Efficient Construction of a Recombinant Adenovirus Vector by an Improved In Vitro Ligation Method

Abstract

An efficient method for constructing a recombinant adenovirus (Ad) vector, based on an in vitro ligation, has been developed. To insert the foreign gene into an adenoviral DNA, we introduced three unique restriction sites, I-CeuI, SwaI, and PI-SceI, into the E1 deletion site of the vector plasmid, which contains a complete E1, E3-deleted adenovirus type 5 genome. I-CeuI and PI-SceI are intron-encoded endonucleases with a sequence specificity of at least 9–10 and 11 bp, respectively. A shuttle plasmid, pHM3, containing multiple cloning sites between the I-CeuI and PI-SceI sites, was constructed. After the gene of interest was inserted into this shuttle plasmid, the plasmid for E1-deleted adenovirus vector could be easily prepared by in vitro ligation using the I-CeuI and PI-SceI sites. SwaI digestion of the ligation products prevented the production of a plasmid containing a parental adenovirus genome (null vector). After transformation into E. coli, more than 90% of the transformants had the correct insert. To make the vector, a PacI-digested, linearized plasmid was transfected into 293 cells, resulting in a homogeneous population of recombinant virus. The large number and strategic location of the unique restriction sites will not only increase the rapidity of production of new first-generation vectors for gene transfer but will allow for rapid further improvements in the vector DNA backbone. One of the limitations of recombinant adenovirus vectors is their construction, which is a time-consuming procedure. This study demonstrates that the plasmid containing recombinant adenovirus DNA can be prepared by a simple in vitro ligation using three unique restriction sites, I-CeuI, SwaI, and PI-SceI, in the E1 deletion region. PacI digestion of the recombinant plasmid generates the DNA for adenovirus vector, which has an inverted terminal repeat at both ends of the genome. Homogeneous recombinant virus could be obtained by the transfection of the linearized plasmid into 293 cells. This improved in vitro ligation system is a simple and efficient method by which to construct a recombinant adenovirus vector for gene therapy.