Abstract:
Hybrid Adenovirus/Adeno-associated viruses (Ad/AAV) combine the capacity, tropism and ease of production of Ad with AAV's ability for site-specific integration (SSI) into chromosome 19 AAVS1. The AAV Rep78 protein is required for SSI although it displays an inhibitory effect on Ad replication, particularly when co-expressed within the Ad backbone. However, strategies to construct an Ad/AAV focused on controlling Rep expression have met with limited success. We hypothesized that Rep's apparently cis-acting inhibitory effect on Ad replication could either be due to increased expression accompanying an increase in copy number, or due to a role for the sequence of the Rep ORF. To elucidate the relative contribution of the Rep ORF sequence and Rep protein levels on inhibition, we modified the 1866bp Rep nucleotide sequence in silico using synonymous codons. We generated two Rep coding sequences, Scrambled and Deoptimized which differed from the wild-type Rep78 nucleotide sequence by 20-30%, while encoding exactly the same protein. The Deoptimized sequence specifically uses codons in underrepresented pairs, expressing Rep78 protein at reduced levels due to codon pair bias. Codon pair bias refers to the preference for some codon pairs over other synonymous codons to encode the same pair of adjacent amino acids. Utilization of underrepresented codon pairs results in an ORF that is expressed at reduced levels, due to inefficient translation. Expression of the Scrambled, Deoptimized and wild-type Rep 78 ORFs within a first generation Adenovirus backbone (Ad/Scr, Ad/Deopt and Ad/wtRep) revealed dramatic results. Where Ad/wtRep was incapable of replication, Ad/Scr and Ad/Deopt replicated as well as any other first generation Ad, indicating a clear role for a sequence specific signal in the inhibition of Ad replication. Modification of this signal allowed tolerance of a high level of Rep protein expression. The signal was localized to a ~135bp sequence within the Rep ORF. The identification of a sequence specific inhibitory signal for AAV Rep mediated inhibition of Ad replication explains the inconsistent and often frustrating results obtained with production of Ad/AAV over the years and paves the way for large scale production of integrating Ad/AAV
