Adenovirus supervector system

Abstract

An adenoviral supervector system is disclosed that is capable of expressing more than 7.5 kilobases of heterologous DNA in a replication defective adenoviral vector. The supervector system comprises an adenoviral vector construct and a helper cell. The vector construct is capable of being replicated and packaged into a virion particle in the helper cell. In particular, the helper cell expresses DNA from the E2 region of the adenovirus 5 genome and complements deletions in that region in the vector construct. In certain embodiments, the disclosed invention comprises tissue specific expression of up to 30 kb of heterologous DNA directed by an adenoviral vector. Also disclosed are methods of transferring heterologous DNA into mammalian cells.

What is claimed is:  
     
         1 . An adenovirus vector construct wherein all or part of the E2 region has been deleted from the adenovirus genome and heterologous DNA is inserted in its place, and wherein said adenovirus vector construct replicates in a helper cell.  
     
     
         2 . The adenovirus vector construct of  claim 1  wherein said vector comprises more than 7.5 kb of heterologous DNA.  
     
     
         3 . The adenovirus vector construct of  claim 2  wherein said vector comprises at least 10 kb of heterologous DNA.  
     
     
         4 . The adenovirus vector construct of  claim 3  wherein said vector comprises at least 20 kb of heterologous DNA.  
     
     
         5 . The adenovirus vector construct of  claim 4  wherein said vector comprises about 30 kb of heterologous DNA.  
     
     
         6 . The adenovirus vector construct of  claim 1  wherein said heterologous DNA comprises one or more structural genes.  
     
     
         7 . The adenovirus vector construct of  claim 6  wherein said one or more structural genes are under the control of a promoter.  
     
     
         8 . The adenovirus vector construct of  claim 7  wherein said one or more structural genes are expressed in a eukaryotic cell.  
     
     
         9 . The adenovirus vector construct of  claim 7 , wherein said promoter is an inducible promoter.  
     
     
         10 . The adenovirus vector construct of  claim 7  wherein said promoter is a tissue specific promoter.  
     
     
         11 . An adenovirus vector construct comprising, at least about 200 base pairs of the left ITR region of the adenovirus genome, more than 7.5 kb of heterologous DNA and at least about 200 base pairs of the right ITR region of the adenovirus genome.  
     
     
         12 . The adenovirus vector construct of  claim 11  further comprising at least 10 kb of heterologous DNA.  
     
     
         13 . The adenovirus vector construct of  claim 12  further comprising at least about 20 kb of heterologous DNA.  
     
     
         14 . The adenovirus vector construct of  claim 13  further comprising about 30 kb of heterologous DNA.  
     
     
         15 . An adenovirus vector construct consisting essentially of map units 0-1.25 of the adenovirus 5 genome, at least 7.5 kb of heterologous DNA and map units 84.5-100 of the adenovirus 5 genome.  
     
     
         16 . A virion particle containing the packaged adenovirus vector construct of  claim 1 ,  11  or  15 .  
     
     
         17 . A recombinant helper cell, wherein said cell expresses one or more genes from the E2 region of the adenovirus genome and is capable of supporting replication of the adenovirus vector construct of  claim 1 ,  11  or  15 .  
     
     
         18 . The cell of  claim 17 , wherein said cell comprises the adenovirus construct of  claim 1 ,  11  or  15 .  
     
     
         19 . The cell of  claim 17 , wherein said cell comprises the virion particle of  claim 16 .  
     
     
         20 . The cell of  claim 17  wherein said cell is derived from a primate cell.  
     
     
         21 . The cell of  claim 20  wherein said cell is derived from a human cell.  
     
     
         22 . The cell of  claim 21  wherein said cell is derived from a human embryonic kidney cell.  
     
     
         23 . The cell of  claim 22  wherein said cell is derived from a 293 cell.  
     
     
         24 . The cell of  claim 23  wherein said cell is an Ad5E2 cell.  
     
     
         25 . A method of expressing a foreign gene in a mammalian cell comprising the following steps: 
 (a obtaining an adenoviral vector construct wherein all or part of the E2 region has been deleted from the adenovirus genome;    (b replicating said adenoviral vector construct in a helper cell;    (c obtaining virion particles produced by said helper cells; and    (d infecting mammalian cells with said virion particles.    
     
     
         26 . The method of  claim 25  wherein said adenoviral vector construct is the adenoviral vector construct of  claim 1 ,  11  or  15 .  
     
     
         27 . The method of  claim 25  wherein said helper cell is an Ad5E2 cell.