene therapy vectors.
• VLPs are proteinaceous particles without a viral genome.
• VLPs are non-infectious and lack virus (or other DNA/RNA) required for replicaton.
• VLPs do not replicate in host cells. Sheep trials have shown that VLPs are more immunogenic than subunit vaccines (viral proteins) or viruses killed by chemical inactivation. In addition, they are more effective at eliciting humoral, cell-mediated and mucosal immunities.
• VLPs are also safe to produce and handle.
• the baculovirus vector and host cells used to make VLPs are not derived from a mammalian source. Hence they do not contain mammalian-derived pathogens (Roy, P 1996 Intervirology 39: 62-71). See OBM 10 spec text page 2, lines 20-30 and page 3, lines 1-10).
• WO 95/22617 suggests using a baculovirus system for retroviral vector production but. importantly, it does not indicate any manner in which this might be achieved.
• retroviral vectors have never before now been used for the production of retroviral vectors.
• One reason for this is that while the protein components of retroviruses had been successfully produced in insect baculovirus systems, it was not expected that the requirements for a functional retroviral vector particle could be met. These requirements include the correct packaging of the vector genome, and an ability of the resulting vector particle to undergo infection of and proviral integration into the target cell.
• a particular consideration is the need for a vector genome capable of undergoing accurate reverse transcription from RNA into proviral DNA in the target cell, and capable of producing proviral DNA which can successfully integrate into the target cell genome.
• RNA contains the appropriate priming and other recognition sites required for reverse transcription (Reviewed in Luciw and Leung: The Retroviridae. Ed.J.A.Levy 1992 Plenum) and that the DNA product has the necessary terminal sequences for achieving integration (Reviewed in Luciw and Leung. Op.cit. ; Cannon et al., 1996 J. Virol. 70, 8234).
• the integrated provirus must contain the necessary control elements for gene expression upon subsequent integration.
• baculovirus expression systems have certain unusual features which make the design of baculovirus vectors for producing retroviral vectors far from straightforward.
• the elements of a baculovirus promoter that are, in part, required for its efficiency are downstream of the RNA transcription start sites (Posse and Howard, 1987 Nucl. Acids Res. 15, 10233; Rankin et al, 1988 Gene 70, 39; Ooi et al., 1989 J.Mol.Biol. 210, 721).
• the presence of such non-retroviral sequences is in principle undesirable, given the need for specific retrovirus terminal sequences for reverse transcription and integration, and can be expected to interfere with normal retroviral vector genome function.
• the present invention overcomes the prior art problems by providing a baculovirus expression system useful for the production of retroviral vectors and/or retroviral particle (which may be capable of acting as a vector).
• the retroviral particles produced by or from the composition of the present invention are useful for the delivery of one or more NOIs to cells in vivo and in vitro, in particular the delivery of therapeutically active NOI(s).
• One or more selected NOI(s) may be incorporated in the vector genome for expression in the target cell.
• the NOI(s) may have one or more expression control sequences of their own, or their expression may be controlled by the vector LTRs.
• a promoter may be included in or between the LTRs which is preferentially active under certain conditions or in certain cell types.
• the NOI may be a sense sequence or an antisense sequence. Furthermore, if there is a plurality of NOIs then those NOIs may be sense sequences or antisense sequences or combinations thereof.
• the retroviral vector genome of the present invention may generally comprise LTRs at the 5' and 3' ends, one or more NOI(s) (including therapeutically active genes and/or marker genes), or suitable insertion sites for inserting one or more NOI(s).
• NOI(s) including therapeutically active genes and/or marker genes
• suitable insertion sites for inserting one or more NOI(s) there may be present a packaging signal to enable the genome to be packaged into a vector particle in a producer ceil.
• primer binding sites and integration sites to allow reverse transcription of the vector RNA to DNA, and integration of the proviral DNA into the target cell genome.
• the retroviral vector particle has a reverse transcription system (compatible reverse transcription and primer binding sites) and an integration system (compatible integrase and integration sites).
• the viral genome or the retroviral vector nucleotide sequence it is possible to manipulate the viral genome or the retroviral vector nucleotide sequence, so that viral genes are replaced or supplemented with one or more NOIs.
• the NOI(s) may be any one or more of selection gene(s), marker gene(s) and therapeutic gene(s). Many different selectable markers have been used successfully in retroviral vectors.
• composition of the present invention may be useful for inter alia medical applications - such as diagnostic or therapeutic
• the NOI can be a therapeutic gene - in the sense that the gene itself may be capable of eliciting a therapeutic effect or it may code for a product that is capable of eliciting a therapeutic effect.
