Intron Splicing–mediated Expression of AAV Rep and Cap Genes and Production of AAV Vectors in Insect Cells

An artificial intron containing the insect cell polyhedrin (polh) promoter was designed, constructed, and inserted into the adeno-associated virus (AAV) Rep and Cap coding sequences to express the Rep and Cap proteins, respectively. The artificial intron was spliced out and full-length Rep78 or VP1 proteins were expressed from the insect promoters located upstream of their respective AUG start codons. The polh promoter located inside the artificial intron was functional, expressed the Rep52 or VP2/VP3 proteins located downstream of the artificial intron, and overlapped with the Rep78 or VP1 proteins. This is the first report that an artificial intron containing an insect cell promoter can be inserted into a coding sequence to express genes with overlapping open-reading frames (ORFs). A method was also established for AAV vector production in insect cells with these intron-containing Rep and Cap coding sequences, and the vectors produced thereby were infectious. These intron-containing AAV Rep and Cap coding sequences were very stable in recombinant baculoviruses and showed no apparent loss of protein expression even after five consecutive amplifications of the plaque-purified recombinant baculoviruses. This newly established AAV production method should prove to be a useful tool for large-scale AAV vector production.