Generation of Replication Competent Viruses for Therapeutic Use

A replication competent virus containing a functional insertion site identified by a method selected from the group consisting of:

• (1) a method comprising steps of:
• (1a) mixing genomic DNA of a target virus with a donor DNA comprising a transposon under conditions that allow transposition wherein the transposon comprises at least one genetic element of interest and inserts into the viral genome in a non-biased manner;
• and
• (1b) isolating replication competent viruses from the step (1a) which express the genetic element of interest;
• (2) a method comprising steps of:
• (2a) mixing a plasmid comprising the genomic DNA of the target virus with a plasmid donor DNA comprising a transposon and an origin of replication under conditions that allow transposition wherein the transposon comprises at least one genetic element of interest operably linked to an expression element and a selectable gene and inserts into the target viral genome in a non-biased manner;
• and
• (2b) isolating replication competent viruses from the step (2a) which express the genetic element of interest;
• and
• (3) a method comprising steps of:
• (3a) mixing a plasmid comprising the genomic DNA of the Ad5 virus with a plasmid donor DNA comprising a Tn-7 based transposon and an R6K origin of replication under conditions that allow transposition wherein the transposon comprises a gene encoding GFP operably linked to an expression element and a chloramphenicol resistance gene and wherein the transposon inserts into the target Ad5 genome in a non-biased manner;
• and
• (3b) isolating replication competent Ad5 viruses which express GFP from the step (3a).