• Non-limiting examples of therapeutic NOIs include genes encoding tumour supressor proteins, cytokines, anti-viral proteins, immunomodulatory molecules, antibodies, engineered immunoglobulin-like molecules, fusion proteins, hormones, membrane proteins, vasoactive proteins or peptides, growth factors, ribozymes, antisense RNA, enzymes, pro- drugs, such as pro-drug activating enzymes, cytotoxic agents, and enzyme inhibitors.
• prodrugs include but are not limited to etoposide phosphate (used with alkaline phosphatase; 5-fluorocytosine (with cytosine deaminase); Doxorubin-N-p- hydroxyphenoxyacetamide (with Penicillin-V-Amidase); Para-N-bis (2- chloroethyl)aminobenzoyl glutamate (with Carboxypeptidase G2); Cephalosporin nitrogen mustard carbamates (with B-lactamase); SR4233 (with p450 reductase); Ganciclovir (with HSV thvmidine kinase); mustard pro-drugs with nitroreductase and cyclophosphamide or ifosfamide (with cytochrome p450).
• etoposide phosphate used with alkaline phosphatase
• 5-fluorocytosine with cytosine deaminase
• Diseases which may be treated include, but are not limited to cancer, heart disease, stroke, neurodegenerative disease, arthritis, viral infection, bacterial infection, parasitic infection, diseases of the immune system, viral infection, tumours, blood clotting disorders, and genetic diseases - such as chronic granulomatosis, Lesch-Nyhan sysndrome, Parkinson's disease, empysema, phenylketonuria, sickle cell anaemia, ⁇ -thalasemia, ⁇ -thalasemia, Gaucher disease.
• Target cells for gene therapy using retroviral vectors include but are not limited to haematopoietic cells, (including monocytes, macrophages, lymphocytes, granulocytes, or progenitor cells of any of these); endothelial cells, tumour cells, stromal cells, astrocytes, or glial cells, muscle cells, epithelial cells, neurons, fibroblasts, hepatocyte. astrocyte, and lung cells.
• haematopoietic cells including monocytes, macrophages, lymphocytes, granulocytes, or progenitor cells of any of these
• endothelial cells including monocytes, macrophages, lymphocytes, granulocytes, or progenitor cells of any of these
• endothelial cells including monocytes, macrophages, lymphocytes, granulocytes, or progenitor cells of any of these
• endothelial cells including monocytes, macrophages, lymphocytes,
• the one or more NOIs can be under the transcriptional control of the viral LTRs.
• a combination of enhancer- promoter elements can be present in order to achieve higher levels of expression.
• the promoter-enhancer elements are preferably strongly active or capable of being strongly induced in the target cells.
• An example of a strongly active promoter-enhancer combination is a human cytomegalovirus (HCMV) major intermediate early (MIE) promoter/enhancer combination.
• the promoter-enhancer combination may be tissue or temporally restricted in their activity. Examples of a suitable tissue restricted promoter-enhancer combinations are those which are highly active in tumour cells such as a promoter-enhancer combination from a MUC1 gene or a CEA gene.
• Hypoxia or ischaemia regulatable expression may also be particularly useful under certain circumstances.
• Hypoxia is a powerful regulator of gene expression in a wide range of different cell types and acts by the induction of the activity of hypoxia-inducible transcription factors such as hypoxia inducible factor-1 (HIF-1) (Wang and Semenza 1993 PNAS. (USA) 90: 430) which bind to cognate DNA recognition sites, the hypoxia responsive elements (HREs) on various gene promoters.
• hypoxia inducible factor-1 HIF-1
• HREs hypoxia responsive elements
• a multimeric form of HRE from the mouse phosphoglycerate kinase-1 (PGK-1) gene has been used to control expression of both marker and therapeutic genes by human fibrosarcoma cells in response to hypoxia in vitro and within solid tumours in vivo (Firth et al 1994, PNAS 91(14): 6496-6500; Dachs et al 1997 Nature Med. 5: 515).
• PGK-1 mouse phosphoglycerate kinase-1
• the fact that glucose deprivation is also present in ischaemic areas of tumours can be used to activate heterologous gene DNA - such as an NOI according to the present invention - expression especially in tumours.
• a truncated 632 base pair sequence of the grp 78 gene promoter known to be activated specifically by glucose deprivation, has been shown to be capable of driving high level expression of a reporter gene in murine tumours in vivo (Gazit et al 1995 Cancer Res. 55: 1660.).
• Retroviral vector genomes of the present invention for subsequent use in gene therapy preferably contain the minimum retroviral material necessary to function efficiently as vectors. The purpose of this is to allow space for the incorporation of the NOI(s), and for safety reasons. Retroviral vector genomes are preferably replication defective due to the absence of functional genes encoding one or more of the structural (or packaging) components encoded by the gag-pol and env genes. The absent components required for particle production are provided in trans in the producer cell. The absence of virus structural components in the genome also means that undesirable immune responses generated against virus proteins expressed in the target cell are reduced or avoided. Furthermore, possible reconstruction of infectious viral particles is preferably avoided where in vivo use is contemplated. Therefore, the viral strucmral components are preferably excluded from the genome as far as possible, in order to reduce the chance of any successful recombination.
