541. Use of Endogenous Viral Elements to Deduce the Sequence of an Ancient Adeno-Associated Virus That Circulated Amongst Australian Marsupials ~30 Million Years Ago

Vectors based on adeno-associated viruses (AAVs) have been successfully applied in clinical trials and pre-clinical settings. AAV capsid proteins are the primary determinants of vector tropism and immunogenicity, and are continually being developed to impart desirable properties, such as immune evasion and specificity for selected target tissues in humans and those of model organisms. One approach that was recently adopted by at least two groups to generate novel capsid variants is the computational deduction of putative ancestral AAV sequences, based upon observable genetic diversity amongst closely related contemporary AAV isolates. Such inferred ancestral sequences potentially recapitulate the evolutionary history of AAVs for hundreds or possibly thousands of years. Germline endogenous viral elements (EVEs) can compensate for the lack of a viral fossil record by genetically preserving viral nucleotide sequences over a geologic time span. Here we describe an AAV-EVE (mAAV-EVE1) that is lineage-restricted to the germlines of members of the Australian marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian marsupial lineages. Orthologous mAAV-EVE1 sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated in silico reconstruction of an inferred ancestral AAV sequence. This sequence represents the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. The deduced ancient AAV bears remarkable resemblance to modern AAVs in its predicted structural and non-structural genes, underscoring the concept that despite their relatively rapid mutation rates, AAVs have evolved under tight constraints imposed by both form and function. Moreover, the evolutionary history of mAAV-EVE1 since its integration into the pro-macropodoid lineage reflects that of the respective host species. In addition to providing an inferred ancient AAV capsid sequence for future transduction studies, this sequence can serve as input for the generation of further novel capsids by approaches such as capsid shuffling and directed capsid engineering/evolution. Furthermore, by comparing it to contemporary AAV capsids, this sequence can inform rational capsid design approaches by providing insights into long-term capsid evolution.