Novel replication deficient adenovirus vectors and methods for making and using them

Abstract

The invention provides novel replication deficient adenovirus vectors and methods for making and using these viruses. The invention also provides vector systems and kits using a serotype specific strategy for making adenoviral vector preparations substantially free of replication competent “helper” virus. The helper virus-free preparations provide novel pharmaceutical compositions substantially free of helper virus for use in gene transfer and gene therapy.

What is claimed is:  
     
         1 . A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);  
 (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
 wherein the helper-dependent adenovirus nucleic acid fails to produce a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs; and  
 (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
   (c) a nucleic acid encoding a polypeptide or a polypeptide having activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence.    
     
     
         2 . The vector system of  claim 1 , wherein the adenovirus capsid, packaging and IVa2 trans-acting protein encoding sequences are human adenovirus sequences.  
     
     
         3 . The vector system of  claim 2 , wherein the helper-dependent and helper adenovirus serotypes are selected from the group consisting of adenovirus type 2 (Ad2), adenovirus type 5 (Ad5), adenovirus type 7 (Ad7), adenovirus type 12 (Ad12), adenovirus type 17 (Ad17), and adenovirus type 40 (Ad40).  
     
     
         4 . The vector system of  claim 2 , wherein the helper-dependent adenovirus serotype is adenovirus type 5 and the helper adenovirus serotype is adenovirus type 7.  
     
     
         5 . The vector system of  claim 2 , wherein the helper-dependent adenovirus serotype is adenovirus type 7 and the helper adenovirus serotype is adenovirus type 5.  
     
     
         6 . The vector system of  claim 1 , wherein the helper-dependent adenovirus sequence fails to produce a complete adenovirus capsid.  
     
     
         7 . The vector system of  claim 6 , wherein the helper-dependent adenovirus sequence is encapsidated in a capsid comprising at least one polypeptide encoded by the helper adenovirus sequence.  
     
     
         8 . The vector system of  claim 6 , wherein the helper-dependent adenovirus sequence is packaged in a capsid encoded by the helper adenovirus sequence.  
     
     
         9 . The vector system of  claim 1 , further including a nucleic acid sequence encoding a 52/55 kDa protein, or a 52/55 kDa protein, wherein the polypeptide is from an adenovirus having the same serotype as the helper-dependent adenovirus serotype-specific cis-acting packaging sequence and different from the serotype of the helper adenovirus serotype-specific cis-acting packaging sequence.  
     
     
         10 . The vector system of  claim 1 , further including a nucleic acid sequence encoding a 52/55 kDa protein, or a 52/55 kDa protein, wherein the polypeptide is from an adenovirus having a different serotype then the helper-dependent adenovirus serotype-specific cis-acting packaging sequence and different from the serotype of the helper adenovirus serotype-specific cis-acting packaging sequence.  
     
     
         11 . The vector system of  claim 1 , wherein the failure to produce a functional IVa2 trans-acting protein is due to a mutation in the sequence encoding the protein.  
     
     
         12 . The vector system of  claim 11 , wherein the mutation is a missense mutation, a point mutation, a frameshift mutation or a deletion mutation.  
     
     
         13 . The vector system of  claim 1 , wherein the helper adenovirus sequence further comprises the nucleic acid sequence encoding the polypeptide having the activity of the IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid.  
     
     
         14 . The vector system of  claim 1 , wherein the nucleic acid encoding the polypeptide having the activity of the IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence is functionally-associated with the genome of an adenovirus replication competent host cell.  
     
     
         15 . The vector system of  claim 14 , wherein adenovirus replication competent host cell is a 293 cell line.  
     
     
         16 . The vector system of  claim 1 , wherein the polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence is a IVa2 trans-acting protein.  
     
     
         17 . The vector system of  claim 1 , wherein the helper-dependent adenovirus sequence lacks at least one nucleic acid sequence needed to produce a capsid and further comprises a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence.  
     
     
         18 . The vector system of  claim 1 , wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, penton gene, fiber gene or hexon polypeptide gene or combination thereof.  
     
     
         19 . A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs;  
 (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs;  
 (ii) a helper adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence,  
 wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.  
   
     
     
         20 . A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs;  
 (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs; and  
 (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
   (c) a cell comprising a nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein, 
 wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.  
   
     
     
         21 . A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs;  
 (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs; and  
 (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
   (c) an expression cassette comprising a nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein, 
 wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.  
   
