Self-rearranging DNA vectors

Abstract

Disclosed are replicatable viral DNA vectors encoding a site-specific DNA-altering enzyme and a DNA target recognized by the enzyme, the enzyme selectively converting, in a cell expressing the enzyme, the DNA vector to a rearranged form. The invention further relates to methods for assembling recombinant adenoviral DNAs. These methods include the steps of: (a) providing a first linearized DNA vector including a restriction site and a cos site and a second linearized DNA vector including the restriction site, an adenoviral nucleic acid molecule, and a cos site; and (b) ligating the first and second linearized DNA vectors, the ligation assembling a recombinant adenoviral DNA.

1 . A method of gene therapy comprising the administration to a patient in need of gene therapy a therapeutically effective amount of a vector which is expressed in said patient, wherein said vector comprises, in the 5′ to 3′ direction, 
 a first genetically engineered cis-acting target recognized by a recombinase or an integrase;    a gene of interest;    a bi-directional promoter, comprising a second genetically engineered cis-acting target recognized by said recombinase or said integrase; and    a nucleic acid molecule encoding said recombinase or said integrase.    
     
     
         2 . The method of  claim 1 , wherein said first genetically engineered cis-acting target is recognized by a recombinase.  
     
     
         3 . The method of  claim 1 , wherein said first genetically engineered cis-acting target is recognized by an integrase.  
     
     
         4 . The method of  claim 1 , wherein said vector comprises an origin of replication functioning in a mammalian cell.  
     
     
         5 . The method of  claim 1 , wherein said vector comprises an adenoviral sequence.  
     
     
         6 . The method of  claim 1 , wherein said vector is transfected in vitro into a host cell or a population of host cells, wherein said host cells are isolated from said patient.  
     
     
         7 . The method of  claim 1 , wherein the route of administration is selected from the group consisting of aerosolization, intravenous, intratracheal, intraperitoneal, intramuscular, subcutaneous, intratumoral, intrapulmonary, and intracranial administration.  
     
     
         8 . A method of gene therapy comprising the administration to a patient in need of gene therapy a therapeutically effective amount of a pharmaceutical composition, said composition comprising a biologically suitable vehicle for administration and a vector, said vector comprising, in the 5′ to 3′ direction, 
 a first genetically engineered cis-acting target recognized by a recombinase or an integrase;    a gene of interest;    a bi-directional promoter, comprising a second genetically engineered cis-acting target recognized by said recombinase or said integrase; and    a nucleic acid molecule encoding said recombinase or said integrase.    
     
     
         9 . The method of  claim 8 , wherein said first genetically engineered cis-acting target is recognized by a recombinase.  
     
     
         10 . The method of  claim 8 , wherein said first genetically engineered cis-acting target is recognized by an integrase.  
     
     
         11 . The method of  claim 8 , wherein said vector comprises an origin of replication functioning in a mammalian cell.  
     
     
         12 . The method of  claim 8 , wherein said vector comprises an adenoviral sequence.  
     
     
         13 . The method of  claim 8 , wherein said vector is transfected in vitro into a host cell or a population of host cells, wherein said host cells are isolated from said patient.  
     
     
         14 . The method of  claim 8 , wherein the route of administration is selected from the group consisting of aerosolization, intravenous, intratracheal, intraperitoneal, intramuscular, subcutaneous, intratumoral, intrapulmonary, and intracranial administration.  
     
     
         15 . A method of gene therapy comprising the administration to a patient in need of gene therapy a therapeutically effective amount of a population of cells transfected with a vector, said vector comprising, in the 5′ to 3′ direction, 
 a first genetically engineered cis-acting target recognized by a recombinase or an integrase;    a gene of interest;    a bi-directional promoter, comprising a second genetically engineered cis-acting target recognized by said recombinase or said integrase; and    a nucleic acid molecule encoding said recombinase or said integrase.    
     
     
         16 . The method of  claim 15 , wherein said first genetically engineered cis-acting target is recognized by a recombinase.  
     
     
         17 . The method of  claim 15 , wherein said first genetically engineered cis-acting target is recognized by an integrase.  
     
     
         18 . The method of  claim 15 , wherein said vector comprises an origin of replication functioning in a mammalian cell.  
     
     
         19 . The method of  claim 15 , wherein said vector comprises an adenoviral sequence.  
     
     
         20 . The method of  claim 15 , wherein the route of administration is intravenous administration.