• the retroviral vector particles of the present invention are typically generated in a suitable producer cell.
• the present invention also relates to a producer cell comprising the composition of the present invention.
• a producer cell may be a packaging cell containing the virus structural genes, normally integrated into its genome.
• the packaging cell is then transfected with a nucleic acid encoding the vector genome, for the production of infective, replication defective vector particles.
• the producer cell may be co-transfected with nucleic acid sequences encoding the vector genome and the structural components, and/or with the nucleic acid sequences present on one or more expression vectors such as plasmids, adeno virus vectors, herpes viral vectors, vaccinia viral vectors, or any method known to deliver functional DNA into target cells.
• Suitable producer cells include insect cells and mammalian cells, but can be other suitable cells.
• the producer cells are insect cells.
• the present invention also provides a pharmaceutical composition for treating an individual by gene therapy, wherein the composition comprises a therapeutically effective amount of the composition according to the present invention or a viral particle produced by or from same.
• the pharmaceutical composition may be for human or animal usage. Typically, a physician will determine the actual dosage which will be most suitable for an individual subject and it will vary with the age, weight and response of the particular patient.
• the composition may optionally comprise a pharmaceutically acceptable carrier, diluent, excipient or adjuvant.
• a pharmaceutically acceptable carrier diluent, excipient or adjuvant.
• the choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.
• the pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s), and other carrier agents that may aid or increase the viral entry into the target site (such as for example a lipid delivery system).
• the pharmaceutical compositions can be administered by any one or more of: inhalation, in the form of a suppository or pessary, topically in the form of a lotion, solution, cream, ointment or dusting powder, by use of a skin patch, orally in the form of tablets containing excipients such as starch or lactose, or in capsules or ovules either alone or in admixture with excipients, or in the form of elixirs, solutions or suspensions containing flavouring or colouring agents, or they can be injected parenterally, for example intracavernosally, intravenously, intramuscularly or subcutaneously.
• compositions may be best used in the form of a sterile aqueous solution which may contain other substances, for example enough salts or monosaccharides to make the solution isotonic with blood.
• compositions may be administered in the form of tablets or lozenges which can be formulated in a conventional manner.
• the present invention provides in one aspect an expression vector comprising a polynucleotide sequence which encodes a retroviral vector genome having a 5' and a 3' end, which retroviral vector genome is capable of being expressed and packaged into a retroviral vector particle in a baculovirus expression system.
• the 5' and 3' ends function in reverse transcription and integration.
• Three strategies are described herein for overcoming the problem of the baculovirus promoter havmg components upstream and downstream of the RNA transcription start site These strategies are illustrated in Figures 1 to 3
• the downstream component of the promoter is incorporated into the R region at the upstream end of the sequence encodmg the retroviral vector genome (referred to as the 5' R region)
• a counterpart to the downstream component of the promoter is also incorporated into the R region at the downstream end of the sequence encoding the retroviral vector genome (referred to as the 3' R region), so that the two R regions are identical or sufficiently similar for conversion of the vector RNA genome to the DNA provirus, the vector genome will function correctly
• a sequence capable of being cleaved in the RNA transcript is included in the vector between the baculovirus promoter and the sequence encoding the retroviral vector genome
• the downstream component of the baculovirus promoter can thus be cleaved off once the primary RNA transcript has been produced
• a cleavage site is present immediately adjacent the sequence encodmg the retroviral vector genome, so that suitable vector genome ends are produced
• Ribozymes are RNA enzymes which can perform such a function and can be engmeered into DNA constructs
• suitable ribozymes has been well-studied (e g Cech 1992 Curr Op Struct Biol 2, 605) Examples include hammerhead, hairpin and hepatitis delta antigenomic ribozymes Inclusion of a ribozyme sequence as the RNA cleavage sequence, with a cleavage site at the end of the sequence encodmg the retroviral vector genome
• a non-baculovirus promoter is employed
• suitable promoters include the T7 phage promoter and the sp ⁇ salmonella phage promoter
• the terminal G residue of the 5' R region could be positioned precisely at the transcription start site of the T7 promoter, to give an authentic 5' end residue in the retroviral vector genome.
• a source of T7 polymerase would need to be provided, for example by a recombinant baculovirus expressing T7 polymerase (Polkinghorn and Roy 1995 Nucl. Acids Res. 23, 188).
