REPLICATION OF REP-CAP GENES IS ESSENTIAL FOR THE HIGH-EFFICIENCY PRODUCTION OF RECOMBINANT AAV

Adenoassociated virus (AAV) has been developed as a vector for gene tr ansfer because of its advantageous features: it is nonpathogenic, natu rally replication-defective; it infects growth-arrested cells, and can transfer the therapeutic gene without co-delivery of any viral genes. However, a major obstacle in conducting systematic studies of AAV-med iated gene transfer in animal models is the difficulty of obtaining la rge quantities of recombinant virus. Recent development of AAV packagi ng cell lines has simplified the procedure of producing recombinant AA V (rAAV). However, the efficacy of producing large quantities of rAAV with these cell lines is yet to be demonstrated. In this study we have analyzed the difference between the replication of wild-type AAV and the production of rAAV. Using a combined single-plasmid system that ca rries both an AAV vector and the rep-cap genes, we have demonstrated t hat the AAV vector replicates to high number of copies whereas the rep -cap sequences remain unamplified in the virus-producing cells. When t he copy number of rep-cap genes was increased by varying the vector/re p-cap ratio in the transfection mixture, the titer of rAAV increased p roportionally. Thus, the titer of rAAV is limited by the low copy numb er of the rep-cap genes that results in an insufficient expression of the Rep and Cap proteins. We have also shown that generation of double -stranded replicating form of the vector DNA is accompanied by an ampl ified transgene expression. We propose that the increased gene express ion from the accumulating double-stranded viral DNA is likely to be th e mechanism by which wild-type AAV produces a large number of particle s necessary to package the self-replicating AAV genomes. We conclude t hat mimicking this amplified expression of rep-cap genes may provide t he key to produce high titers of rAAV.