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Flavivirus expression and delivery systemRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Process Of Mutation, Cell Fusion, Or Genetic Modification, Introduction Of A Polynucleotide Molecule Into Or Rearrangement Of Nucleic Acid Within An Animal Cell, The Polynucleotide Is Encapsidated Within A Virus Or Viral CoatFlavivirus expression and delivery system description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060088937, Flavivirus expression and delivery system. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a continuation of U.S. Ser. No. 09/580,476, filed May 26, 2000, which is a continuation of International Application No. PCT/AU98/00993 (published as International Publication No. WO 99/28487), filed 30 Nov. 1998 and designating the United States, which in turn claims priority from Australian Application Nos. PP 0627, filed 28 Nov. 1997 and PP 6096, filed 23 Sep. 1998, the teachings of all of which are incorporated herein by reference. [0002] The present invention generally relates to the field of gene expression and in particular to Flavivirus gene expression and delivery systems and to virus like particles produced from such systems. [0003] The present invention generally relates to the field of gene expression and in particular to Flavivirus gene expression and delivery systems and to virus like particles produced from such systems. [0004] Improved methodologies for maximising recombinant gene expression are an on-going effort in the art. Of particular interest is the development of methodologies that maximise recombinant expression of mammalian genes in safe vectors suitable for producing commercially useful quantities of biologically active proteins. [0005] Currently, there are numerous expression systems available for the expression of genes. While procaryotic and yeast expression systems are extremely efficient and easy to use, these systems suffer from a number of disadvantages, including an inability to glycosylate proteins, inefficient cleavage of "pre" or "prepro" sequences from proteins (eg., inefficient post translational modification), and a general inability to secrete proteins. [0006] Another expression system widely available is the baculovirus expression system. This system is arguably one of the most efficient in protein production, but is limited only to use in insect cell lines. Unfortunately, insect cell lines glycosylate proteins differently from mammalian cell lines thus this system has not proven useful for the production of many mammalian proteins. Another disadvantage of this system is that it relies on the use of homologous recombination for the construction of recombinant virus stocks. Thus, this system often proves very laborious when large numbers of genetic variants have to be analysed. [0007] In view of these problems the art has sought eucaryotic host systems, typically mammalian host cell systems, for mammalian protein production. One feature of such systems is that the protein produced has a structure most like that of the natural protein species and purification often is easier since the protein can be secreted into the culture medium in a biologically active form. [0008] One of the most efficient mammalian cell expression systems is based on Vaccinia virus. The main problem with this system, however, is that it uses recombinant viruses that express the heterologous gene upon infection. Thus there is no control over the virus once it has been release. [0009] Recently researchers have started to explore the use of positive strand RNA viruses such as Semliki Forest Virus (SFV), Sindbis (SIN) virus, and poliovirus, as vectors for expression of heterologous genes in vitro and in vivo. The success of these expression systems has been mainly based on each virus' ability to produce high titer stocks of "pseudo" infectious particles containing recombinant replicon RNA packaged by structural proteins. In commercially available Semliki Forest virus (SFV) and Sindbis virus expression systems this is achieved by co-transfection of replicon RNA with defective helper RNA(s) expressing structural genes, but lacking the packaging signal. Replicon RNA expression provides enzymes for RNA replication and transcription of both RNA's, whereas helper RNA supports the production of structural proteins for packaging of replicon RNA via expression of its subgenomic region. The main problem with these expression systems is that the viruses used in the expression system are cytopathic and often compete out the host protein synthesis. Another major disadvantage of these systems includes possible contamination with infectious particles containing packaged full-length genomic RNA (in other words, infectious virus) due to the high probability of recombination between replicon and helper RNAs. [0010] The present invention seeks to provide an improved expression and delivery system that at least ameliorates some of the problems associated with prior art systems. [0011] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers including method steps. SUMMARY OF THE INVENTION [0012] The present invention provides a gene expression system comprising: [0013] a) a replicon of flavivirus origin, which is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities and which is unable to express at least part or all of a structural protein and or another protein(s) required for packaging of a flavivirus genome into a virus particle; and [0014] b) at least a second vector that is capable of expressing flavivirus structural protein(s) and/or any other proteins required for packaging of the self-replicating expression vector into flavivirus viral particles which vector is engineered to prevent recombination with the self-replicating vector when in its presence. [0015] Any replicon (self-replicating expression vector) derived from any flavivirus RNA may be used in the present invention. The replicon should however encode a sufficient amount of a flavivirus 5' UTR and at least a portion of the 5' flavivirus coding region for core protein, each of which is required for RNA replication. Both the 5' UTR and the 5' core protein coding region of a flavivirus genome contains regulatory elements that are required for flavivirus RNA replication. It will be appreciated that the flavivirus 5' UTR and the 5' core protein coding region may contain mutations or deletions in these regions and still be able to replicate. Preferably, the replicon should contain 5' UTR and at least about between 60 and 80 nucleotides from the 5' coding region for flavivirus core protein. The relative number of nucleotides from the 5' core protein coding region that will be required in the replicon for RNA replication will largely depend on the type of flavivirus used in the vector. For example when the replicon is derived from Kunjin virus it must contain at least 60 nucleotides of the 5' core protein coding region. [0016] In one particular embodiment of the invention there is provided a gene expression system comprising: [0017] a) a replicon of flavivirus origin which includes the nucleotide sequence for a flavivirus 5' untranslated region (UTR), at least a portion of the 5' coding region for flavivirus core protein, the nucleotide sequence coding for the flavivirus non-structural proteins, and part or all of the 3'-terminal sequence of a flavivirus 3' UTR, required