High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H

Production of high-titer rAAV is essential for in vivo clinical application . One limiting factor may be the failure of existing systems to replicate t he packaging genome in such a way that expression of Rep and Cap proteins i s coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genet ically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In t his study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen i n wtAAV infection by incorporating both these and vector sequences in HSV a mplicons, Facilitated production of AAV Rep and Cap proteins translates int o a considerably improved recovery of rAAV, which transduces cells of the n euroretina in vivo with high efficiency, The potential for contamination wi th infectious herpes particles is eliminated by the use of noncomplementing (gH(-)) cell lines to propagate the virus, and by standard purification me thods. The use of DISC-HSV and herpes-derived amplicons for production of r AAV may be a useful strategy for future in vivo studies and for clinical ap plication.