• the expression vector encoding the retroviral vector genome is a baculovirus expression vector, that is, a vector based on a recombinant baculovirus genome.
• Virus vectors are in general easier to handle than non-viral vectors. For example, the transfer of a virus vector into a cell is more reliably performed than transfection of a cell with a DNA plasmid.
• the expression vector may alternatively be a non-baculovirus expression vector, or a non-viral expression vector, such as a DNA plasmid.
• RNA cleavage sequence located downstream of the sequence encoding the retroviral vector, to ensure correct termination of the RNA transcript and a correct 3' end for the vector genome.
• the cleavage sequence has a cleavage site immediately adjacent the sequence encoding the retroviral vector genome, for cleaving at the 3' end of the genome.
• Ribozymes can provide suitable RNA cleavage sequences, as already discussed herein. Ribozymes may have a cleavage site either 5 ' or 3' to the ribozyme sequence. Thus, where there is a ribozyme for each end of the vector genome, these will usually be different.
• the hammerhead ribozyme cleaves to the right of itself and will thus be suitable for the 5' end of the vector genome, while the hairpin ribozyme cleaves to the left and will be suitable for the 3' end of the vector genome.
• the invention provides in further aspects, a retroviral vector particle production system comprising an expression vector as described herein, in an insect cell, and retroviral vector particles produced by such systems.
• packaging components will need to be provided. These will usually be gag-pol and env, which may be provided on one or more suitable expression vectors.
• the expression vector or vectors will need to be capable of expressing the packaging components in a baculovirus expression system.
• the packaging components will be under the expression control of one or more baculovirus promoters.
• the expression vector or vectors carrying the packaging components are preferably baculovirus expression vectors. Alternatively they may be non-baculovirus or non-viral expression vectors, such as plasmids. Some or all of the packaging components may be provided on the same expression vector as the retroviral vector genome.
• Suitable baculovirus systems for use in the invention are easily available and well known in the art. Baculovirus expression vectors are described in detail in O'Reilly et al. 1994 (Op. cit).
• One suitable baculovirus for use in a recombinant baculovirus vector is the well- studied Autographa californica nuclear polyhedrosis virus (AcMNPV).
• a commercially available recombinant baculovirus vector such as pBAC4 x- 1 (Novagen) may be used.
• pBAC4x-l contains four insertion sites, and could be engineered to accommodate nucleic acid sequences encoding the retroviral vector genome and the packaging components.
• suitable baculovirus promoters for use in the invention are well known in the art.
• Commonly-used baculovirus promoters are the polyhedrin and plO promoters.
• the DNA sequence for a polyhedrin promoter is shown in Figure 18.
• the present invention also includes mutants, variants, homologues or fragments of that sequence.
• variant in relation to the nucleotide sequence coding for the preferred enzyme of the present invention include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence providing the resultant nucleotide sequence codes for or is capable of coding for a promoter, preferably being at least as biologically active as the sequence shown as in Figure 18.
• homologue covers homology with respect to structure and/or function providing the resultant nucleotide sequence codes for or is capable of coding for a promoter.
• sequence homology preferably there is at least 75% , more preferably at least 85%, more preferably at least 90% homology to the sequence shown as SEQ ID No. 1. More preferably there is at least 95%, more preferably at least 98%, homology to the sequence shown in Figure 18.
• sequence identity as used herein may be equated with the term "identity”.
• Relative sequence homology (1 e. sequence identity) can be determined by commercially available computer programs that can calculate % homology between two or more sequences.
• a typical example of such a computer program is CLUSTAL.
• variant also encompasses sequences that are complementary to sequences that are capable of hybridising to the nucleotide sequence presented herein
• the present invention also covers nucleotide sequences that can hybridise to the nucleotide sequence of the present mvention (mcludmg complementary sequences of those presented herem)
• the present mvention covers nucleotide sequences that can hybridise to the nucleotide sequence of the present invention under strmgent conditions (e g 65°C and 0 lxSSC) to the nucleotide sequence presented herem (mcludmg complementary sequences of those presented herem)
• baculovirus promoters - as well as mutants, variants, homologues or fragments of those promoters - may also be used
• Insect cell lines for use with the expression vectors according to the invention are also readily available
• One example is the sf8 insect cell line.
• the insect cells used need to be compatible with the particular expression vector chosen.
• the insect cells need to support replication of the expression vector encoding the retroviral vector genome, and to support expression of the retroviral vector genome
• Certain glycosylation-deficient cell lines may be particularly suitable, because of differences in glycosylation between mammalian and insect systems The glycosylation differences are not expected to present a problem, examples of clinical use such as clinical trials with HIV gpl20 produced in insect cells, indicate that any differences in secondary modifications are not significant
• the types of retrovirus which can be used in the invention are not limited to any specific retrovirus.
