Recombinase-Based System for Expression of Foreign Proteins Using Adenovirus Vectors

Abstract

In the present invention, viruses, plasmids or both are constructed which contain viral DNA and lox sites positioned such that site-specific recombination between lox sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express the Cre recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system.

1 . A method for introducing and expressing a foreign nucleic acid sequence in a host cell, comprising: 
 (a) introducing into a mammalian host cell a first plasmid comprising a modified adenovirus genome having a modification within early region 1 (E1) sufficient to render said first plasmid unable to form viable viral particles in mammalian host cells, said modification eliminating susceptibility of said first plasmid to being encapsidated into a viral particle, said first plasmid further comprising at least one nucleic acid sequence for (A) encoding antibiotic resistance and (B) for replication of said modified adenovirus genome in bacterial host cells, and said first nucleic acid sequence further comprising at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase;    (b) introducing into said host cell recited in (a), an E1 shuttle plasmid that comprises a sequence comprising an adenovirus genome E1 region, comprising a packaging signal, and further comprising at least one recombinase recognition target site sufficiently identical with said recombinase recognition target site in said first nucleic acid as to be recognized by the same site-specific recombinase which recognizes said site-specific recombinase recognition target site in said first nucleic acid, and an inserted foreign nucleic acid sequence comprising a coding sequence having an open reading frame inserted into the E1 region and comprising sequences that regulate the expression of said open reading frame; and wherein a site-specific recombinase which recognizes said site-specific recombinase recognition target sites is either (i) expressed by a cell into which said first and said second nucleic acids are introduced, (ii) operatively encoded by said first nucleic acid, said second nucleic acid or both, or (iii) is provided in trans through expression from a third nucleic acid or is provided in trans as an active protein;    (c) isolating virus particles comprising a recombined modified adenoviral genome having elements of said first plasmid plus the packaging signal and the inserted foreign nucleic acid sequence of the E1 shuttle plasmid;    (d) introducing said recombinant modified viral genome into a host cell; and    (e) expressing the coding sequences contained in said modified recombinant viral genome.    
     
     
         2 . The method according to  claim 1  wherein said at least one site-specific recombinase recognition target site of said first nucleic acid sequence and said at least one site-specific recombinase recognition target site of said second nucleic acid sequence each is comprised of a loxP sequence, and wherein said site-specific recombinase is comprised of Cre.  
     
     
         3 . The method according to  claim 1  wherein said at least one site-specific recombinase recognition target site of said first nucleic acid sequence and said at least one site-specific recombinase recognition target site of said second nucleic acid sequence each is comprised of an frt sequence, and wherein said site-specific recombinase is comprised of FLP.  
     
     
         4 . A method for eliciting an immune response in a mammalian host to protect against an infection comprising administering a vaccine composition comprising the recombined modified adenoviral genome of  claim 1 .  
     
     
         5 . A vaccine for protecting a mammalian host against infection comprising: (a) the isolated virus particle of  claim 1  comprising the recombined modified adenoviral genome wherein the inserted foreign nucleic acid encodes an antigenic determinant produced by a disease organism responsible for the infection; and (b) a pharmaceutically acceptable excipient.  
     
     
         6 . A composition comprising the recombination product of a first plasmid comprising a modified adenovirus genome having a modification within early region 1 (E1) sufficient to render said first plasmid unable to form viable viral particles in mammalian host cells, said modification eliminating susceptibility of said first plasmid to being encapsidated into a viral particle, said first plasmid further comprising at least one nucleic acid sequence for (A) encoding antibiotic resistance and (B) for replication of said modified adenovirus genome in bacterial host cells, and said first nucleic acid sequence further comprising at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase and a second plasmid that comprises a sequence comprising an adenovirus genome E1 region, comprising a packaging signal, and further comprising at least one recombinase recognition target site sufficiently identical with said recombinase recognition target site in said first nucleic acid as to be recognized by the same site-specific recombinase which recognizes said site-specific recombinase recognition target site in said first nucleic acid, and an inserted foreign nucleic acid sequence comprising a coding sequence having an open reading frame inserted into the E1 region and comprising sequences that regulate the expression of said open reading frame; and wherein a site-specific recombinase which recognizes said site-specific recombinase recognition target sites is either (i) expressed by a cell into which said first and said second nucleic acids are introduced, (ii) operatively encoded by said first nucleic acid, said second nucleic acid or both, or (iii) is provided in trans through expression from a third nucleic acid or is provided in trans as an active protein.