     
     
         22 . A vector comprising a replication defective adenovirus sequence comprising: 
 (a) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence;    (b) a nucleic acid sequence encoding a functional helper adenovirus serotype-specific IVa2 trans-acting protein, wherein the helper adenovirus serotype-specific IVa2 trans-acting protein does not have the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein, lacking the ability to produce a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein, 
 wherein the replication defective adenovirus sequence comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.  
   
     
     
         23 . The vector of  claim 22 , further comprising at least one adenoviral nucleic acid sequence needed to produce an adenoviral capsid.  
     
     
         24 . The vector of  claim 23 , further comprising sufficient adenoviral nucleic acid sequence to produce a complete adenoviral capsid when the vector is expressed in an adenovirus replication-competent host cell.  
     
     
         25 . The vector of  claim 22 , wherein the helper-dependent and helper adenovirus serotypes are selected from the group consisting of adenovirus type 2 (Ad2), adenovirus type 5 (Ad5), adenovirus type 7 (Ad7), adenovirus type 12 (Ad12), adenovirus type 17 (Ad17), and adenovirus type 40 (Ad40).  
     
     
         26 . The vector of  claim 25 , wherein the helper-dependent adenovirus serotype is adenovirus type 5 and the helper adenovirus serotype is adenovirus type 7.  
     
     
         27 . The vector of  claim 25 , wherein the helper-dependent adenovirus serotype is adenovirus type 7 and the helper adenovirus serotype is adenovirus type 5.  
     
     
         28 . A transformed or isolated infected cell comprising the vector system of  claim 1  or the vector of  claim 22 .  
     
     
         29 . A kit for making adenovirus encapsidated replication defective nucleic acid sequences comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);  
 (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
 wherein the helper-dependent adenovirus nucleic acid fails to produce a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs; and  
 (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
   (c) a nucleic acid encoding a polypeptide or a polypeptide having activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence.    
     
     
         30 . The kit of  claim 29 , wherein the nucleic acid sequence encoding a polypeptide having the activity of the IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence is functionally-associated with the genome of an adenovirus replication competent host cell.  
     
     
         31 . The kit of  claim 29 , wherein the nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein further comprises an expression cassette.  
     
     
         32 . The kit of  claim 29 , wherein the helper adenovirus sequence further comprises the nucleic acid sequence encoding a polypeptide having the activity of a helper-dependent adenovirus serotype IVa2 trans-acting protein.  
     
     
         33 . A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps: 
 (a) transforming or infecting into adenovirus replication competent host cells 
 (i) a helper-dependent adenovirus nucleic acid sequence comprising: 
 5′ and 3′ adenovirus inverted terminal repeats (ITRs);  
 a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 a heterologous gene;  
 
 (ii) a helper adenovirus nucleic acid sequence comprising: 
 5′ and 3′ adenovirus ITRs;  
 a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
 
 (iii) a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence; and  
   (b) culturing the cells under conditions where the helper-dependent replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector.    
     
     
         34 . A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps: 
 (a) transforming or infecting into an adenovirus replication competent host cell two adenovirus replication defective sequences, wherein the cell comprises a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype IVa2 trans-acting protein that supports packaging of a helper-dependent adenovirus nucleic acid sequence and fails to support packaging of a helper adenovirus nucleic acid sequence, 
 (i) a helper-dependent adenovirus nucleic acid sequence comprising: 
 5′ and 3′ adenovirus inverted terminal repeats (ITRs);  
 a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 a heterologous gene;  
 
 (ii) a helper adenovirus nucleic acid sequence comprising: 
 5′ and 3′ adenovirus ITRs;  
 a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
 
   (b) culturing the cells under conditions where the helper-dependent replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector, wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.    
     
     
         35 . A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps: 
 (a) transforming or infecting into an adenovirus replication competent host cell two adenovirus replication defective sequences comprising: 
 (i) a helper-dependent adenovirus nucleic acid sequence comprising: 
   5 ′ and 3′ adenovirus inverted terminal repeats (ITRs);  
 a helper-dependent adenovirus serotype-specific cis-acting packaging sequence;  
 a heterologous gene; and  
 a nucleic acid sequence encoding a polypeptide having the activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence; and  
 
 (ii) a helper adenovirus nucleic acid sequence comprising: 
 5′ and 3′ adenovirus ITRs;  
 a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
 
   (b) culturing the cells under conditions where the helper-dependent replication defective adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector, wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.    
     