for self-replication of flavivirus genomic material, which vector is adapted to receive at least a nucleotide sequence without disrupting its replication capabilities and which is unable to express at least part or all of a structural protein(s) region and or a protein(s) or part thereof required for packaging of a flavivirus genome into a virus-like particle; and [0018] b) at least a second vector that is capable of expressing flavivirus structural protein(s) and/or any other proteins required for packaging of the self-replicating expression vector into flavivirus viral particles which vector is engineered to prevent recombination with the self-replicating vector when in its presence. [0019] According to the present invention, the replicon of flavivirus origin is adapted to receive at least a nucleotide sequence. Insertion of such a nucleotide sequence, into the replicon may be achieved at any point in the replicon that does not effect processing of flavivirus proteins. For example, heterologous genes may be inserted into the 3' UTR of the flavivirus replicon, within a structural gene or within the locality of deleted structural genes. Preferably, heterologous genes are inserted into structural genes or in place of deleted structural genes since such insertions generally produce higher levels of expression and generally do not affect replication efficiency of the replicon. If, however, the nucleotide sequence(s) are inserted into the 3' UTR they may be preceded by an internal ribosomal entry site (IRES) sequence. In an embodiment of the invention, the 3' UTR is used only for insertion of IRES-Neo (neomycin transferase) or IRES-pac (puromycin N-acetyl transferase) sequences. Such insertions allow the generation of stable cell lines persistently expressing foreign genes via antibiotic (eg Geneticin or puromycin) selection. [0020] In another preferred embodiment of the invention there is provided a gene expression system comprising: [0021] a) a replicon of flavivirus origin which includes a nucleotide sequence for a flavivirus 5' UTR, at least a portion of a 5' coding region for flavivirus core protein, a nucleotide sequence coding for a flavivirus non-structural proteins, the complete or most of the 3'-terminal region of a flavivirus 3' UTR required for self-replication of the genomic material and the nucleotide coding sequence for flavivirus structural proteins, wherein (i) the vector is adapted to receive at least a nucleotide sequence without disrupting the replication capabilities of the vector, (ii) the nucleotide sequence is inserted into the vector in a manner which deactivates expression of at least a gene that would otherwise code for a flavivirus structural protein and (iii) the inserted nucleotide sequence does not encode for the structural protein sequence that it deactivates; and [0022] b) at least a second vector that is (i) capable of expressing the flavivirus structural protein(s) that is not expressed by the replicon and (ii) engineered to prevent recombination with the self-replicating vector when in its presence. [0023] When the nucleotide sequence is inserted into the replicon it should be introduced into the vector in a manner which avoids a frame shift in the open reading frame of the vector coding sequence. This may be achieved by either adapting the foreign nucleotide sequence or the vector to ensure the reading frame of the vector coding sequence is maintained. In an alternative arrangement foreign nucleotide sequence can be inserted without preserving open reading frame of the vector if it is followed by a termination codon and an internal ribosomal entry site (IRES) sequence to ensure initiation of translation of the vector's nonstructural proteins. [0024] A replicon which encodes flavivirus structural and non-structural proteins may be either RNA or DNA based provided it is capable of self-replication and encodes flavivirus structural and non-structural protein coding information. Where the replicon is an RNA sequence the flavivirus genome is first reverse transcribed into complementary DNA sequence and cloned into appropriate plasmid vector containing prokaryotic (bacteriophage) DNA-dependent RNA polymerase promotor. The nucleotide sequence is then inserted into the resulting plasmid containing replicon complementary DNA sequence and the genomic sequence is then transcribed back into RNA prior to delivery to a host cell. Where the vector is DNA based the flavivirus genome is first reverse transcribed into complementary DNA sequence and cloned into appropriate plasmid vector containing eucaryotic expression promoter. A nucleotide sequence can then be inserted into the resulting plasmid containing replicon complementary DNA sequence, which is then introduced into a host cell as plasmid DNA. [0025] While the replicon will in most circumstances be prepared from a single strain of flavivirus it should be appreciated that in some circumstances nucleotide sequences from more than one flavivirus strain may be brought together in a single vector. Preferably the replicon is derived from the genomic sequence of a single flavivirus species. Most preferably the replicon is derived from a single flavivirus species (such as Kunjin virus (KUN)) and includes the entire or a substantial portion of the genome of that strain, the genome being modified in at least one of its structural proteins to accept a nucleotide sequence such that the insertion of the nucleotide sequence into the structural protein nucleotide sequence disrupts coding for part or all of the structural protein. [0026] Nucleotide sequences that may be inserted into the replicon include, for example, parts of flavivirus or non-flavivirus cDNA gene sequences. Nucleotide sequence(s) that are inserted into the replicon must, however, disrupt the expression of at least a structural protein thus preventing viral genome packaging. Desirably the inserted nucleotide sequence is a non-flavivirus nucleotide sequence (hereinafter referred to as a "heterologous nucleotide sequence"). The heterologous nucleotide sequence is not limited only to a sequence that encodes an amino acid sequence, but may also include sequences appropriate for promoting replication and or expression of a sequence that encodes an amino acid sequence. [0027] Insertion of a heterologous nucleotide sequence into the replicon may occur at any point in a flavivirus structural protein(s) or in any region of the nucleotide sequence where such a protein would normally be expressed in the native flavivirus sequence had the protein not been deleted. In one embodiment of the invention the heterologous nucleotide sequence is inserted into at least one of the structural genes deactivating that gene. In another embodiment at least a structural gene is deleted from the vector and the deletion site is adapted to serve as the insertion site for heterologous genetic sequences. Most preferably, the nucleotide sequence is inserted into the locality from where at least a structural gene was deleted. [0028] By positioning heterologous nucleotide sequences within the locality of one or more sites in the replicon that might otherwise code for structural genes in a native flavivirus, the replicon is unable to produce structural proteins for viral packaging. Continue reading about Flavivirus expression and delivery system... 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