New adenovirus vectors for protein production and gene transfer

Based on two new adenovirus expression cassettes, we have constructed a ser ies of Ad transfer vectors for the overexpression of one or two genes eithe r in a dicistronic configuration or with separate expression cassettes. Inc lusion of the green or blue fluorescent protein in the vectors accelerates the generation of adenovirus recombinants and facilitates the functional ch aracterization of genes both in vitro and in vivo by allowing easy quantifi cation of gene transfer and expression. With our optimized tetracycline-reg ulated promoter (TR5) we have generated recombinant adenoviruses expressing proteins, that are either cytotoxic or which interfere with adenovirus rep lication, at levels of 10-15% of total cell protein. Proteins that are not cytotoxic can be produced at levels greater than 20% of total cell protein. As well, these levels of protein production can be achieved with or withou t adenovirus replication. This yield is similar to what can be obtained wit h our optimized human cytomegalovirus-immediate early promoter-enhancer (CM V5) for constitutive protein expression in non-complementing cell lines. Us ing the green fluorescent protein as a reporter, we have shown that a pAdCM V5-derived adenovirus vector expresses about 6-fold more protein in complem enting 293 cells and about 12-fold more in non-complementing HeLa cells tha n an adenovirus vector containing the standard cytomegalovirus promoter. Mo reover, a red-shifted variant of green fluorescent protein incorporated in one series of vectors was 12-fold more fluorescent than the S65T mutant, ma king the detection of the reporter protein possible at much lower levels of expression.