     
         36 . The method of  claim 33 ,  34 , or  35  wherein the helper adenovirus sequence further comprises an adenoviral nucleic acid sequence encoding a complete adenoviral viral capsid.  
     
     
         37 . A vector for selectively packaging replication defective nucleic acid sequences in adenovirus capsids, the vector comprising: 
 (a) a replication defective adenovirus sequence comprising an adenovirus serotype 7 (Ad7) cis-acting packaging sequence;    (b) a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype 5 (Ad5) IVa2 trans-acting protein; and    (c) an adenoviral nucleic acid sequence that encodes a viral capsid and fails to encode or produce a polypeptide having the activity of an adenovirus 7 serotype IVa2 trans-acting protein.    
     
     
         38 . A pharmaceutical composition comprising an encapsidated replication defective adenovirus, made using the vector system of  claim 1 , substantially free of helper virus, and a pharmaceutically acceptable excipient.  
     
     
         39 . The pharmaceutical composition of  claim 38 , wherein the pharmaceutical composition is 99% free of helper virus.  
     
     
         40 . A method of delivering a heterologous nucleic acid to a cell comprising transforming or infecting a cell with the pharmaceutical composition of  claim 38 .  
     
     
         41 . The method of  claim 40 , wherein the pharmaceutical composition is administered to a patient systemically, regionally or locally.  
     
     
         42 . A packaging cell line for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the cell line comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);  
 (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs;  
 (ii) a helper adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce a polypeptide having the activity of the helper adenovirus serotype IVa2 trans-acting protein; and  
   (c) a polypeptide having activity of a IVa2 trans-acting protein that supports packaging of the helper-dependent adenovirus nucleic acid sequence and fails to support packaging of the helper adenovirus nucleic acid sequence, 
 wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.  
   
     
     
         43 . A packaging cell line for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the cell line comprising: 
 (a) a nucleic acid sequence encoding a polypeptide having the activity of an adenovirus serotype-specific IVa2 trans-acting protein;    (b) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus inverted terminal repeats (ITRs);  
 (ii) a helper-dependent adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
   (c) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs;  
 (ii) a helper adenovirus serotype-specific cis-acting packaging sequence that fails to support the activity of the polypeptide having the activity of an adenovirus serotype-specific IVa2 trans-acting protein,  
 wherein the replication defective adenovirus comprises a defective or modified adenovirus E1 gene, E2A gene, E2B gene, E3 gene, E4 gene, E4 promoter, a penton gene, a fiber gene or a hexon gene, or a combination thereof.  
   
     
     
         44 . A vector system for selectively packaging a replication defective nucleic acid sequence in a virus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ viral inverted terminal repeats (ITRs);  
 (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid,  
 wherein the helper-dependent adenovirus nucleic acid fails to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the first adenovirus serotype-specific cis-acting packaging sequence;  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ virus ITRs;  
 (ii) a second adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the second adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the second adenovirus serotype-specific cis-acting packaging sequence; and  
   (c) a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific IVa2 trans-acting protein that supports packaging of the first adenovirus serotype-specific cis-acting packaging sequence and fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence and a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific 52/55 kDa trans-acting protein that supports packaging of the first adenovirus serotype-specific cis-acting packaging sequence and fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence.    
     
     
         45 . A vector system for selectively packaging a replication defective nucleic acid sequence in a virus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ viral inverted terminal repeats (ITRs);  
 (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid,  
 wherein the helper-dependent adenovirus nucleic acid fails to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the first adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the first adenovirus serotype-specific cis-acting packaging sequence;  
   (b) a helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ virus ITRs;  
 (ii) a second adenovirus serotype-specific cis-acting packaging sequence,  
 wherein the helper adenovirus nucleic acid fails to produce to produce (1) a polypeptide having the activity of a serotype-specific IVa2 trans-acting protein specific for the second adenovirus serotype-specific cis-acting packaging sequence or (2) a polypeptide having the activity of a serotype-specific 52/55 kDa protein specific for the second adenovirus serotype-specific cis-acting packaging sequence; and  
   (c) a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific IVa2 trans-acting protein that supports packaging of the first adenovirus serotype-specific cis-acting packaging sequence and fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence and a nucleic acid encoding a polypeptide or a polypeptide having an activity of a serotype-specific 52/55 kDa trans-acting protein that is derived from any adenovirus serotype that fails to support packaging of the second adenovirus serotype-specific cis-acting packaging sequence.    
     