• Oncoretroviruses such as the murine type-C virus MLV, or lentiviruses such as HIV, or other well-known retroviruses such as ASLV, SNV and RSV could be used.
• the invention is particularly useful for retroviral vectors based on lentiviruses, as these have been very difficult to produce in high titres.
• Titres of lentiviral vectors are commonly two orders of magnitude lower than the murine vectors such as MLV (e.g. Naldini et al. 1996 Science 272, 263).
• retroviral RNA can be so efficiently produced in baculovirus expression systems, it may be possible to omit from the retroviral vector genome lentiviral elements such as HIV Rev and RRE (Gheysen et al. 1989 Op. cit.). It is advantageous to avoid unnecessary retroviral elements in retroviral vectors, particularly when using HIV, or other lentiviruses, because of the possible adverse effects of these elements.
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• Figure 28 is a schematic diagram
• Figure 29 is a schematic diagram.
• the strategy is to first insert the envelope sequences into pBAC4x-l (Novagen), followed by insertion of the MLV gag-pol sequences, which will first be reconstructed in pBluescript (Stratagene).
• Any viral envelope gene could be inserted into this vector in similar ways using technology known in the field of recombinant DNA.
• Envelope genes might include those from any retrovirus or any other virus capable of pseudotyping or otherwise being incorporated into particles produced from retroviral gag-pol genes.
• 5' and 3' fragments from pgagpolgpt are produced by PCR (see primer list).
• the 5' fragment is amplified up to the Xhol site and it creates Kpnl and NofI sites at the beginning of the coding sequence, to generate a 940bp fragment.
• the 3 1 fragment is amplified from the Sphl site of the gag-pol sequence to the end of the pol coding sequence, generating a 700bp fragment.
• EcoRI sites are created at both ends of this fragment and a ⁇ otI site only at the 3' end, so that when the entire gag- pol sequence is reconstructed, it can be excised by digestion with NotI and inserted into pBAC4x-l (Novagen) at the N ⁇ tl site.
• the 5' fragment is inserted into pBluescript at the KpnI/XhoI site, and the 3' fragment into the EcoRI site.
• the Xhol-Sphl (30bp) fragment is then excised from the above plasmid and replaced with an Xhol-Sphl fragment (3580bp) from pgagpolgpt to reconstruct the entire gag-pol sequence in pBluescript to create pBluescript-gagpol.
• the N ⁇ tl fragment containing the gag-pol sequences from pBluescript-gagpol is isolated and inserted into the N ⁇ tl site of pBAC4-env-e and -v to create pBAC4mgagpol-env-e and pBAC4mgagpol-env-v for the MLV envelope version and the VSV-G version respectively.
• plasmids are used as baculovirus transfer vectors using standard methods as described in O'Reilly et al. (Op. Cit.).
• the resulting virus preparations are designated bBAC4mgagpol-env-e and bBAC4mgagpol-env-v respectively.
• bBAC4mgagpol-env-e When these viruses are used to infect insect cells such as sf8 cells large numbers of VLPs are produced.
• the vector genome is expressed from a plasmid that will be used in a transient expression system.
• the genome could be expressed from the same recombinant baculovirus as the gag-pol and env genes.
• the MLV genome derived from pLZSN (Adams et al., 1991 J.Virol. 65, 4985) is inserted into pEc-Hd (Polkinghorn 1996 D.Phil. Thesis, University of Oxford) at the EagllSmal sites.
• the genome that is placed into this vector has the following structure:
• the 5 ' and 3 ' ends of the genome are amplified by PCR so that the T7 promoter sequence is placed immediately upstream of the 5' R region.
• the 3' end is amplified up to the end of the 3' R sequence, generating a blunt-ended product so that it will be precisely fused to the hepatitis delta antigenomic ribozyme motif.
• a proof-reading polymerase is used for PCR to generate the blunt-ended product.
• the 5' end of the genome is amplified up to the E gl site in the packaging signal by PCR.
• An additional ⁇ agI site is created at the very 5' end for insertion into the ⁇ agI site of p ⁇ c- Hd.
• the 3' end of the genome is amplified from the Smal site in the 3' R sequence, up to the very end of the R sequence.
• the 3' fragment is inserted first into p ⁇ c-Hd at the Smal site (a Smal site will not be regenerated at the very 3' end), then the 5' fragment is inserted into the Eagl site.
• the Smal fragment from above is excised and replaced with the Smal fragment (5850bp) from pLZSN to create p ⁇ c-Hd-LZSN.
• the same expression cassette could be assembled into a baculovirus transfer vector with the gag-pol and env sequences by standard procedures.
