ANALYSIS OF RECOMBINANT ADENOASSOCIATED VIRUS PACKAGING AND REQUIREMENTS FOR REP AND CAP GENE-PRODUCTS

Adeno-associated virus (AAV) is a human parvovirus currently being dev eloped as a vector for gene therapy applications. Because the gene tra nsfer vector commonly retains only the AAV terminal repeats, propagati on of recombinant AAV (rAAV) requires that the viral replication (Rep) and capsid (Cap) proteins be supplied in traits. In an effort to opti mize the production of these vectors, a panel of helper plasmids was c onstructed to determine if expression of the rep and/or cap genes is a limiting factor for rAAV packaging. Expression of the Rep and Cap pro teins was increased by replacing the endogenous AAV promoters, p5 and p40, with the Rous sarcoma virus (RSV) long terminal repeat (LTR) and the cytomegalovirus immediate early promoter, respectively. Increased synthesis of the Cap proteins resulted in an approximately 10-fold inc rease in the yield of rAAV, indicating that production of capsid prote ins is one limiting factor for rAAV packaging. Expression of the rep g ene from the RSV LTR not only failed to increase the yield of rAAV but also prevented activation of p40 transcription with adenovirus infect ion, resulting in a reduced level of capsid protein synthesis.