Transposon-based mutagenesis generates diverse adeno-associated viral libraries with novel gene delivery properties.

The engineering of novel properties and functions into viral vectors for improved gene delivery remains a barrier to the development of efficient, customized gene delivery vehicles. Rational methods for designing improved viral vectors are often experimentally challenging and laborious, particularly when knowledge of viral structure-function relationships is limited. As an alternative, high-throughput libraries may be rapidly and efficiently selected for viral variants with a desired function. Here we describe a transposon-based insertional mutagenesis approach to generate large diverse adeno- associated viral (AAV) libraries containing a randomly located peptide. Briefly, a selectable marker is randomly inserted throughout the AAV2 cap gene and the resulting "bookmarked' AAV cap gene is cloned into an AAV packaging vector. The selectable marker is then replaced with a defined oligonucleotide, and the final AAV library is used to package a diverse pool of AAV virions, which can used for functional selection.