• Plasmid pEc-Hd-LZSN can be used to transfect sf21 cells that are coinfected with bBAC4mgagpol-env-e or bBAC4mgagpol-env-v and Bac-T7 which expresses the T7 polymerase. In these cells all the components of a retroviral vector are expressed and so the production of functional vectors can be tested. High titres of functional vectors are produced that transfer the lacZ gene to target cells.
• the source of the HIV gag-pol sequences is plasmid pRV664.
• This is a pWI3 (Kim et al., 1989 J.Virol. 63, 3708) derived gagpol vif expression plasmid.
• the RRE of pWI3 (Genbank Accession number: U26942) is inserted by blunt-ending the Sty l/Sty I fragment (7720-8050) into Sma I cut pBluescript KS+ (Stratagene) thereby creating pBSRRE.
• the Nar II Eco Rl fragment of pWI3 (637-5743) is inserted into pBSRRE cut with Cla I and Eco Rl to create pBSGPRREl .
• the Xho l/Not I fragment (containing gagpol and RRE) is inserted into the expression plasmid pCI-Neo to create pGR-RREl.
• a 5.6kb Xhol-Notl fragment containing the HIV-1 gag-pol sequences from pRV664 is isolated and blunt ended with the Klenow fragment of DNA polymerase I. This is then inserted into the Smal site of pBAC4-env-v to produce pBAC4hgagpol-env-v. This is used in the same way as the plasmids described in Example 1 were used.
• the genome from pH4nZ has the structure as shown in Figure 9. It is produced as follows. HIVdge is made from HIVgpt (Page et al. , 1990 J.Virol. 64, 5270) by blunt-ending the Cla / site (829) to create a frameshift mutation. HIVdge is then cut with Bgl II and Pst I (473- 1414) and inserted into pTIN406 (Cannon et al. Op. Cit). pTIN406 has an LTR structure of CMV, R (HIV) and U5 (MLV). This creates a hybrid LTR containing CMV, and R, U5 from HIV called pBS5'.
• HIVdge is made from HIVgpt (Page et al. , 1990 J.Virol. 64, 5270) by blunt-ending the Cla / site (829) to create a frameshift mutation. HIVdge is then cut with Bgl II and Pst I (473- 1414) and inserted into pTIN406
• the Eco llXho I fragment (5743-8897) is cut from HIVdge 1.2 which is a HIVdge derivative containing a deletion from NJe I to Bgl II (6403-7621) and is inserting into pBS5' to create pBS5'R.
• the 3' LTR is provided by inserting the Not II Xho I fragment of pBS3' into pBS5'R creating pH2.
• pBS3' is created by a three way ligation of the Xho II Hind III fragment of pWI3, the Hind lll/Kpn I fragment of pTIN408 (Cannon et al. Op.
• pSPCMV is created by inserting the Pst l/Hind III fragment from pLNCX (Genbank Accession number: M28246) into pSP72 (Promega).
• the ⁇ -galactosidase gene is inserted from pTIN414 (Cannon et al. Op. Cit.) into pSP72 (Xho HSph I) to make pSPlacZ.
• a Xho l/Sal I digest of pSPlacZ gives the ⁇ -galactosidase coding region which is inserted into pH2-CMV to give pH3Z.
• pH4Z is constructed to create tat-deficient vector. The first 50 bp of the tat-coding region is removed by replacing EcoRI (5743)I-5 eI fragment in pH3 with EcoRI (5881)-5pel PCR product amplified using PCR primers
• the Nsi HSpe I fragment from pH4 is inserted into pH3Z to generate pH4Z.
• Two PCR reactions are performed to amplify the 5' and 3' ends of the genome for baculovirus expression. Subsequently, the entire genome is reconstructed by inserting the missing intervening sequences.
• a PCR reaction is performed to amplify the 3' sequences of the genome.
• the Seal site in the 3' U3 sequence is changed to a Smal site and amplified to the very end of the R sequence. This change creates a 3 base pair mutation but should not affect integration.
• a proof-reading polymerase is used to create the 900bp blunt-ended product. This product is then inserted into the Smal site of pEc-Hd ( Figure 10). This destroys the Smal site at the junction of the R sequence and pEc-Hd.
• the 5' sequences are first amplified from the beginning of the R sequence up to the first EcoRI sequence located in the packaging signal. This produces a 900bp fragment. An ⁇ agI site is placed at both ends of the product and a Xhol site at the very 5' end of the fragment. This product is inserted into the Xh ⁇ l-EcoRl site of pBluescript KS (Stratagene) ( Figure 11).
• the ⁇ agl-Spel fragment containing the genome sequences is then isolated from pBluescript KS and inserted into the Eagl-Spel sites of p ⁇ c-Hd ( Figure 12).
• the entire genome is finally reconstituted by insertion of the Spel-Scal fragment (3.9kb) isolated from pH4nZ into the Spel-Smal site of p ⁇ c-Hd ( Figure 12) to produce p ⁇ c-Hd- H4nZ.
