Flavivirus expression and delivery system

Abstract

The present invention provides a gene expression system comprising: a) a self-replicating expression vector of flavivirus origin which includes the 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 the complete or most of the 3′-terminal sequence of the 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 b) at least a second vector that is capable of expressing flavivirus structural protein(s) and 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.

1 . A gene expression and delivery system comprising: 
 (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(s) region and or a protein(s) or part thereof required for packaging of a flavivirus genome into a virus-like particle; and    (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 replicon into infectious flavivirus-like particles which vector is engineered to prevent recombination with the replicon when in its presence.    
     
     
         2 . A gene expression and delivery system according to  claim 1  wherein the replicon of flavivirus origin 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.  
     
     
         3 . A gene expression and delivery system according to  claim 1  wherein the replicon contains a sufficient amount of flavivirus 5′ UTR and a sufficient amount of 5′ flavivirus coding region for core protein required for RNA replication.  
     
     
         4 . A gene expression and delivery system according to claim I wherein the replicon contains a flavivirus 5′ UTR and at least about between 60 and 80 nucleotides from the 5′ coding region for flavivirus core protein.  
     
     
         5 . A gene expression and delivery system according to  claim 1  wherein the replicon is derived from Kunjin virus and contains the Kunjin virus 5′ UTR and at least 60 nucleotides of the Kunjin virus 5′ core protein coding region.  
     
     
         6 . A gene expression and delivery system comprising: 
 (a) a self-replicating expression vector of flavivirus origin which includes the nucleotide sequence for the flavivirus 5′ UTR, at least a portion of the 5′ nucleotide coding region for flavivirus core protein, the nucleotide coding region for flavivirus non-structural proteins, a sufficient amount of the 3′-terminal region of the flavivirus 3′ UTR required for self-replication of flavivirus genomic material 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    (b) at least a second vector that is (i) capable of expressing the flavivirus structural protein(s) that is not expressed by the self-replicating expression vector described in (a) and (ii) engineered to prevent recombination with the self-replicating vector described in (a) when in its presence.    
     
     
         7 . A gene expression and delivery system according to  claim 1  wherein the replicon is adapted to receive at least a nucleotide sequence at any point in the replicon that does not effect processing of flavivirus proteins and RNA replication.  
     
     
         8 . A gene expression and delivery system according to  claim 7  wherein the nucleotide sequence is inserted into the 3′ UTR of the replicon.  
     
     
         9 . A gene expression and delivery system according to  claim 8  wherein the nucleotide sequence that is inserted into the 3′ UTR of the replicon is preceded by an IRES sequence.  
     
     
         10 . A gene expression and delivery system according to  claim 6  wherein the nucleotide sequence is inserted within a structural gene.  
     
     
         11 . A gene expression and delivery system according to  claim 6  wherein the nucleotide sequence is inserted within the locality of at least a deleted gene encoding a structural protein.  
     
     
         12 . A gene expression and delivery system according to  claim 11  wherein the nucleotide sequence that is inserted possesses at its 3′ end a termination codon and an IRES sequence.  
     
     
         13 . A gene expression and delivery system according to  claim 11  wherein the inserted nucleotide sequence possesses at its 3′ end a 2A autoprotease sequence of foot and mouth disease virus.  
     
     
         14 . A gene expression and delivery system according to  claim 13  wherein the inserted nucleotide sequence possesses at its 5′ end a mouse ubiquitin sequence.  
     
     
         15 . A gene expression and delivery system according to  claim 1  wherein the replicon includes after the 3′ terminal sequence of a flavivirus 3′ UTR a sequence cassette containing antigenomic ribozyme of the hepatitis delta virus and SV 40 polyadenylation signal, required for production of vector RNA with precise 3′ terminus possessing high replication efficiency.  
     
     
         16 . A gene expression and delivery system according to  claim 1  wherein the replicon is an RNA based vector.  
     
     
         17 . A gene expression and delivery system according to  claim 16  wherein the replicon is an RNA based vector, which is capable of producing replicon RNA in in vitro transcription reactions by bacteriophage DNA-dependent RNA polymerases from plasmid DNAs incorporating corresponding bacteriophage promoters preceding the replicon sequence.  
     
     
         18 . A gene expression and delivery system according to  claim 1  wherein the replicon is a DNA based vector.  
     
     
         19 - 42 . (canceled)  
     
     
         43 . A method for producing a stable cell line capable of persistently producing replicon RNA's, comprising the steps of: 
 (i) introducing into a cell 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(s) region and or a protein(s) or part thereof required for packaging of a flavivirus genome into a virus-like particle; and    (ii) culturing that cell line under conditions which permit cell growth and replication.    
     
     
         44 . A method for producing a flavivirus like particles comprising the steps of: 
 (i) introducing into a cell 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(s) region and or a protein(s) or part thereof required for packaging of a flavivirus genome into a virus-like particle;    (ii) introducing into a replicon-containing cell a second vector that is capable of expressing flavivirus structural protein(s) and any/or 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; and    (iii) harvesting virus like particles containing the replicon.    
     
     
         45 - 65 . (canceled)