PRODUCTION AND CHARACTERIZATION OF IMPROVED ADENOVIRUS VECTORS WITH THE E1, E2B, AND E3 GENES DELETED

Adeno virus (Ad)-based vectors have great potential for use in the gen e therapy of multiple diseases, both genetic and nongenetic. While cap able of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems, Most Ad vectors are engineered such that a transgene replaces the rid E1a, E1b, and E3 ge nes; subsequently the replication-defective vector can be propagated o nly in human 293 cells that supply the deleted El gene functions in tr ans, Unfortunately, the use of high titers of El deleted vectors has b een repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of re plication-competent Ad (RCA) by recombination events with the E1 seque nces residing in 293 cells further limits the usefulness of E1-deleted Ad vectors, We addressed these problems by isolating new Ad vectors d eleted for the E1, E3, and the E2b gene functions, The new vectors can be readily grown to high titers and have several improvements, includ ing an increased carrying capacity and a theoretically decreased risk for generating RCA, We have also demonstrated that the further block t o Ad vector replication afforded by the deletion of both the E1 and E2 b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the m odified vector genome, The results suggested that these modified vecto rs may be very useful both for in vitro and in vivo gene therapy appli cations.