• This plasmid can be used in a similar way to pEc-Hd-LZSN but with a baculovirus vector derived from pBAC4hgagpol-env-v to produce HIV-based retroviral vectors at very high titre.
• the starting molecule is pSPEIAV19 (AC:U01866) which is a proviral clone of EIAV.
• the envelope encoding region is disrupted by cutting pSPEIAV19 with Hind III (5835/6571) within the envelope region and self ligating to produce pSPEIAV19dH. This creates an envelope minus proviral clone.
• pSPEIAV19dH is then cut with Mlu I (216/8124) and the resulting fragment inserted into pCI-Neo (Promega) cut with Mlu I (216) to make pONY3.
• the MM fragment from pONY3 containing the EIAV gag-pol, tat and rev coding sequences is isolated and blunt ended with the Klenow fragment of DNA polymerase I. This is then inserted into pBAC4-VSVenv at the Smal site (blunt-ended) to produce pBAC4egagpol-env-v.
• This plasmid is used in a similar way to that described in Example 1 to produce a recombinant baculovirus that will produce high levels of EIAV VLPs.
• the genome of plasmid pONY2.1nlslacZ has the structure as shown in Figure 14. This is constructed as follows.
• the 5' LTR of EIAV clone pSPEIAV19 is PCR amplified using pfii polymerase with the following primers:
• a Bss HII digest (619/792) of pBluescript II KS+ is carried out to obtain the multiple cloning site. This is blunt ended by 5' overhang fill-in and ligated to pONY2 cut with Bgl II and Nco I (1901/4949) and blunt ended by 5' overhang fill-in. The orientation is 3' to 5' in relation to the EIAV sequence. This is called pONY2.1 Plasmid pSPCMV is created by inserting the Psti/Hindlll fragment from pLNCX (Genbank Accession number: M28246) into pSP72 (Promega). The ⁇ - galactosidase gene is inserted from pTIN414 (Cannon et al. Op.
• pSP72 into pSP72 cut with Xho I and Sph I to make pSPlacZ.
• the 5' end to the ⁇ -galactosidase gene is replaced by the SV40 T antigen nuclear localisation signal from pAD.RSVBgal (Bloggs et al.. 1992 J.Clin.Invest. 90, 626).
• pAD.RSVbgal is cut with Xho II Cla I and the fragment inserted into Xho 1/ Cla I cut pSPlacZ to make pSPnlslacZ.
• the Pst I fragment containing the CMV promoter driving the lacZ gene from pSPnlslacZ is inserted into the Pst I site of pONY2.1 in the 5' to 3' orientation of EIAV. This is designated pONY2.1nlslacZ .
• Two PCR reactions are then performed using pONY2.1nlslacZ as template to amplify the 5' and 3' ends of the genome for the baculovirus expression cassette. Subsequently, the entire genome is reconstructed by inserting the missing intervening sequences.
• the 3' PCR product is amplified from the Smal site in the env sequence to the very end of the R sequence using a proof-reading polymerase to produce blunt-ended products.
• the product is then inserted into the Smal site of pEc-Hd. The very 3' end loses the Smal site. ( Figure 15).
• the Smal fragment containing the CMVnlslacZ sequences is isolated from pONY2.1nlslacZ and inserted into the Smal site ( Figure 16).
• the 5' PCR product is amplified up to the EagI site in the MCS and an EagI site is added at die 5' end.
• This product is then inserted into the EagI site to reconstitute the pONY genome in pEc-Hd ( Figure 17) thereby creating pEc-Hd-ONY2.1nlslacZ.
• This can be used in conjunction with pBAC4egagpol-env-v to produce high titres of EIAV -based vectors in a similar way to that described in Examples 2 and 4.
• the plasmid containing the LZSN retroviral vector genome expressed from the polyhedrin promoter is called pBHLZSN ( Figure 19).
• This plasmid is constructed as follows: The starting template for a series of PCR reactions is pHITlll (Soneoka et al. , 1996 Op. Cit.). The 5'LTR containing the polyhedrin promoter is created using PCR primers in reaction A to produce a PCR product shown as PCR: A ( Figure 19). A second reaction (B) creates a fragment that overlaps with PCR:A. Reaction C then produces a combined fragment that is cleaved with Sail and Hindlll.
• This plasmid is constructed as follows: The starting template for a series of PCR reactions is pONY2.1nlslacZ (see Example 6 and GB patent application number 9727135.7 and GB patent application number 9711578.6). The 5' LTR containing the polyhedrin promoter is created using PCR primers in reaction AE to produce a PCR product shown as PCR:AE. A second reaction (BE) creates a fragment that overlaps with PCR:AE.
