RECOMBINANT ADENOASSOCIATED VIRUS TYPE-2 REPLICATION AND PACKAGING ISENTIRELY SUPPORTED BY A HERPES-SIMPLEX VIRUS TYPE-1 AMPLICON EXPRESSING REP AND CAP

Recombinant adeno-associated virus (AAV) type 2 (rAAV) vectors have re cently been shown to have great utility as gene transfer agents both i n vitro and in vivo. One of the problems associated with the use of rA AV vectors has been the difficulty of large-scale vector production, L ow-efficiency plasmid transfection of the rAAV vector and complementin g AAV type 2 (AAV-2) functions (rep and cap) followed by superinfectio n with adenovirus has been the standard approach to rAAV production, T he objectives of this study were to demonstrate the ability of a recom binant herpes simplex virus type 1 (HSV-1) amplicon expressing AAV-2 R ep and Cap to support replication and packaging of rAAV vectors, HSV-1 amplicon vectors were constructed which contain the AAV-2 rep and cap genes under control of their native promoters (p5, p19, and p40). An HSV-1 amplicon vector, HSV-RC/KOS or HSV-RC/d27, was generated by supp lying helper functions with either wild-type HSV-1 (KOS strain) or the ICP27-deleted mutant of HSV-1, d27-1, respectively, Replication of th e amplicon stocks is not inhibited by the presence of AAV-2 Rep protei ns, which highlights important differences between HSV-1 and adenoviru s replication and the mechanism of providing helper function for produ ctive AAV infection, Coinfection of rAAV and HSV-RC/KOS resulted in th e replication and amplification of rAAV genomes. Similarly, rescue and replication of rAAV genomes occurred when rAAV vector plasmids were t ransfected into cells followed by HSV-RC/KOS infection and when two rA AV proviral cell lines were infected with HSV-RC/KOS or HSV-RC/d27, Pr oduction of infectious rAAV by rescue from two rAAV proviral cell line s has also been achieved with HSV-RC/KOS and HSV-RC/d27, The particle titer of rAAV produced with HSV-RC/d27 is equal to that achieved by su pplying rep and cap by transfection followed by adenovirus superinfect ion, Importantly, no detectable wild-type AAV-2 is generated with this approach, These results demonstrate that an HSV-1 amplicon expressing the AAV-2 genes rep and cap along with HSV-1 helper functions support s the replication and packaging of rAAV vectors in a scaleable process .