484. Murine Liver as a Model for AAV Replication, Recombination and Evolution

Abstract

Recent isolation and characterization of a family of novel adeno- associated viruses (AAVs) by our laboratory suggested that AAVs are highly prevalent in primate populations and undergo evolution through mechanisms such as intra-genome recombination. In an attempt to further understand the process of AAV evolution, we evaluated experimental models for AAV recombination. Murine liver is a good target for transduction by AAV and adenovirus vectors and, therefore, could be a potential model for our study. We proposed that the recombination could occur during and after replication of incoming single stranded AAV genomes. The ability of murine liver to support AAV replication becomes a critical qualification for its use as a model to study AAV recombination. Preliminary data generated from immune competent mice indeed suggested that wild type AAV genomes could be sufficiently replicated and possibly packaged in mouse liver (as measured by virus genome and infectivity titration, and DNA hybridization analysis) when co-injected with human adenovirus serotype 5. Experiments to characterize kinetics of AAV replication and dose response to helper adenovirus are under way. Co-administration of either AAV 1 and 8 or AAV1 and AAV2 together with adenovirus helper to the liver of C57BL/6 mice and passage of the resulted liver homogenate onto 293 cells led to recovery of AAV sequences that with either of the two input AAVs (90% of all genomes characterized) indicating that both viruses replicated and were packaged. Importantly, about 10% of the genomes were hybrids between the two AAV serotypes indicating in vivo intertypic recombination. The AAV clones were primarily screened by restriction mapping and representative clones were characterized by sequencing. Our preliminary data suggested that variant clones contained AAV genomic sequences derived from either point mutations or recombination/domain swapping of input parental viral genomes. Our study suggested that murine liver could be a potential model to study AAV biology and evolution.