Conditional replication and expression system

Abstract

The present invention relates to the utilization of conditionally replicating recombinant nucleic acid molecules rescued from the integrated state for the expression of foreign proteins. The usefulness of the system is illustrated with a conditionally replicating recombinant nucleic acid molecule encoding the adeno-associated virus (AAV) capsid proteins. The present invention also relates to methods employing and conditionally replicating recombinant nucleic acid molecules for the packaging of recombinant AAV nucleic acid molecule into AAV capsids. The present invention also relates to packaging cell lines for recombinant AAV, expressing both the AAV rep and cap-genes.

What is claimed is:  
     
         1 . A process for producing a packaging cell for packaging recombinant vectors from AAV, said process comprising providing said packaging cell with a vector encoding at least a functional part of a rep-gene and an integrated vector encoding at least a functional part of a cap-gene, wherein said cap-gene is functionally excisable from a host cell genome for replication.  
     
     
         2 . The process according to  claim 1 , wherein the rep-gene is under control of a combination of activator/repressor sequences.  
     
     
         3 . The process according to  claim 1 , wherein the integrated vector comprising the functional part of the cap-gene includes functional AAV-ITR's.  
     
     
         4 . The process according to  claim 3 , wherein the integrated vector comprising the functional part of the cap-gene is too large for packaging into a viral (AAV) particle.  
     
     
         5 . The process of according to  claim 1 , wherein the integrated vector comprising the functional part of the cap-gene is under control of at least a functional part of a rep-protein.  
     
     
         6 . A packaging cell produced by the process of  claim 1 .  
     
     
         7 . A cell line comprising the packaging cells of  claim 6 .  
     
     
         8 . The process according  claim 1 , wherein the integrated vector comprises a gene of interest.  
     
     
         9 . A method of producing a recombinant adeno-associated virus (AAV) comprising a gene of interest, said method comprising: 
 providing a packaging cell including an integrated vector encoding at least a functional part of a cap-gene, which cap-gene is functionally excisable from said packaging cell for replication; and    inducing a lytic cycle in said packaging cell, wherein said inducing produces said recombinant AAV and said gene of interest.    
     
     
         10 . The method according to  claim 9 , further comprising introducing into said packaging cell a vector encoding at least a functional part of a rep-gene.  
     
     
         11 . The method according to  claim 9 , wherein said inducing comprises infecting said packaging cell with a helper virus.  
     
     
         12 . The method according to  claim 9 , wherein said gene of interest is under the control of an inducible promoter.  
     
     
         13 . A kit of parts for producing a recombinant adeno-associated virus (AAV) comprising a gene of interest, said kit of parts comprising: 
 a packaging cell produced by a process comprising providing a packaging cell with a vector encoding at least a functional part of a rep-gene, and an integrated vector encoding at least a functional part of a cap-gene, which cap-gene is functionally excisable from a eukaryotic host cell genome for replication; and    a recombinant vector derived from AAV comprising two functional AAV-ITR's, an AAV-packaging signal and the gene of interest.    
     
     
         14 . The kit of parts of  claim 13 , wherein said vector derived from AAV comprises elements from a helper virus genome necessary for AAV replication and/or production.  
     
     
         15 . The kit of parts of  claim 14 , wherein said elements are functional E 1 , E 2A , E 4 , and VA genes.  
     
     
         16 . The kit of parts of  claim 13 , further comprising a vector having helper virus functions for AAV replication and/or production.  
     
     
         17 . A vector encoding at least a functional part of a cap gene of AAV wherein the cap sequence includes an overlap with the AAV rep gene to the extent that the cap gene sequence includes the splice sites in said overlap.  
     
     
         18 . A cell comprising the vector according to  claim 17 .  
     
     
         19 . A packaging cell line for producing recombinant AAV, wherein said packaging cell line comprises at least one cell according to  claim 18 .  
     
     
         20 . A cell line for producing recombinant AAV, said cell line comprising a packaging cell line according to  claim 19 , wherein said packaging cell line has been provided with at least one additional vector comprising two functional AAV-ITR's, an AAV-packaging signal, and a gene of interest.