CHARACTERIZATION OF WILD-TYPE ADENOASSOCIATED VIRUS TYPE 2-LIKE PARTICLES GENERATED DURING RECOMBINANT VIRAL VECTOR PRODUCTION AND STRATEGIES FOR THEIR ELIMINATION

The pSub201-pAAV/Ad plasmid cotransfection system was developed to eli minate homologous recombination which leads to generation of the wild- type (wt) adeno-associated virus type 2 (AAV) during recombinant vecto r production. The extent of contamination with wt AAV has been documen ted to range between 0.01 and 10%. However, the precise mechanism of g eneration of the contaminating wt AAV remains unclear. To characterize the wt AAV genomes, recombinant viral stocks were used to infect huma n 293 cells in the presence of adenovirus, Southern blot analyses of v iral replicative DNA intermediates revealed that the contaminating AAV genomes were not authentic wt but rather wt AAV-like sequences derive d from recombination between (i) AAV inverted terminal repeats (ITRs) in the recombinant plasmid and (ii) AAV sequences in the helper plasmi d. Replicative AAV DNA fragments, isolated following amplification thr ough four successive rounds of amplification in adenovirus-infected 29 3 cells, were molecularly cloned and subjected to nucleotide sequencin g to identify the recombinant junctions. Following sequence analyses o f 31 different ends of AAV-like genomes derived from two different rec ombinant vector stocks, we observed that all recombination events invo lved 10 nucleotides in the AAV D sequence distal to viral hairpin stru ctures. We have recently documented that the first 10 nucleotides in t he D sequence proximal to the AAV hairpin structures are essential for successful replication and encapsidation of the viral genome (X.-S. W ang et al., J. Virol. 71:3077-3082, 1997), and it was noteworthy that in each recombinant junction sequenced, the same 10 nucleotides were r etained. We also observed that adenovirus ITRs in the helper plasmid w ere involved in illegitimate recombination with AAV ITRs, deletions of which significantly reduced the extent of wt AAV-like particles. Furt hermore, the combined use of recombinant AAV plasmids lacking the dist al 10 nucleotides in the D sequence and helper plasmids lacking the ad enovirus ITRs led to complete elimination of replication-competent wt AAV-like particles in recombinant vector stocks. These strategies shou ld be useful in producing clinical-grade AAV vectors suitable for huma n gene therapy.