Efficient production of adenovirus vector lacking genes of virus-associated RNAs that disturb cellular RNAi machinery

First-generation adenovirus vectors (FG AdVs) are widely used in basic studies and gene therapy. However, virus-associated (VA) RNAs that act as small-interference RNAs are indeed transcribed from the vector genome. These VA RNAs can trigger the innate immune response. Moreover, VA RNAs are processed to functional viral miRNAs and disturb the expressions of numerous cellular genes. Therefore, VA-deleted AdVs lacking VA RNA genes would be advantageous for basic studies, both in vitro and in vivo. Here, we describe an efficient method of producing VA-deleted AdVs. First, a VA RNA-substituted “pre-vector” lacking the original VA RNA genes but alternatively possessing an intact VA RNA region flanked by a pair of FRTs was constructed. VA-deleted AdVs were efficiently obtained by infecting 293hde12 cells, which highly express FLP, with the pre-vector. The resulting transduction titers of VA-deleted AdVs were sufficient for practical use. Therefore, VA-deleted AdVs may be substitute for current FG AdV.