Efficient generation of adenovirus-based libraries by positive selection of adenoviral recombinants through ectopic expression of the adenovirus protease

Abstract

Disclosed is a new system for generating recombinant adenovirus vectors and adenovirus-based expression libraries, by positive selection of recombinants deleted for the endogenous protease gene, which gene is expressibly cloned into another region of the adenoviral genome. In a preferred embodiment, the invention allows positive selection of E1-deleted, protease-deleted recombinant adenovirus vectors comprising an exogenous gene or an expressible piece of exogenous DNA, by providing an expression cassette comprising the protease gene and the exogenous DNA inserted in place of E1 region in a shuttle vector. In vivo recombination of the shuttle vector with a protease-deleted adenoviral genome in suitable non-complementing cells generates viable recombinants only when rescuing the protease cloned in E1 region. Non-recombinant viral genomes are not able to grow due to the deletion of the protease gene, ensuring that only recombinant viral plaques are generated. This positive selection can be used for the generation of a large number of high purity recombinant adenovirus vectors and allows generation of adenovirus-based libraries with diversity exceeding 10 6 clones.

1 . An adenoviral expression library comprising a plurality of recombinant adenoviruses, each recombinant adenovirus being deleted for an essential gene of a late transcriptional region of adenoviral genome and having the essential gene expressibly cloned in a second transcriptional region of adenoviral genome, each recombinant adenovirus further comprising an expressible piece of exogenous DNA.  
     
     
         2 . An adenoviral expression library according to claim  50 , wherein the essential gene of a late transcriptional region is the gene of adenovirus protease.  
     
     
         3 . An adenoviral expression library according to claim  51 , wherein the second transcriptional region is an early transcriptional region.  
     
     
         4 . An adenoviral expression library according to claim  52 , wherein the early transcriptional region is selected from the group consisting of E1, E2, E3 and E4 transcriptional regions.  
     
     
         5 . An adenoviral expression library according to claim  53 , wherein the early transcriptional region is E1 transcriptional region.  
     
     
         6 . An adenoviral expression library according to claim  51 , wherein the essential gene is a part of a first expression cassette.  
     
     
         7 . An adenoviral expression library according to claim  55 , wherein the expressible piece of exogenous DNA is a part of the first expression cassette.  
     
     
         8 . An adenoviral expression library according to claim  56 , wherein the first expression cassette is a dicistronic cassette.  
     
     
         9 . An adenoviral expression library according to claim  57 , wherein the first expression cassette comprises a regulatable promoter.  
     
     
         10 . An adenoviral expression library according to claim  58 , wherein the regulatable promoter is an inducible promoter.  
     
     
         11 . An adenoviral expression library according to claim  59 , wherein the inducible promoter is a tetracycline-inducible promoter.  
     
     
         12 . An adenoviral expression library according to claim  51 , wherein the expressible piece of exogenous DNA is a part of a second expression cassette.  
     
     
         13 . An adenoviral expression library according to claim  61 , wherein the second expression cassette comprises a regulatable promoter.  
     
     
         14 . An adenoviral expression library according to claim  62 , wherein the regulatable promoter is an inducible promoter.  
     
     
         15 . An adenoviral expression library according to claim  63 , wherein the inducible promoter is a tetracycline-inducible promoter.  
     
     
         16 . An adenoviral expression library according to claim  51 , wherein the expressible piece of exogenous DNA is derived from a DNA library.  
     
     
         17 . An adenoviral expression library according to claim  51 , wherein the expressible piece of exogenous DNA is a DNA fragment expressing antisense RNA fragment for a protein gene or a cis-acting element regulating gene expression.  
     
     
         18 . An adenoviral expression library according to claim  66 , wherein the cis-acting element regulating gene expression is selected from the group consisting of promoters (TATA boxes), enhancers, suppressers, IRES, polyA, termination sequences, and UTR sequences of messages that regulate the stability and/or transport of mRNA.  
     
     
         19 . An adenoviral expression library according to claim  51 , said library having diversity of at least 10 3  clones.  
     
     
         20 . An adenoviral expression library according to claim  62 , said library having diversity of at least 10 6  clones.