     
         46 . A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ ITRs from a first adenovirus serotype;  
 (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
 wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype;  
   (b) a chimeric helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;  
 (ii) a second adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,  
 wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype.  
   
     
     
         47 . The vector system of  claim 46 , wherein the first adenovirus serotype is Ad7.  
     
     
         48 . The vector system of  claim 46 , wherein the second adenovirus serotype is Ad5.  
     
     
         49 . A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ ITRs from a first adenovirus serotype;  
 (ii) a first adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
 wherein the helper-dependent adenovirus nucleic acid fails to encode a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype;  
   (b) a chimeric helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;  
 (ii) a second adenovirus serotype-specific cis-acting packaging sequence; and  
 (iii) an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,  
 wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype; and  
   (c) a nucleic acid encoding a polypeptide or a polypeptide having activity of a IVa2 trans-acting protein having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype.    
     
     
         50 . A method of producing a replication defective encapsidated adenovirus gene transfer vector, comprising the following steps: 
 (a) transforming or infecting into an adenovirus replication competent host cell with adenovirus replication defective sequences comprising: 
 (i) a helper-dependent adenovirus nucleic acid sequence comprising: 
 5′ and 3′ ITRs from a first adenovirus serotype;  
 a first adenovirus serotype-specific cis-acting packaging sequence; and  
 a heterologous nucleic acid;  
 
 wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from a first adenovirus serotype;  
 (ii) a chimeric helper adenovirus nucleic acid sequence comprising: 
 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;  
 a second adenovirus serotype-specific cis-acting packaging sequence; and  
 an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,  
 
 wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype; and  
   (b) culturing the cells under conditions where the helper-dependent adenovirus sequence is encapsidated to produce a replication defective adenovirus gene transfer vector.    
     
     
         51 . The method of  claim 50 , wherein the first adenovirus serotype is Ad7.  
     
     
         52 . The method of  claim 50 , wherein the second adenovirus serotype is Ad5.  
     
     
         53 . The method of  claim 50 , wherein the host cell is a 293 cell.  
     
     
         54 . A chimeric helper adenovirus nucleic acid sequence comprising: 
 (a) 5′ and 3′ adenovirus ITRs from a second adenovirus serotype;    (b) a second adenovirus serotype-specific cis-acting packaging sequence; and    (c) an adenovirus serotype-specific nucleic acid sequence from a non-second adenovirus serotype,    wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype.    
     
     
         55 . The chimeric helper adenovirus nucleic acid sequence of  claim 54 , wherein the non-second adenovirus serotype is Ad7.  
     
     
         56 . The chimeric helper adenovirus nucleic acid sequence of  claim 54 , wherein the second adenovirus serotype is Ad5.  
     
     
         57 . The chimeric helper adenovirus nucleic acid sequence of  claim 54 , wherein the nucleic acid is designated Ad7/5ITR Ψ -GFP.  
     
     
         58 . A cell line for packaging the chimeric helper adenovirus nucleic acid sequence of  claim 54 , wherein the cell line comprises a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from a second adenovirus serotype.  
     
     
         59 . The cell line of  claim 58 , wherein the second adenovirus serotype is Ad5.  
     
     
         60 . The cell line of  claim 58 , wherein the cell line is a 293 cell line.  
     
     
         61 . The cell line of  claim 58 , wherein the cell line is designated 293-L1.  
     
     
         62 . A vector system for selectively packaging a replication defective adenovirus nucleic acid sequence in an adenovirus capsid, the vector system comprising: 
 (a) a helper-dependent adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ ITRs from adenovirus serotype Ad7;  
 (ii) an adenovirus serotype Ad7 cis-acting packaging sequence; and  
 (iii) a heterologous nucleic acid;  
 wherein the helper-dependent adenovirus nucleic acid encodes a polypeptide having the activity of a IVa2 trans-acting protein specific for the cis-acting packaging sequence from adenovirus serotype Ad7;  
   (b) a chimeric helper adenovirus nucleic acid sequence comprising: 
 (i) 5′ and 3′ adenovirus ITRs from adenovirus serotype Ad5;  
 (ii) an adenovirus serotype Ad5 cis-acting packaging sequence; and  
 (iii) an adenovirus serotype-specific nucleic acid sequence from adenovirus serotype AD7,  
 wherein the chimeric helper adenovirus nucleic acid fails to encode a polypeptide having the activity of IVa2 trans-acting protein specific for the cis-acting packaging sequence from adenovirus serotype Ad5.