• Reaction CE then produces a combined fragment that is cleaved with Sal I and Hind III. The resulting fragment is then inserted into pBluescript KS+ to produce plasmid pBEHR.
• PCR reactions DE and EE are carried out. The products of these reactions are then used to produce a combination PCR product (PCR:FE) which is cut with Xba I and Not I and the resulting fragment is inserted into pBEHR to produce pBEHU3HR.
• Plasmid pBONY2.1nlslacZ can be used in conjunction with pBAC4egagpol-env-v to produce high titres of EIAV -based vectors. (Reference may also be made to Figure 21).
• This PCR will give part of polyhedrin with R.
• This PCR will give U3 with part of polyhedrin promoter.
• This PCR will give part of polyhedrin promoter with R.
• the plasmid containing the pH4Z retroviral vector genome expressed from the polyhedrin promoter is called pBH4Z.
• This plasmid is constructed as follows: The starting template for a series of PCR reactions is pH4Z (see PCT/GB 97/02857 and GB patent application number: 9711578.6).
• the 5' LTR containing the polyhedrin promoter is created using PCR primers in reaction AH to produce a PCR product shown as PCR: AH.
• a second reaction (BH) creates a fragment that overlaps with PCR;AH.
• Reaction C then produces a combined fragment that is cleaved widi Sal I and Hind III.
• PCR:FH combination PCR product
• Xba I and Not I the resulting fragment is inserted into pBHHR to produce pBHHU3HR.
• the final stage is to then insert the internal region of pH4Z by cutting it with Nar I and Sph I and inserting the resulting fragment into pBHHU3HR cut with the same enzymes (see the commentary below and Figure 22).
• Plasmid pBH4Z can be used in conjunction with pBAC4hgagpol-env-v to produce HIV -based vectors at high titres. (Reference may also be made to Figure 23.)
• This PCR product will give die polyhedrin promoter with part of R
• This PCR will give part of polyhderin with R.
• This PCR will give U3 with part of polyhedrin promoter.
• This PCR will give part of polyhedrin promoter with R.
• RNA transcripts now contain the correct R sequences at their five prime end.
• each of these self-cleaving polyhedrin promoter based retroviral genomic expression vectors will now be outlined in turn.
• Each of these can be used in conjunction with theeeir cognate gagpol and env expression systems to produce high titre retroviral vector preparations.
• their genome expression cassettes can be inserted into baculovirus vectors by standard procedures.
• Primers polyhAM5 and polyhAM3 are used to PCR amplify the 5 'LTR of pEc-Hd-LZSN to incorporate the required changes (polyhedrin promoter/ribozyme sequence addition flush to the 5' R sequence) into a PCR fragment which is then cloned into pEc-Hd-LZSN by EcoRI- Spel digestion to produce the finished vector- pBHz-Hd-aR.
• This PCR will give polyhderin, hammerhead ribozyme, MSV R region, U5 and leader.
• This fragment can be cloned into pEc-Hd- LZSN at the EcoR I and Spe I sites to give pBHz-H ⁇ - ⁇ RLZSN.
• Primers polyhAEM5 and polyhAEM3 are used to PCR amplify the 5 'LTR of pEc-Hd-ONY2.1nlslacZ to incorporate the required changes (polyhedrin promter/ribozyme sequence addition flush to the 5' R sequence) into a PCR fragment which is then cloned into pEc-Hd-ONY2.1nlslacZ by Eag 1 digestion to produce the finished vector- pBEHz-H ⁇ - ⁇ R.
• This PCR will give polyhedrin, hammerhead ribozyme, EIAV R region, U5 and leader.
• This fragment can be inserted into pEc-Hd-ONY2.1nlslacZ at the Eag I and Eag I sites to give pBEHz-H ⁇ - ⁇ R.
• Primers polyhAHM5 and polyhAHM3 are used to PCR amplify the 5 'LTR of pEc-Hd-H4nZ to incorporate the required changes (polyhedrin promter/ribozyme sequence addition flush to the 5' R sequence) into a PCR fragment which is then cloned into pEc-Hd-H4nZ by Eagl-Nar digestion to produce the finished vector- pBHHz-H ⁇ - ⁇ R.
• This PCR will give polyhderin, hammerhead ribozyme, HIV R region, U5 and leader.
• This fragment can be inserted into pEc-Hd- H4nZ at the Eag I and Nar I sites to give pBHHz-H ⁇ - ⁇ RLZSN.
• the present invention provides a novel system for producing retroviral vector particles.
• die novel system uses a baculovirus expression vector encoding a retroviral vector genome.
• the present invention provides a baculovirus expression vector encoding a retroviral vector genome, and to retroviral vector particles produced by the novel system of the invention.

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